Vaccination in newborns and infants

Abstract

To provide vaccines that effectively evoke immune responses in newborns and / or infants.SOLUTION: The present invention relates to vaccines comprising at least one mRNA encoding at least one antigen for use in the treatment of a disease in newborns and / or infants, preferably exhibiting an age of 2 years or less, preferably of 1 year or less, further preferably of 9 months or less, or even 6 months or less, where the treatment comprises vaccination of the newborn or infant, the vaccines eliciting an immune response in the newborn or infant. The present invention further relates to kits and kits of parts comprising such a vaccine and its components, and to methods of applying such a vaccine or kit.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a vaccine comprising at least one mRNA encoding at least one antigen for use in the treatment of a disease in newborns and / or infants, preferably aged 2 years or less, preferably 1 year or less, more preferably 9 months or less or even 6 months or less, wherein said treatment comprises vaccination of the newborn or infant to elicit an immune response in said newborn or infant. The present invention is further directed to kits and kits of parts comprising such vaccines and components thereof, as well as methods of applying such vaccines or kits. [Background technology]

[0002] The diagnosis, prevention, and treatment of infectious diseases and allergies in newborns and infants are topics of great interest and increasingly intense research worldwide. In this context, a deep understanding of the mechanisms of the immune system in newborns and infants is crucial. As is widely known, the primary role of the immune system is to defend living organisms against pathogens, but the immune system's response to such pathogens is not uniform throughout life. It is further known that immune system responses undergo age-related changes. Similarly, the immune system responses of newborns and infants are not equivalent to those of adults. In particular, T cell and B cell responses differ in many aspects, which in fact contribute to the prenatal requirements of the fetal immune system and the transition to external conditions at birth.

[0003] As is well known, all organ systems of the body undergo a dramatic transition at birth from a protected intrauterine life to a radically different environment in the outside world. This acute transition is followed by a gradual, age-dependent maturation. As discussed by [PubMed], fetal and neonatal immune systems are typically engaged in a three-part physiological demand: defending against infections, including viral and bacterial pathogens, at the maternal-fetal interface; avoiding proinflammatory / T helper 1 (Th1) cell-polarizing responses that could potentially induce harmful maternal-fetal alloimmune responses; and mediating the transition from the normally sterile intrauterine environment, including early colonization of the skin and intestinal tract by microorganisms, to an external, foreign antigen-rich environment. Given limited antigen exposure in utero and well-documented defects in neonatal adaptive immunity, neonates must rely to a significant extent on the innate immune system for protection. Because the innate immune system can direct adaptive immune responses, the distinctive functional expression of neonatal innate immunity, including a bias toward Th1 cell-polarizing cytokines, contributes to the distinctive pattern of neonatal adaptive immune responses. Mounting evidence indicates that infection-induced production of proinflammatory / Th1 cell-polarizing cytokines, including tumor necrosis factor (TNF) and interleukin-1β (IL-1β), is associated with preterm labor and birth. In particular, TNF production is thought to support abortion through the induction of apoptosis in placental and fetal cells. The ability of inflammatory cytokines to induce spontaneous abortion appears to be an important reason for the strong bias toward Th2 cell-polarizing cytokines in the maternal and fetal immune systems of several mammalian species (see Non-Patent Document 2, supra).

[0004] Because of this impaired production of Th1 cell-associated cytokines, it was initially thought that the innate immune system of newborns was generally impaired or weakened. However, the production of certain cytokines (e.g., IL-6, IL-10, and IL-23) induced by stimulation of neonatal monocytes and antigen-presenting cells (APCs) actually exceeds that of adults (see Non-Patent Documents 3-5). Nevertheless, a bias toward Th1 cell-polarizing cytokines still appears, leaving newborns susceptible to microbial infections and contributing to the impaired immune response of newborns to most vaccines, thereby hindering efforts to protect this susceptible population. After birth, there is an age-dependent maturation of the immune response. As is known, prenatal and postnatal exposure to environmental microbial products that can activate innate immunity can accelerate this maturation process, reducing Th2 cell polarization and / or enhancing Th1 cell polarization, particularly if exposure occurs repeatedly over time, potentially reducing allergies and atopy according to the hygiene hypothesis (see again Non-Patent Document 2, supra).

[0005] T cell-mediated immune responses in young adults, particularly neonates, have been reviewed by Non-Patent Document 6. As described there, circulating neonatal T lymphocytes are fundamentally different from adult naive T cells and have the properties of thymic emigrant cells. They contain high concentrations of T cell receptor excision circles (TRECs), which are episomal DNA by-products of TCR α chain rearrangement that are not replicated but diluted during cell division. Like adult naive cells, most neonatal T lymphocytes are CD45RA +Neonatal lymphocytes express CD38 isoforms, as well as the costimulatory molecules CD27 and CD28. In contrast to adult naive lymphocytes, neonatal lymphocytes express the CD38 molecule. In addition, a high proportion of circulating neonatal T cells circulate and show increased susceptibility to apoptosis, indicative of high cell turnover. Naive T lymphocyte proliferation can also be detected during fetal life and can persist up to 5 years of age. The high cell turnover observed in early life likely plays a central role in establishing the T cell repertoire. Despite their high turnover, T cells retain long telomere sequences through high constitutive telomerase activity. In vitro apoptosis of neonatal T lymphocytes can be prevented by cytokine signaling via the γ chain of the IL-2 receptor, namely IL-2, IL-4, IL-7, and IL-15. Among these cytokines, IL-7 and IL-15 also induce neonatal T lymphocyte proliferation in the absence of other stimuli. IL-7 is involved in thymocyte development at a stage preceding T cell receptor rearrangement. IL-15 stimulates the proliferation of CD8 cells preferentially over CD4+ T cells. In contrast to IL-7, IL-15 induces the differentiation of CD8+ T lymphocytes in vitro (see Non-Patent Document 6, supra).

[0006] Several mechanisms limit T helper 1 (Th1)-type responses in early life. In utero, Th1 responses are toxic to the placenta and inhibited by trophoblast-derived IL-10 and progesterone. At birth, Th1 responses are still smaller in magnitude than later in life. In vitro, neonatal CD4+ T cells produce lower levels of IFNγ than naive adult T cells, and CpG and non-CpG sites within the IFNγ promoter are hypermethylated. In the presence of suboptimal CD28 costimulation, IL-12 stimulates the production of both IL-4 and IFNγ by neonatal CD4 T lymphocytes, whereas adult cells do not produce IL-4 under similar conditions. In response to polyclonal or superantigen activation, postnatal thymocytes develop into Th2 cytokine-producing CD4 T cells, but IL-12 is required to stimulate IFNγ production. In contrast, neonatal CD8+ T cells produce similar levels of IFNγ and have IFNγ promoter methylation patterns comparable to those of naive adult cells. Additionally, neonatal CD8 T lymphocytes strictly depend on the presence of IL-4 during priming to differentiate into IL-4-producing cells. The ability of neonatal CD4 T lymphocytes to express CD40 ligand (CD154), a molecule that plays a key role in the support of B lymphocytes and CD8+ T cells, remains controversial (see also Non-Patent Document 6, supra).

[0007] At the same time, available data indicate that naive T lymphocytes are programmed differently in neonates and adults. In summary, the ability of neonatal CD4+ T cells to produce IFNγ and the ability of neonatal DCs to promote Th1 responses are lower in in vitro studies compared with adults. Additionally, in vivo Th1 responses to many vaccines and infectious pathogens are low during early life. However, mature Th1 responses can be developed under certain conditions, such as neonatal BCG vaccination and Bordetella pertussis infection, possibly in association with more efficient DC activation. Therefore, the classic paradigm that neonates have incompetent T lymphocytes that only develop weak or tolerogenic responses needs to be reconsidered. The finding that mature cellular immune responses can be developed at an early age suggests that neonatal T lymphocytes can be directed to combat intracellular pathogens under appropriate stimulatory conditions (see also Non-Patent Document 6, supra).

[0008] Differences in not only T cell responses but also B cell responses appear to have profound effects on immune defense in neonates and / or infants compared with adults. The vulnerability of children younger than 18 to 24 months to encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae B (Hib), and Neisseria meningitidis has long been thought to reflect a general dysfunction in the generation of T cell-independent B cell responses to most bacterial polysaccharides (see Non-Patent Document 7). However, the immaturity of the immune system in neonates and / or infants also has a direct impact on the magnitude of antibody responses to T cell-dependent protein antigens. The mechanisms shaping B cell responses in early life have been investigated using a neonatal mouse immunization model developed to mimic the major limitations of immune responses to vaccines administered at an early age (see Non-Patent Document 7, supra). While there are few differences in human peripheral B cells, there are many differences between splenic B cells in neonates and adult mice. Specifically, human neonatal B cells express low levels of the costimulatory molecules CD40, CD80, and CD86, which diminishes their response to CD40 ligand (CD40L) and interleukin-10 (IL-10) expressed by their T cells. Infant B cells in the splenic marginal zone express low levels of CD21, which limits their ability to respond to polysaccharide-complement complexes. Expression of the key costimulatory receptor TACI (transmembrane activator and calcium-regulating cyclophilin ligand interactor, also known as TNFRSF13B) is also reduced in both neonatal mouse and human B cells, particularly in premature infants. Additionally, B cell responses in early life are influenced by many external factors. Maternal antibodies bind epitope-specifically to vaccine antigens, thus preventing infant B cells from accessing immunodominant vaccine epitopes. Furthermore, human and mouse neonates have low levels of serum complement component C3, which limits their response to antigen-C3d complexes. Human spleens contain fewer marginal zone macrophages (which play a key role in inducing antibody responses through the capture of particulate antigens) in newborns compared with adults, and the cellular cytokine-producing capacity differs.In infant mice, B cell responses are limited by a significant delay in the maturation of the follicular dendritic cell (FDC) network, resulting from the failure of FDC precursors to respond to B cell-mediated lymphotoxin-α signaling. Nuclear germinal center responses of FDCs, which attract antigen-specific B cells, retain antigens in the form of highly stimulatory immune complexes and provide signals that lead to somatic hypermutation and class switch recombination. The immaturity of the FDC network therefore delays the induction and limits the magnitude of germinal center responses, even when using potent adjuvants that induce adult-like B cell, T cell, and DC activation patterns. In addition to postnatal maturation of antibody responses, in vivo antibody persistence plays a key role. Long-term maintenance of specific antibodies with short half-lives requires the persistence of antibody-producing B cells, which can be continuously produced from the memory B cell pool or persist as long-lived plasma cells. Antibody-mediated memory B cell depletion, without affecting plasma cells, demonstrates that the plasma cell stage is independent of the memory B cell pool. It has also been shown that antibody persistence in vivo can be influenced by environmental factors, supporting the hypothesis that the limited persistence of antibody responses in early life is due to exposure to a large load of environmental antigens, which leads to competition for access to a limited set of plasma cells in the bone marrow for survival sites (see Non-Patent Document 7, supra).

[0009] As previously known, a single dose of vaccine administered at birth may fail to induce specific antibodies while simultaneously priming subsequent secondary responses, indicating that the neonatal differentiation pathway favors memory B cells over plasma cells. Several factors appear to contribute to this B cell differentiation pattern. The fate of antigen-specific naive B cells and their differentiation into short-lived plasma cells, long-lived plasma cells, or memory B cells are controlled by early B cell activation signals. While intermediate-affinity B cells persist as memory B cells in secondary lymphoid organs, high-affinity B cells are actively recruited to the plasma cell pool. Therefore, reduced affinity of early B cell receptors and / or delayed affinity maturation of neonatal naive B cells may reduce the strength of signals supporting memory B cell differentiation.

[0010] The restricted expression of CD21 by infant B cells also supports the generation of memory B cells and impairs plasma cell differentiation. This is supported by CD40-mediated signaling, cytokines such as IL-21, and interactions with ligands such as B cell-activating factor (BAFF, also known as TNFSF13B) and APRIL. Surprisingly, all of these plasma cell-supporting factors are expressed at low levels in early life unless additional activation signals are provided to enhance DC and T cell activation. Furthermore, early life B cells must compete for limited resources within germinal centers, which impairs antibody responses. Plasma cell differentiation is thus "forced" in early life by providing additional DC activation signals. Thus, a combination of factors appears to result in a pattern of preferential differentiation of early life B cells toward memory B cells instead of long-lived plasma cells. Importantly, although it has been shown that a pool of memory B cells can be formed early in life, this should not be taken as evidence that its size or persistence is similar to that induced in immunologically mature hosts. The recent finding that booster vaccines fail to induce memory responses in adolescents or young adults primed against hepatitis B virus in infancy suggests that memory B cells induced in infants do not persist throughout life. Whether this reflects a smaller pool of B cells in infants and / or the influence of as yet undefined homeostatic factors remains to be determined. In summary, numerous B cell internal and extrinsic determinants appear to cooperate to limit the induction and persistence of antibody-secreting plasma cells in early life while simultaneously supporting the preferential induction of memory B cell responses (see Non-Patent Document 7, supra).

[0011] Changes in B cell responses in addition to T cell responses, as mentioned above, have a significant effect not only on immune defense against pathogens, but also on vaccination strategies in newborns and / or infants in combating infectious diseases, and possibly allergies, autoimmune diseases, or further diseases. Many attempts have been made to provide efficient vaccines that may be able to overcome at least some of the limitations mentioned above.

[0012] One previous effort to overcome the deficiencies inherent in virus-derived influenza vaccines involved the administration of naked DNA plasmids (pHA) expressing hemagglutinin (HA) from the neuropathogenic strain A / WSN / 33 of influenza virus to prime protective immune responses by vaccinating newborn and adult mice (see Non-Patent Document 8). As shown by Non-Patent Document 8, continued exposure to small doses of antigen after DNA immunization of newborns can lead to the priming of specific B and Th cells rather than the induction of tolerance. However, pHA immunization of adult mice only primed a strongly biased Th1 response, whereas in newborns it induced a mixed Th1 / Th2 response. A further, very similar effort from the same research group involved the combined administration of plasmids expressing influenza virus nucleoprotein (NP) or hemagglutinin (HA). Neonatal immunization of BALB / c mice was followed by the priming of B cells, Th cells, and CTL rather than tolerance (see Non-Patent Document 9). However, complete protection in terms of survival against lethal challenge with homologous or heterologous strains has not been reported. Furthermore, in the case of NP-expressing plasmids, the protective immunity induced by neonatal immunization requires a longer time to develop compared to adult immunization. Neither [Non-Patent Document 8] nor [Non-Patent Document 9] demonstrated favorable Th1 responses in neonates. Furthermore, both papers emphasize that DNA vaccines represent an efficient and safe means for generating broad humoral and cellular immune responses to influenza viruses in the earliest stages of life. However, DNA poses risks due to unwanted insertions into the genome. Such DNA-based vaccinations may lead to disruption of functional genes and even cancer or the formation of anti-DNA antibodies, and therefore are no longer a focus of current research.

[0013] Further efforts have been directed to improving delivery systems and administration of immunostimulants to optimize vaccine responses in early life. Non-Patent Document 8 focuses on the problems arising from attempts to vaccinate against pathogens at a very early age and the role of selective adjuvants that can be used to (i) rapidly induce strong antibody responses of appropriate isotypes, (ii) elicit sustained antibody responses extending beyond infancy, (iii) induce efficient Th1 and CTL responses despite the predominant Th2 polarization of early-life responses, (iv) avoid inhibition of vaccine responses mediated by maternal antibodies, (v) demonstrate acceptable reactogenicity in early life, and (vi) allow the incorporation of multiple vaccine antigens into a single formulation to reduce the number of injections required (see Non-Patent Document 8). (2003) discusses, inter alia, the use of different antigen delivery systems, such as particulates, emulsions, liposomes, virosomes, microspheres, live vaccines, vector and DNA vaccine administration, as well as immunostimulants, such as MPL derivatives, QS21 derivatives, MDP derivatives, cytokines, interferons, and oligodeoxynucleotides, and combinations of antigen-presenting systems with immunostimulants. However, as also indicated in (2003) many of these combinations are hypothetical and may not even provide an efficient Th1 response or may even lead to undesirable side effects.

[0014] Similarly, Non-Patent Document 11 suggests the use of bacterial CpG motifs to activate immature antigen-presenting cells and enhance the neonatal immunogenicity of DNA vaccines. In addition, Non-Patent Document 11 suggests the combination with subsequent booster immunization using conventional vaccines. Nevertheless, the method outlined in this paper does not lead to a conclusive Th1 response. Furthermore, the method is based on the use of DNA vaccines, which can be considered potentially dangerous as outlined above.

[0015] Another promising, yet highly specific, approach relies on the use of a specific novel adjuvant, IC31. As is known in the art, only a few adjuvants are approved for human use. One major adjuvant approved for human use is alum, an aluminum salt-derived adjuvant. However, while approved for human use, aluminum salts and other adjuvants have failed to provide sufficient immune response enhancement to seasonal influenza vaccines in early human clinical trials, an effect that could be expected for other vaccination methods as well. Additional licensed adjuvanted influenza vaccines include Fluad® (Novartis Vaccines), which contains MF59 in combination with a subunit vaccine formulation; the virosome vaccine Inflexal® V (Berna Biotech, a Crucell company); and Invivac® (Solvay). Although animal studies and human clinical trials have demonstrated a higher immunogenicity profile, defined as increased antibody responses, with MF59-adjuvanted influenza vaccines, MF59 is not a potent adjuvant for the induction of type 1-induced cellular immune responses. Unlike Fluad®, virosome vaccines represent a reconstituted influenza virus envelope containing functional influenza surface proteins hemagglutinin and neuraminidase within a phospholipid bilayer. The immunogenicity and local tolerance of virosome-derived influenza vaccines have been demonstrated in several studies. However, the development of virosome formulations is highly complex and the cost of goods is high.

[0016] In this regard, Non-Patent Document 12 reported the use of a specific adjuvant, IC31, in combination with an Ag85b-ESAT-6 fusion protein for immunization of neonatal and adult mice. In contrast to alum, IC31H induced strong Th1 and Th17 responses, characterized by polyfunctional T cells expressing IL-2 and TNFα with or without IFNγ, in both age groups. In draining lymph nodes, similarly low numbers of DCs contained adjuvant and / or antigen following immunization of neonatal or adult mice. Expression of CD40, CD80, CD86, and IL-12p40 production were concentrated in the adjuvant-bearing DC population, where DC targeting / activation was similar in adult and neonatal mice. These DC / T cell responses resulted in a comparable reduction in bacterial growth following infection with mycobacterial BCG, whereas no protection was observed when alum was used as an adjuvant. However, no further adjuvants are presented in Non-Patent Document 12 that would allow the extension of this embodiment to other vaccines.

[0017] To summarize the above, none of the existing prior art vaccines allow for the effective induction of an immune response in newborns and / or infants, and exhibit at least similar characteristics to the immune response in adults. In particular, many vaccines are unable to provide an efficient Th1 immune response in newborns and / or infants. Therefore, there is an urgent need to optimize vaccines for such patients. More precisely, vaccines are needed that do not have the problems exhibited by the prior art, or at least alleviate these problems to a significant extent. Furthermore, it is highly anticipated to provide a vaccine that allows for the induction of a Th1 immune response in newborns and / or infants, and preferably does not induce a shift from a Th1 immune response to a Th2 immune response after administration. Similarly, the administration of DNA-based vaccines should be avoided due to possible DNA integration into the genome, possible genetic interference, and the formation of anti-DNA antibodies. [Prior art documents]

Non-licensed literature

[0018] [Non-licensed document 1] Ofer, NATURE REVIEWS | IMMUNOLOGY VOLUME 7 | MAY 2007 | 379 [Non-licensed document 2] Levy, 2007 [Non-licensed document 3] Angelone, D. et al., Pediatr. Res. 60, 205-209 (2006)

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[0019] The object underlying the present invention is more preferably solved by the subject matter of the appended claims as outlined below.

[0020] According to a first embodiment, the object underlying the present invention is solved by a vaccine comprising at least one mRNA encoding at least one antigen for use in the prevention and / or treatment of diseases in newborns and / or infants preferably representing an age of 2 years or less, preferably 1 year or less, more preferably 9 months or less or 6 months or less, characterised in that said treatment comprises vaccination of the newborn or early infant and induces an immune response in said newborn or infant.

[0021] Without being bound by theory, RNA vaccines precisely integrate adjuvant properties and antigen expression, thereby mimicking relevant aspects of viral infection. This improves their efficacy and simplifies handling and production compared to other inactivated (killed) vaccines, which require the use of advanced adjuvants in neonates or infants. RNA can address a range of dedicated immunological pattern recognition receptors, such as Toll-like receptor 3, Toll-like receptor 7, Toll-like receptor 8, RIG-I, MDA5, PKR, and others, which may act synergistically to enhance the induction of antigen-specific adaptive B and T cell responses. Importantly, by synthesizing antigens in transfected host cells, mRNA vaccines directly introduce antigens into the cellular antigen processing and presentation pathway, allowing access to MHC molecules and eliciting T cell responses, regardless of the host's MHC haplotype. This allows for the induction of polyclonal T cell responses, which can act synergistically with other immune responses, including B cells. Moreover, presentation of a full spectrum of MHC-binding epitopes may circumvent limitations imposed by the immature immune system of newborns or infants. Internal production of antigens also ensures accurate post-translational modifications (e.g., proteolytic processing and glycosylation) that can positively influence immunogenicity. RNA vaccines also exhibit safety features that make them superior for use in newborns and / or infants. For example, the increased reactogenicity of live attenuated vaccines generally precludes their use in this highly relevant target population. However, given the short persistence and traceless decay of vaccine vectors over a few days, the observed favorable immunogenicity is unexpected and contrasts with the requirement of plasmid DNA vaccines, whose efficacy is variably related to persistent antigen expression.

[0022] The at least one mRNA encoding at least one antigen of the vaccine of the present invention defined in the first embodiment of the present invention can be selected from any antigen known to those skilled in the art, preferably suitable for eliciting an antigen-specific immune response in a patient. According to the present invention, the term "antigen" refers to a substance that can be recognized by the immune system and induce an antigen-specific immune response, for example, by the formation of antibodies or antigen-specific T cells as part of the adaptive immune response. In this context, the first step in the adaptive immune response is the activation of different immune cells capable of inducing an antigen-specific immune response, either naive antigen-specific T cells or antigen-presenting cells. This occurs in lymphoid tissues and organs through which naive T cells continuously pass. Three types of cells that can function as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a different function in eliciting an immune response. Dendritic cells in tissues take up antigens by phagocytosis and macropinocytosis and, upon stimulation, for example, by contact with foreign antigens, migrate to local lymphoid tissues and differentiate into mature dendritic cells. Macrophages ingest particulate antigens, such as bacteria, and are induced by infectious agents or other appropriate stimuli to express MHC molecules. The unique ability of B cells to bind and absorb soluble protein antigens via their receptors is also important for inducing T cells. Presenting antigens on MHC molecules activates T cells, which induces their proliferation and differentiation into armed effector T cells. The most important functions of effector T cells are to cooperate to create cell-mediated immunity: killing of infected cells by CD8+ cytotoxic T cells and activation of macrophages by Th1 cells, and activation of B cells by both Th2 and Th1 cells to produce different classes of antibodies, thereby driving the humoral immune response. T cells do not directly recognize and bind antigens; instead, they recognize antigens via their T cell receptors, which recognize short peptide fragments, such as pathogen proteins, bound to MHC molecules on the surface of other cells.

[0023] In the context of the present invention, the antigen encoded by at least one mRNA of the vaccine of the present invention typically includes any antigen falling within the above definition, more preferably protein and peptide antigens. According to the present invention, the antigen encoded by at least one mRNA of the vaccine of the present invention may be an antigen produced extracellularly, more typically an antigen not derived from the host organism (e.g., human) itself (i.e., a non-self antigen), or more precisely, an antigen derived from a host cell outside the host organism, such as a pathogenic antigen, in particular a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an animal antigen (preferably selected from the animals or organisms disclosed herein), and an allergy antigen. The antigen encoded by at least one mRNA of the vaccine of the present invention may also be an antigen produced within a cell, tissue, or body, for example, by protein secretion, degradation, and metabolism. Such antigens include antigens derived from the host organism (e.g., human) itself, such as self-antigens or auto-antigens, such as tumor antigens and autoimmune autoantigens, as well as the above (non-self) antigens that originally originate from host cells outside the host organism but are fragmented or degraded within the body, tissues, or cells, for example, by (protease) degradation and metabolism.

[0024] Examples of pathogenic antigens include, in particular, antigens derived from influenza, preferably antigens derived from influenza A, influenza B, influenza C or Thogotovirus, preferably at least one of influenza antigens hemagglutinin (HA) and neuraminidase (NA), preferably influenza antigens derived from hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14 or H15 and neuraminidase subtypes N1, N2, N3, N4, N5, N6. , N7, N8 or N9 influenza antigens, or preferably those selected from influenza A subtypes H1N1, H1N2, H2N2, H2N3, H3N1, H3N2, H3N3, H5N1, H5N2, H7N7 or H9N2, or any further combinations, or those selected from matrix protein 1 (M1), ion channel protein M2 (M2) or nucleoprotein (NP), or antigens from respiratory syncytial virus (RSV), including for example F protein or G protein.

[0025] A further class of antigens encoded by at least one mRNA of the vaccine of the present invention includes allergy antigens. Allergy antigens are antigens that typically cause allergies in humans and may be derived from humans or other sources. Such allergy antigens can be selected from antigens derived from different sources, such as animals, plants, fungi, and bacteria. Related allergens also include antigens derived from grasses, pollen, molds, drugs, or numerous environmental factors. Allergy antigens typically belong to different classes of compounds, such as proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, and phospholipids. Of particular interest in the present invention are antigens encoded by at least one mRNA of the vaccine of the present invention, i.e., protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments encoding such protein antigens or protein antigens and their fragments or epitopes.

[0026] Particularly preferred animal-derived antigens encoded by at least one mRNA of the vaccine of the present invention include, but are not limited to, those derived from insects such as mites (e.g., house dust mites), mosquitoes, wasps (e.g., honeybees, bumblebees), cockroaches, ticks, moths (e.g., silkworm moths), midges, bugs, fleas, hunting wasps, caterpillars, fruit flies, migratory locusts, grasshoppers, ants and aphids; those derived from crustaceans such as shrimp, crab, krill, lobster, prawn, crayfish and scampi; duck, goose, seagull, turkey, ostrich and chicken. and other birds; fish such as eel, herring, carp, sea bream, cod, halibut, catfish, beluga, salmon, flounder, mackerel, cuttlefish and perch; mollusks such as scallops, octopus, abalone, snails, whelks, squid, clams and mussels; spiders; mammals such as cows, rabbits, sheep, lions, jaguars, leopards, rats, pigs, buffalo, dogs, loris, hamsters, guinea pigs, fallow deer, horses, cats, mice, ocelots and servals; arthropods such as spiders or silverfish; helminths such as nematodes; Trichinella species or roundworms; amphibians such as frogs; or sea squirts. Antigens of animal origin are also antigens contained in animal products, preferably antigens contained in animal products derived from the animals defined above, such as milk, eggs and meat, but may also include antigens derived from any kind of excreta or deposits from any of these animals.

[0027] Most preferably, particularly preferred antigens of animal origin encoded by at least one mRNA of the vaccine of the present invention may include antigens of such animals that cause a disease as defined herein, preferably an infectious disease or an autoimmune disease as defined herein, or any further disease as defined herein.

[0028] Plant-derived antigens encoded by at least one mRNA of the vaccines of the present invention include, but are not limited to, kiwi, pineapple, jackfruit, papaya, lemon, orange, mandarin, melon, persimmon, strawberry, lychee, apple, cherry paradise apple, and the like. Fruits such as apple, mango, passion fruit, plum, apricot, nectarine, pear, passion fruit, raspberry and grapes; vegetables such as garlic, onion, leek, soybean, celery, cauliflower, turnip, pepper, chickpea, fennel, zucchini, cucumber, carrot, yam, bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley, horseradish, chirimoya, beet, pumpkin and spinach; spices such as mustard, coriander, saffron, pepper and anise; oats, buckwheat, barley, rice, wheat, corn, rapeseed and sesame. from nuts such as cashew, walnut, butternut, pistachio, almond, hazel, peanut, Brazil nut, pecan and chestnut; from trees such as alder, hornbeam, cedar, birch, hazel, beech, ash, privet, oak, sycamore, cypress and palm; from flowers such as ragweed, carnation, forsythia, sunflower, lupine, chamomile, lilac and passionflower; from grasses such as quackgrass, common bentgrass, bromegrass, bermudagrass, sweetgrass and ryegrass; or from other plants such as poppy, pyrethrium, plantain, tobacco, asparagus, mugwort and cress.

[0029] Fungal antigens encoded by at least one mRNA of the vaccines of the present invention may include, but are not limited to, antigens derived from the genera Alternia sp., Aspergillus sp., Beauveria sp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp., Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillium sp., Pleospora sp., and Saccharomyces sp.

[0030] Antigens derived from bacteria encoded by at least one mRNA of the vaccine of the present invention may include, but are not limited to, antigens derived from Bacillus tetani, Staphylococcus aureus, and Streptomyces griseus.

[0031] A further class of antigens encoded by at least one mRNA of the vaccine of the present invention includes tumor antigens. "Tumor antigens" are preferably located on the surface of (tumor) cells. Tumor antigens may also be selected from proteins overexpressed in tumor cells compared to normal cells. Furthermore, tumor antigens also include antigens expressed in cells associated with a potential tumor, but not in (or not originally in) the degenerated cells themselves. Antigens associated with tumor-supplying blood vessels or their (re)formation, particularly antigens associated with their neovascularization, such as growth factors such as VEGF and bFGF, are also included herein. Tumor-associated antigens also include antigens derived from cells or tissues, particularly those derived from cells or tissues that embed tumors. Furthermore, certain substances (usually proteins or peptides) are expressed in patients (whether diagnosed or not) with cancer, resulting in increased concentrations of these substances in the patient's body fluids. These substances are also referred to as "tumor antigens," but are not antigens in the strict sense of an immune response inducer. The class of tumor antigens is further divided into tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are presented only by tumor cells, not by normal "healthy" cells. They typically result from tumor-specific mutations. More common TAAs are usually presented by both tumor cells and healthy cells. These antigens can be recognized, and antigen-presenting cells can be destroyed by cytotoxic T cells. In addition, tumor antigens can also occur on the surface of tumors, for example, in the form of mutated receptors. In this case, they can be recognized by antibodies. According to the present invention, the terms "cancer disease" and "tumor disease" are used interchangeably herein.

[0032] Examples of tumor antigens encoded by at least one mRNA of the vaccine of the present invention include, but are not limited to, 5T4, 707-AP (707 alanine proline), 9D7, AFP (alpha-fetoprotein), AlbZIP HPG1, alpha5beta1-integrin, alpha5beta6-integrin, alpha-methylacyl-coenzyme A racemase, ART-4 (adenocarcinoma antigen recognized by T cells 4), B7H4, BAGE-1 (B antigen), BCL-2, BING-4, CA 15-3 / CA 27-29, CA 19-9, CA 72-4, CA125, calreticulin, CAMEL (CTL-recognized antigen of melanoma), CASP-8 (caspase-8), cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen), CLCA2 (calcium-activated chloride channel-2), CML28, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6 (bromodomain testis-specific protein), Cten (C-terminal tensin-like protein), cyclin B1, cyclin D1, cyp-B (cyclophilin B), CYPB1 (cytochrome P450 1B1), DAM-10 / MAGE-B1 (differentiation antigen melanoma 10), DAM-6 / MAGE-B2 (differentiation antigen melanoma 6), EGFR / Her1, EMMPRIN (tumor cell-associated extracellular matrix metalloproteinase-inducer / ), EpCam (epithelial cell adhesion molecule), EphA2 (ephrin type A receptor 2), EphA3 (ephrin type A receptor 3), ErbB3, EZH2 (enhancer of zeste homolog 2), FGF-5 (fibroblast growth factor-5), FN (fibronectin) , Fra-1 (Fos-related antigen-1), G250 / CAIX (glycoprotein 250), GAGE-1 (G antigen 1), GAGE-2 (G antigen 2), GAGE-3 (G antigen 3), GAGE-4 (G antigen 4), GAGE-5 (G antigen 5), GAGE-6 (G antigen 6), GAGE-7b (G antigen 7b), GAGE-8 (G antigen 8), GDEP (gene differentially expressed in the prostate), GnT-V (N-acetylglucosaminyltransferase V), gp100 (glycoprotein 100)kDa), GPC3 (Glypican 3), HAGE (Helicase Antigen), HAST-2 (Human Signet Ring Tumor-2), Hepsin, Her2 / neu / ErbB2 (Human Epithelial Receptor-2 / Neurological), HERV-K-MEL, HNE (Human Neutrophil Elastase), Homeobox NKX 3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT (Human Telomerase Reverse Transcriptase), iCE (intestinal carboxylesterase), IGF-1R, IL-13Ra2 (interleukin-13 receptor α2 chain), IL-2R, IL-5, immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205, KK-LC-1 (Kitakyushu lung cancer antigen 1), KM-HN-1, LAGE-1 (L antigen), livin, MAGE-A1 (melanoma antigen-A1), MAGE-A10 ( Melanoma antigen-A10), MAGE-A12 (melanoma antigen-A12), MAGE-A2 (melanoma antigen-A2), MAGE-A3 (melanoma antigen-A3), MAGE-A4 (melanoma antigen-A4), MAGE-A6 (melanoma antigen-A6), MAGE-A9 (melanoma antigen-A9), MAGE-B1 (melanoma antigen-B1), MAGE-B10 (melanoma antigen-B10), MA GE-B16 (melanoma antigen-B16), MAGE-B17 (melanoma antigen-B17), MAGE-B2 (melanoma antigen-B2), MAGE-B3 (melanoma antigen-B3), MAGE-B4 (melanoma antigen-B4), MAGE-B5 (melanoma antigen-B5), MAGE-B6 (melanoma antigen-B6), MAGE-C1 (melanoma antigen-C1), MAGE-C2 (melanoma antigen-C) 2), MAGE-C3 (melanoma antigen-C3), MAGE-D1 (melanoma antigen-D1), MAGE-D2 (melanoma antigen-D2), MAGE-D4 (melanoma antigen-D4), MAGE-E1 (melanoma antigen-E1), MAGE-E2 (melanoma antigen-E2), MAGE-F1 (melanoma antigen-F1), MAGE-H1 (melanoma antigen-H1), MAGEL2 (MAGE-like2), mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1 / melanoma antigen A), MART-2 (melanoma antigen recognized by T cells-2), matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor gene), mesothelin, MG50 / PXDN, MMP 11 (M-phase phosphoprotein 11), MN / CA IX-antigen, MRP-3 (multidrug resistance-associated protein 3), MUC1 (mucin 1), MUC2 (mucin 2), NA88-A (NA of patient M88) cDNA clone), N-acetylglucosaminyltransferase-V, Neo-PAP (Neo-poly(A) polymerase), NGEP, NMP22, NPM / ALK (nucleophosmin / anaplastic lymphoma kinase fusion protein), NSE (neuron-specific enolase), NY-ESO-1 (New York esophageal carcinoma (esophageous) 1), NY-ESO-B, OA1 (ocular albinism type 1 protein), OFA-iLRP (oncofetal antigen-immature laminin receptor), OGT (O-linked N-acetylglucosaminyltransferase), OS-9, osteocalcin, osteopontin, p15 (protein 15), p15, p190 minor bcr-a bl, p53, PAGE-4 (prostate GAGE-like protein-4), PAI-1 (plasminogen activator inhibitor 1), PAI-2 (plasminogen activator inhibitor 2), PAP (prostatic acic phosphatase), PART-1, PATE, PDEF, Pim-1-kinase, Pin1 (propyl isomerase), POTE, PRAME (selectively expressed antigen in melanoma), prostein, proteinase-3, PSA (prostate-specific antigen), PSCA, PSGR, PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal antigen), RHAMM / CD168 (receptor for hyaluronan-mediated motility), RU1 (renal ubiquitous 1), RU2 (renal ubiquitousubiquitous)1), S-100, SAGE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen rejection tumor 1), SART-2 (squamous cell carcinoma antigen rejection tumor 1), SART-3 (squamous cell carcinoma antigen rejection tumor 1), SCC (squamous cell carcinoma antigen), Sp17 (sperm protein 17), SSX-1 (synovial sarcoma X breakpoint 1), SSX-2 / HOM-MEL-40 (synovial sarcoma X breakpoint), SSX-4 (synovial sarcoma X breakpoint 4), STAMP-1, STEAP (six-transmembrane epithelial antigen of the prostate), survivin, survivin-2B (intron 2-retaining survivin), TA-90, TAG-72, TARP, TGFb (TGFβ), TGFbRII (TGFβ receptor II), TGM-4 (prostate-specific transglutaminase), TRAG-3 (taxol resistance-associated protein 3), TRG (testin-related gene), TRP-1 (tyrosine-related protein 1), TRP-2 / 6b (TRP-2 / novel exon 6b), TRP-2 / INT2 (TRP-2 / intron 2), Trp-p8, tyrosinase, UPA (urokinase-type plasminogen activator), VEGF (vascular endothelial growth factor), VEGFR-2 / FLK-1 (vascular endothelial growth factor receptor The antibody may comprise an antigen selected from the group including, but not limited to, α-actinin-4 / m, ARTC1 / m, bcr / abl (breakpoint cluster region-Abelson fusion protein), β-catenin / m (β-catenin), BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m (cell division cycle 27), CDK4 / m (cyclin-dependent kinase 4), CDKN2A / m, CML66, COA-1 / m, DEK-CAN (fusion protein), EFTUD2 / m, ELF2 / m (elongation factor 2), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene fusion protein), FN1 / m (fibronectin 1), GPNMB / m, HLA-A *0201-R170I (arginine to isoleucine substitution at residue 170 of the α-helix of the α2-domain of the HLA-A2 gene), HLA-A11 / m, HLA-A2 / m, HSP70-2M (mutated heat shock protein 70-2), KIAA0205 / m, K-Ras / m, LDLR-FUT (LDR-fucosyltransferase fusion protein), MART2 / m, ME1 / m, MUM-1 / m (melanoma ubiquitous mutation 1), MUM-2 / m (melanoma ubiquitous mutation 2), MUM-3 / m (melanoma ubiquitous mutation 3), myosin class I / m, neo-PAP / m, NFYC / m, N-Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa (promyelocytic leukemia / retinoic acid receptor alpha), PRDX5 / m, PTPRK / m (receptor protein tyrosine phosphatase kappa), RBAF600 / m, SIRT2 / m, SYT-SSX-1 (synaptotagmin I / synovial sarcoma X fusion protein), SYT-SSX-2 (synaptotagmin I / synovial sarcoma X fusion protein), TEL-AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1 fusion protein), TGFbRII (TGF beta receptor II), and TPI / m (triosephosphate isomerase). However, according to certain aspects, mRNA encoding at least one of the antigens gp100, MAGE-A1, MAGE-A3, MART-1 / Melan-A, survivin and tyrosinase, more preferably mRNA encoding at least one of the antigens gp100, MAGE-A1, MAGE-A3, MART-1 / Melan-A, survivin and tyrosinase, which forms a complex with or is stabilized by protamine (e.g., a ratio of approximately 80 μg of mRNA and 128 μg of protamine), may be excluded from the scope of the present invention.In a preferred embodiment, the tumor antigen encoded by at least one mRNA of the vaccine of the present invention is 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 15-3 / CA 27-29, CA 19-9, CA72-4. CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD40, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-1 0, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE -2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A *0201-R17I, HLA-A11 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A 9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESO-B, OA1, O FA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, p15, p190 minor, bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml / PARα, POTE, PRAME, PRDX5 / m, prostein, protein -3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, S ART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP, Survivin, Selected from the group consisting of survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFβ, TGFβRII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2 / FLK-1, and WT1.

[0033] According to a particularly preferred embodiment, the tumor antigen encoded by at least one mRNA of the vaccine of the present invention is selected from the group consisting of MAGE-A1 (e.g., MAGE-A1 of accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 of accession number NM_005363), MAGE-C1, MAGE-C2, Melan-A (e.g., Melan-A of accession number NM_005511), GP100 (e.g., GP100 of accession number M77348), tyrosinase (e.g., tyrosinase of accession number NM_000372), survivin (e.g., survivin of accession number AF077350), CEA (e.g., CEA of accession number NM_004363), Her-2 / neu (e.g., Her-2 / neu of accession number M11730), WT1 (e.g., WT1 under accession number NM_000378), PRAME (e.g., PRAME under accession number NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g., EGFRI (epidermal growth factor receptor 1) under accession number AF288738), MUC1, mucin-1 (e.g., mucin-1 under accession number NM_002456), SEC61G (e.g., accession number NM_014 302), hTERT (e.g., hTERT of Accession No. NM_198253), 5T4 (e.g., 5T4 of Accession No. NM_006670), NY-Eso-1 (e.g., NY-Eso1 of Accession No. NM_001327), TRP-2 (e.g., TRP-2 of Accession No. NM_001922), STEAP, PCA, PSA, and PSMA, and the like.

[0034] Particularly preferred antigens are influenza A virus (HA antigen, NA antigen, NP antigen, M2 antigen, M1 antigen), influenza B virus (HA antigen, NA antigen), respiratory syncytial virus (F antigen, G antigen, M antigen, SH antigen), parainfluenza virus (glycoprotein antigen), Streptococcus pneumoniae (pPht antigen, PcsB antigen, StkP antigen), Corynebacterium diphtheriae, Clostridium tetanus, measles, mumps, rubella, rabies virus (G antigen, N antigen), Staphylococcus aureus (toxin antigen), Clostridium difficile (toxin antigen), Mycobacterium tuberculosis (acute antigen and inactive antigen), Candida albicans, Haemophilus influenzae B (HiB), poliovirus, hepatitis B virus (surface antigen and core antigen), human papillomavirus (L1, L2, E6, E7), human immunodeficiency virus (gp120 antigen, gag antigen, env antigen), SARS Selected from CoV (spike protein), Staphylococcus aureus (IsdA antigen, IsdB antigen, toxin antigen), pertussis toxin, poliovirus (VP1-4), malaria parasite (NANP antigen, CSP protein antigen, ssp2 antigen, ama1 antigen, msp142 antigen), Staphylococcus aureus (IsdA, IsdB, and toxin), Bordetella pertussis (toxin), poliovirus VP1-4, malaria parasite (NANP antigen, CSP protein antigen, ssp2 antigen, ama1 antigen, msp142 antigen).

[0035] The antigens encoded by at least one mRNA of the vaccines of the invention described herein may further comprise fragments of the antigens described herein, particularly protein or peptide antigens. Such antigen fragments in the context of the present invention may include fragments preferably about 6 to about 20 or more amino acids in length, e.g., fragments processed and presented by MHC class I molecules, preferably about 8 to about 10 amino acids in length, e.g., 8, 9, or 10 (or even 11 or 12) amino acids in length, or fragments processed and presented by MHC class II molecules, preferably about 13 or more amino acids in length, e.g., 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids in length, and these fragments may be selected from any portion of the amino acid sequence. Such fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule; i.e., the fragments are not recognized in their native form.

[0036] Fragments of antigens as defined herein may also comprise epitopes of those antigens. Epitopes (also called "antigenic determinants") are fragments that are typically located on the surface of (native) protein or peptide antigens as defined herein and that can be recognized by antibodies (i.e. in their native form), preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, even more preferably 6 to 9 amino acids.

[0037] According to a further particularly preferred embodiment, the tumor antigens encoded by at least one mRNA of the vaccine of the invention may form a mixture of antigens, e.g., an active (immunostimulatory) composition or a kit of parts (preferably, each antigen is included as part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of a disease or disorder as defined herein. To this end, the vaccine of the invention may comprise at least one mRNA, each of which may encode at least one, preferably two, three, four or more (preferably different) antigens as described herein. Alternatively, the vaccine of the invention may comprise at least one, two, three, four or more (preferably different) mRNAs, each of which encodes at least one antigen as described herein.

[0038] Such a mixture of antigens encoded by at least one mRNA of the vaccine of the present invention may be used, for example, in the treatment of prostate cancer (PCa), preferably as a neoadjuvant and / or hormone-refractory prostate cancer, and diseases or disorders related thereto. To this end, the vaccine of the present invention may comprise at least one mRNA, each of which may encode at least one, preferably two, three, four, or more (preferably different) antigens as described herein. Alternatively, the vaccine of the present invention may comprise at least one, two, three, four, or more (preferably different) mRNAs, each of which encodes at least one antigen as described herein. Preferably, the antigen is selected from PSA (prostate-specific antigen) = KLK3 (kallikrein-3), PSMA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), and / or STEAP (six-transmembrane epithelial antigen of the prostate).

[0039] Furthermore, a mixture of antigens encoded by at least one mRNA of such a vaccine of the present invention may be used, for example, to treat non-small cell lung cancer (NSCLC), preferably selected from the three major subtypes of lung cancer: squamous cell carcinoma, adenocarcinoma, and large cell lung cancer, or disorders related thereto. To this end, a vaccine of the present invention may comprise at least one mRNA, each of which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve (preferably different) antigens as described herein. Alternatively, a vaccine of the present invention may comprise at least one, preferably two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve (preferably different) mRNAs, each of which encodes at least one antigen as described herein. Preferably, such antigens are selected from at least one of hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2 / neu, NY-ESO-1, CEA, survivin, MAGE-C1, and MAGE-C2.

[0040] In the above embodiment, each of the above-defined antigens may be encoded by one (monocistronic) mRNA, in other words, in this case, the at least one mRNA of the vaccine of the invention may be at least two (such as three and four) (monocistronic) mRNAs, where each of these at least two (such as three and four) (monocistronic) mRNAs may for example comprise the mRNA of only one (preferably different) antigen, preferably selected from one of the above antigen combinations.

[0041] According to a particularly preferred embodiment, the at least one mRNA of the vaccine of the present invention may comprise (at least) one bicistronic or multicistronic mRNA, preferably an mRNA, i.e., (at least) one mRNA carrying two or more coding genes for at least two (preferably different) antigens, e.g., preferably selected from one of the above-mentioned antigen combinations. For example, such coding sequences for the at least two (preferably different) antigens of the (at least) one bicistronic or multicistronic mRNA may be separated by at least one IRES (internal ribosome entry site) as defined below. Thus, the term "encoding at least two (preferably different) antigens" may mean, but is not limited to, that (at least) one (bicistronic or multicistronic) mRNA may encode, for example, at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more (preferably different) antigens from the above-mentioned group of antigens, or fragments or variants thereof. In this context, the so-called IRES (internal ribosome entry site) sequence as defined herein can function as a single ribosome binding site, but it can also serve to provide a bicistronic or multicistronic RNA as defined herein, which encodes multiple proteins that are translated by the ribosome independently of each other. Examples of IRES sequences that can be used according to the invention are those derived from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV), or cricket paralysis viruses (CrPV).

[0042] According to a further particularly preferred embodiment, at least one mRNA of the vaccine of the invention may comprise at least one monocistronic mRNA and at least one bicistronic or multicistronic RNA, preferably a mixture of mRNAs, as defined herein. The at least one monocistronic RNA and / or at least one bicistronic or multicistronic RNA preferably encode different antigens, or fragments or variants thereof, preferably selected from one of the antigens described above, more preferably in one of the combinations described above. However, the at least one monocistronic RNA and at least one bicistronic or multicistronic RNA may also preferably encode (partially) identical antigens, preferably selected from one of the antigens described above, preferably in one of the combinations described above, provided that the at least one mRNA of the vaccine of the invention as a whole provides at least two (preferably different) antigens as defined herein. Such an embodiment may be advantageous for, for example, alternate, e.g., time-dependent, administration of at least one mRNA of one or several vaccines of the invention to a patient in need thereof. The components of such a vaccine may be included in a kit of (different) parts of the composition or may be administered separately, for example as a composition of the same inventive vaccine as defined in accordance with the present invention.

[0043] In a further preferred embodiment, at least one mRNA (or any further nucleic acid as defined herein) of the vaccine of the invention may also occur in the form of a modified nucleic acid.

[0044] According to a first aspect, at least one mRNA (or any further nucleic acid as defined herein) of the vaccine of the invention may be provided as a "stabilised nucleic acid" that is substantially resistant to in vivo degradation (e.g. by exonucleases or endonucleases).

[0045] In this context, at least one mRNA (or any further nucleic acid as defined herein) of a vaccine of the invention may comprise a backbone modification, a sugar modification or a base modification. A backbone modification in the context of the present invention is a modification in which the phosphate of the backbone of a nucleotide comprised in at least one mRNA (or any further nucleic acid as defined herein) of a vaccine of the invention is chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of a nucleotide of at least one mRNA (or any further nucleic acid as defined herein) of a vaccine of the invention. Furthermore, a base modification in the context of the present invention is a chemical modification of the base moiety of a nucleotide of at least one mRNA (or any further nucleic acid as defined herein) of a vaccine of the invention.

[0046] According to a further aspect, at least one mRNA (or any further nucleic acid as defined herein) of the vaccine of the present invention can comprise a lipid modification. Such lipid-modified nucleic acids typically comprise a nucleic acid, e.g., mRNA or any further nucleic acid, as defined herein. Such lipid-modified mRNA (or any further lipid-modified nucleic acid as defined herein) of the vaccine of the present invention typically further comprises at least one linker covalently linked to the nucleic acid molecule, and at least one lipid covalently linked to each linker. Alternatively, the lipid-modified mRNA (or any further lipid-modified nucleic acid as defined herein) of the vaccine of the present invention comprises at least one nucleic acid molecule, e.g., mRNA, or any further nucleic acid, as defined herein, and at least one (bifunctional) lipid covalently linked to the nucleic acid molecule (without a linker). According to a third alternative, the lipid-modified mRNA (or any further lipid-modified nucleic acid as defined herein) of the vaccine of the invention comprises a nucleic acid molecule as defined herein, e.g., mRNA, or any further nucleic acid, at least one linker covalently linked to said nucleic acid molecule, and at least one lipid covalently linked to each said linker, and further at least one (bifunctional) lipid covalently linked (without a linker) to said nucleic acid molecule.

[0047] The at least one mRNA (or any further nucleic acid as defined herein) of the vaccine of the present invention may also be stabilized by various techniques to prevent degradation of the mRNA (or any further nucleic acid molecule). It is known in the art that general RNA instability and (rapid) degradation present serious problems for the application of RNA-based compositions. This RNA instability is typically due to the RNA-degrading enzyme "RNAase" (ribonuclease), and the combination with such ribonuclease sometimes completely degrades RNA in solution. Therefore, the natural degradation of RNA in the cytoplasm of cells is very precisely controlled, and RNase contamination can generally be removed by special treatment, in particular diethyl pyrocarbonate (DEPC), before using the composition. Many mechanisms of natural degradation are known in the prior art in this regard and can be used similarly. For example, the terminal structure is typically very important, especially for mRNA. As an example, the 5' end of natural mRNA usually has a so-called "cap structure" (modified guanosine nucleotides) and the 3' end typically has a sequence of up to 200 adenosine nucleotides (the so-called poly-A tail).

[0048] According to another embodiment, at least one mRNA of the vaccine of the invention may be modified and thus stabilized by modifying the G / C content of the mRNA, preferably its coding region.

[0049] In a particularly preferred embodiment of the invention, the G / C content of the coding region of at least one mRNA of the vaccine of the invention is modified, in particular increased, compared to the G / C content of the coding region of the particular wild-type coding sequence, i.e., unmodified mRNA, and the encoded amino acid sequence of the mRNA is preferably unmodified compared to the encoded amino acid sequence of the particular wild-type mRNA.

[0050] The modification of the G / C content of at least one mRNA of the vaccine of the present invention is based on the fact that the sequence of any mRNA region to be translated is important for the efficient translation of that mRNA. Therefore, the composition and sequence of various nucleotides are important. Specifically, sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased (adenosine) / U (uracil) content. According to the present invention, the codons of a coding sequence or mRNA are altered compared to the wild-type coding sequence or mRNA so that they contain an increased amount of G / C nucleotides, while maintaining the translated amino acid sequence. Given the fact that several codons encode the same amino acid (the so-called degeneracy of the genetic code), it is possible to determine the codons that are most advantageous for stability (the so-called alternative codon usage).

[0051] Preferably, the G / C content of the coding region of at least one mRNA of the vaccine of the invention is increased by at least 7%, more preferably at least 15%, and particularly preferably at least 20% compared to the G / C content of the coding region of the wild-type mRNA. According to a particular embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%, 95%, or 100% of the replaceable codons in the region encoding a protein or peptide or fragment thereof or variant thereof as defined herein, or the wild-type mRNA sequence, or coding sequence, are substituted, thereby increasing the G / C content of said sequence.

[0052] In this regard, it is particularly preferred to increase the G / C content of at least one mRNA of the vaccine of the invention, particularly in the protein-coding region, to the maximum extent (i.e., 100% of substitutable codons) compared to the wild-type sequence.

[0053] According to the present invention, a further preferred modification of at least one mRNA of the vaccine of the invention, particularly when the nucleic acid is in the form of an mRNA or encoding an mRNA, is based on the finding that the efficiency of translation is also determined by the different frequencies of occurrence of tRNAs in the cell. Thus, if so-called "rare codons" are present to an increased extent in at least one mRNA of the vaccine of the invention, the corresponding modified mRNA will be translated to a significantly lesser extent than if codons encoding relatively "frequent" tRNAs are present.

[0054] Preferably, the coding region of at least one mRNA of the vaccine of the invention is modified compared to the corresponding region of the wild-type mRNA or coding sequence in such a way that at least one codon of the wild-type sequence encoding a tRNA that is relatively rare in the cell is replaced with a codon encoding a tRNA that is relatively frequent in the cell and carries the same amino acid as said relatively rare tRNA. By this modification, the sequence of at least one mRNA of the vaccine of the invention is modified in such a way that a codon that results in a frequently occurring tRNA is inserted, particularly when the nucleic acid is in the form of an mRNA or one that encodes an mRNA. In other words, according to the invention, by this modification, all codons of the wild-type sequence encoding a tRNA that is relatively rare in the cell can in each case be replaced with codons encoding a tRNA that is relatively frequent in the cell and in each case carries the same amino acid as said relatively rare tRNA.

[0055] Those skilled in the art know which tRNAs occur relatively frequently in cells, and conversely, which tRNAs occur relatively rarely in cells (see, e.g., Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666). Particularly preferred are codons that use the most frequently occurring tRNA for a particular amino acid, e.g., the Gly codon that uses the most frequently occurring tRNA in (human) cells.

[0056] According to the invention, it is particularly preferred to link the increased, in particular maximized, sequence G / C content in at least one modified mRNA of the vaccine of the invention with "frequent" codons without modifying the amino acid sequence encoded by the coding region of said mRNA. This preferred embodiment allows to provide at least one stabilized (modified) mRNA of the vaccine of the invention that is particularly efficiently translated.

[0057] According to a further preferred embodiment of the present invention, at least one mRNA of the vaccine of the present invention as defined herein, or any further nucleic acid molecule as defined herein, preferably comprises 5' and / or 3' stabilizing sequences. These stabilizing sequences in the 5' and / or 3' untranslated regions have the effect of increasing the half-life of the nucleic acid in the cytosol. These stabilizing sequences can have 100% sequence identity with sequences occurring naturally in viruses, bacteria, and eukaryotes, but can also be partially or completely synthetic. For example, the untranslated sequences (UTRs) of the (α-)globin genes from Homo sapiens or Xenopus laevis can be cited as examples of stabilizing sequences that can be used to stabilize nucleic acids in the present invention. Other examples of stabilizing sequences are contained in the 3'UTRs of highly stable RNAs encoding (α-)globin, type I collagen, 15-lipoxygenase, or tyrosine hydroxylase (see Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410-2414), and have the general formula (C / U)CCAN. x CCC(U / A)Py x UC(C / U)CC (SEQ ID NO: 383). Such stabilizing sequences can, of course, be used alone or in combination with another stabilizing sequence or with stabilizing sequences known to those skilled in the art.

[0058] Nevertheless, substitutions, additions, or deletions are preferably made with at least one mRNA or any additional nucleic acid molecule of the vaccine of the present invention defined herein, particularly when the nucleic acid is in the form of mRNA, using well-known site-directed mutagenesis or oligonucleotide ligation methods to prepare a DNA matrix for the nucleic acid molecule (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, NY, 2001). In such a process, for the preparation of at least one mRNA of the vaccine of the present invention defined herein, the corresponding DNA molecule may be transcribed in vitro. This DNA matrix preferably contains a suitable promoter for in vitro transcription, such as a T7 promoter or an SP6 promoter, followed by the desired nucleic acid sequence of the at least one mRNA to be prepared, and a termination signal for in vitro transcription. The DNA molecule forming the matrix for at least one mRNA of interest may also be prepared by fermentative growth and subsequent isolation as part of a replicable plasmid in bacteria. Plasmids that may be mentioned as being suitable for the present invention are, for example, the plasmid pT7Ts (GenBank accession number U26404; Lai et al., Development 1995, 121: 2349-2360), the pGEM™ series, such as pGEM™-1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327) (see also Mezei and Storts, Purification of PCR Products, in Griffin and Griffin (eds.), PCR Technology: Current Innovation, CRC Press, Boca Raton, FL, 2001).

[0059] The nucleic acid molecules as defined herein used in accordance with the present invention, such as at least one mRNA of a vaccine of the present invention, or any further nucleic acid molecules as defined herein, may be modified as outlined above for at least one mRNA of a vaccine of the present invention.

[0060] Additionally, nucleic acid molecules as defined herein for use in accordance with the present invention, e.g. at least one mRNA of a vaccine of the present invention, or any further nucleic acid molecules as defined herein, may be prepared using any method known in the art, including synthetic methods such as, for example, solid phase synthesis, as well as in vitro methods such as in vitro transcription reactions.

[0061] According to one preferred embodiment of the present invention, the at least one mRNA of the vaccine of the present invention may be administered naked, without any additional excipients, transfection agents or complexing agents to increase the transfection efficiency of the at least one mRNA.

[0062] In a further preferred embodiment of the present invention, the at least one mRNA of the vaccine of the present invention is accompanied by any further excipient, transfection agent or complexing agent to increase the transfection efficiency of the at least one mRNA. Particularly preferred agents in this context suitable for increasing the transfection efficiency are cationic or polycationic compounds including protamine, nucleolin, spermine or spermidine, or poly-L-lysine (PLL), polyarginine, basic polypeptides, HIV-binding peptides, HIV-1 and other cationic peptides or proteins such as Tat (HIV), Tat-derived peptides, penetratin, VP22-derived or similar peptides, plague virus Erns, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, buforin 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histone-containing cell-penetrating peptides (CPPs). Additionally, preferred cationic or polycationic proteins or peptides may be selected from the following proteins or peptides having the general formula: (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x wherein l+m+n+o+x=8 to 15, where l, m, n, or o are, independently of one another, any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (with the proviso that Arg, Lys, His, and Orn represent at least 50% of the total amino acids of the oligopeptide), Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except Arg, Lys, His, or Orn, and x may be any number selected from 0, 1, 2, 3, or 4 (with the proviso that the total content of Xaa does not exceed 50% of the total amino acids of the oligopeptide). Particularly preferred cationic peptides in this regard include, for example, Arg7, Arg8, Arg9, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, and Y(RKH)2R. Further preferred cationic or polycationic compounds that can be used as transfection agents include cationic polysaccharides such as chitosan and polybrene, cationic polymers such as polyethyleneimine (PEI), for example DOTMA ([1-(2,3-dioleyloxy)propyl)]-N,N,N-trimethylammonium chloride), DMRIE, diC14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE (dioleylphosphatidylethanolamine), DOSPA, DODAB, DOIC, DMEPC, DOGS (dioctadecylamidoglycylspermine), DIMRI (D-dimyristoyloxypropyldimethylhydroxyethylammonium bromide), DOTAP (dioleoyloxy-3-(trimethylammonio)propane), DC-6-14 (O,O-ditetradecanoyloxypropylmethylammonium bromide), Cationic lipids such as N-(α-trimethylammonioacetyl)diethanolamine chloride), CLIP1 (rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride), CLIP6 (rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium), CLIP9 (rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium) and oligofectamine, or modified polyamino acids such as β-amino acid polymers or reverse polyamides, modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified amidoamines such as pAMAM (poly(amidoamine)), and diamine-terminated 1,Examples of suitable cationic or polycationic polymers include modified poly-β-aminoesters (PBAEs) such as 4-butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, polyimines such as PEI (poly(ethyleneimine)) and poly(propyleneimine), polyallylamine, cyclodextrin-based polymers, dextran-based polymers, and sugar-based polymers such as chitosan, silane-based polymers such as PMOXA-PDMS copolymers, block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above), and block polymers consisting of a combination of one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0063] At least one mRNA of the vaccine of the present invention, encoding at least one antigen, may also be complexed with a polymeric carrier formed by disulfide-bridged cationic moieties. The term "cationic moiety" typically refers to a charged molecule (cation) that has a positive charge at a pH value of about 1 to 9, preferably below 9, below 8, or below 7, most preferably at a physiological pH value of, for example, 7.3 to 7.4. Thus, cationic peptides, proteins, or polymers according to the present invention have a positive charge under physiological conditions, preferably under physiological saline conditions, particularly in vivo in cells. The definition of "cationic" may also refer to "polycationic" moieties.

[0064] In this context, the cationic component which by disulfide bridges forms the basis of the polymeric carrier of the vaccine of the invention is typically selected from any cationic or polycationic peptide, protein or polymer suitable for this purpose, in particular any cationic or polycationic peptide, protein or polymer capable of complexing, and thereby preferably condensing, nucleic acids as defined in accordance with the invention. The cationic or polycationic peptide, protein or polymer is preferably a linear molecule, although branched cationic or polycationic peptides, proteins or polymers may also be used.

[0065] Each cationic or polycationic protein, peptide or polymer of the polymeric carrier that can be used to complex at least one mRNA of the present invention comprises at least one -SH moiety, most preferably at least one cysteine ​​residue, or any further chemical group presenting an -SH moiety capable of forming a disulfide bond by condensation with at least one further cationic or polycationic protein, peptide or polymer as the cationic component of the polymeric carrier referred to herein.

[0066] Each cationic or polycationic protein, peptide, or polymer, or any other polymeric carrier component that can be used to complex at least one mRNA of the vaccine of the present invention, is preferably linked to its neighboring component (cationic protein, peptide, polymer, or other component) via a disulfide bridge. Preferably, the disulfide bridge is a (reversible) disulfide bond (-SS-) between at least one cationic or polycationic protein, peptide, or polymer and at least one further cationic or polycationic protein, peptide, or polymer, or other component of the polymeric carrier. Disulfide bridges are typically formed by condensation of -SH moieties of components of the polymeric carrier, particularly cationic components. Such -SH moieties may be part of the structure of the cationic or polycationic protein, peptide, or polymer, or any other polymeric carrier component, prior to disulfide bridging, or may be added by the below-defined modifications prior to disulfide bridging. In this regard, the sulfur adjacent to one component of the polymeric carrier necessary to provide a disulfide bond may be provided by the component itself, for example, by an -SH moiety as defined herein, or by modifying the component to present an -SH moiety. These -SH groups are typically provided by each component, for example, via cysteine ​​or any additional (modified) amino acid of the component bearing an -SH moiety. When the cationic component or any additional component of the polymeric carrier is a peptide or protein, the -SH moiety is preferably provided by at least one cysteine ​​residue. Alternatively, the component of the polymeric carrier may be modified with an -SH moiety, preferably via chemical reaction with a compound bearing an -SH moiety, so that each component of the polymeric carrier bears at least one such -SH moiety. Such a compound bearing an -SH moiety may be, for example, an (additional) cysteine ​​or any additional (modified) amino acid or compound of the component of the polymeric carrier bearing an -SH moiety.Such compounds may also be any non-amino compound or moiety that allows the introduction of an -SH moiety into a component as defined herein, such as by coupling of 3-thiopropionic acid or 2-iminothiolane (Traut's reagent), by amide formation (e.g., carboxylic acids, sulfonic acids, amines, etc.), by Michael addition (e.g., maleimide moieties, α,β-unsaturated carbonyls, etc.), by click chemistry (e.g., azides or alkynes), alkene / alkyne metathesis (e.g., alkenes or alkynes), imine or hydrazone formation (aldehydes or ketones, hydrazines, hydroxylamines, amines), conjugation reactions (avidin, biotin, protein G), or S. n The -SH moiety may be linked to the polymeric carrier component of the present invention via chemical reaction or attachment of the compound with a compound that allows for a substitution reaction (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonate esters, oxyphosphonium salts) or other chemical moieties that can be used to attach additional components. In some cases, the -SH moiety may be covered by a protecting group during chemical attachment to the component. Such protecting groups are known in the art and may be removed after chemical attachment. In either case, the -SH moiety, e.g., cysteine, or any additional (modified) amino acid or compound, may be present at any position in the polymeric carrier component, either terminally or internally. As defined herein, each component of the polymeric carrier typically presents at least one -SH group, but may contain two, three, four, five, or more -SH moieties. In addition to binding cationic moieties, the -SH moiety may be used to link further components of the polymeric carrier of the vaccine of the invention as defined herein, particularly amino acid components such as antigenic epitopes, antigens, antibodies, cell-penetrating peptides (e.g., TAT), and ligands.

[0067] As defined above, the polymeric carriers that can be used to complex at least one mRNA of the vaccine of the invention may be formed by disulfide-bridged cationic (or polycationic) components.

[0068] According to one first alternative, at least one cationic (or polycationic) component of the polymeric carrier that can be used to complex at least one mRNA of the vaccine of the present invention can be selected from cationic or polycationic peptides or proteins. Such cationic or polycationic peptides or proteins are preferably about 3 to 100 amino acids in length, preferably about 3 to 50 amino acids in length, more preferably about 3 to 25 amino acids in length, e.g., about 3 to 10, 5 to 15, 10 to 20, or 15 to 25 amino acids in length. Alternatively or additionally, such cationic or polycationic peptides or proteins may exhibit a molecular weight of about 0.01 kDa to 100 kDa, including a molecular weight of about 0.5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa, and even more preferably about 10 kDa to about 30 kDa.

[0069] In the particular case where the cationic component of the polymeric carrier that can be used to complex at least one mRNA of the vaccine of the present invention comprises a cationic or polycationic peptide or protein, if the polymeric carrier is entirely composed of a cationic or polycationic peptide or protein, the cationic character of the cationic or polycationic peptide or protein, or of the entire polymeric carrier, can be determined by its cationic amino acid content. Preferably, the cationic amino acid content in the cationic or polycationic peptide or protein and / or the polymeric carrier is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60%, or 70%, but also at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99%, or 100%, and most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 10%. % to 90%, more preferably in the range of about 15% to 75%, and even more preferably in the range of 20% to 50%, such as 20%, 30%, 40% or 50%, or the range formed by any two of the aforementioned values ​​(however, the content of all amino acids, e.g., cationic amino acids, lipophilic amino acids, hydrophilic amino acids, aromatic amino acids, and further amino acids, in a cationic or polycationic peptide or protein, or in any polymeric carrier, is taken to be 100% when the polymeric carrier is a cationic or polycationic peptide or protein).

[0070] Preferably, such cationic or polycationic peptides or proteins of the polymeric carrier, which contain at least one -SH moiety or are additionally modified to contain at least one -SH moiety, include, but are not limited to, cationic peptides or proteins such as protamine, nucleolin, spermine or spermidine, oligo- or poly-L-lysine (PLL), basic polypeptides, oligo- or polyarginine, cell penetrating peptides (CPPs), chimeric CPPs such as transportan or MPG peptides, HIV-binding peptides, Tat, HIV-1 CPPs derived from Tat (HIV), Tat-derived peptides, such as penetratin, Antennapedia-derived peptides (particularly from Drosophila Antennapedia), members of the penetratin family such as pAntp and pIsl, and antibacterial agents such as Buforin 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, oligomers, FGF, lactoferrin, histones, VP22-derived or similar peptides, Erns of plague virus, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, and calcitonin peptides.

[0071] Alternatively or additionally, such cationic or polycationic peptides or proteins of polymeric carriers containing at least one -SH group or additionally modified to contain at least one -SH moiety are selected from, but not limited to, the following cationic peptides having the following general formula (I):

[0072] {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x}

[0073] wherein l+m+n+o+x=3 to 100, and l, m, n, and o are each independently any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, and 91 to 100 (provided that the total content of Arg (arginine), Lys (lysine), His (histidine), and Orn (ornithine) is less than the total amino acid content of the oligopeptide). Xaa is selected from native (naturally occurring) or non-native amino acids excluding Arg, Lys, His, or Orn, where x is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90 (provided that the total Xaa content does not exceed 90% of the total amino acids in the oligopeptide). Any of the amino acids Arg, Lys, His, Orn, and Xaa may be located at any position in the peptide. In this regard, cationic peptides or proteins ranging from 7 to 30 amino acids are particularly preferred. Further preferred peptides of this formula include, for example, Arg7, Arg8, Arg9, Arg10, Arg11, Arg12, Arg13, Arg14, Arg15, Arg16, Arg17, Arg18, Arg19, Arg19, Arg20, Arg21, Arg22, Arg23, Arg24, Arg25, Arg26, Arg27, Arg28, Arg29 ...9, Arg21, Arg22, Arg23, Arg24, 12 , His3Arg9, Arg9His3, His3Arg9His3, His6Arg9His6, His3Arg4His3, His6Arg4His6, TyrSer2Arg9Ser2Tyr, (ArgLysHis)4, and Tyr(ArgLysHis)2Arg.

[0074] According to certain preferred embodiments, the cationic or polycationic peptide or protein of the polymeric carrier having the above empirical sum formula (I) includes, but is not limited to, at least one of the subgroups of the following formulae:

[0075] Arg7, Arg8, Arg9, Arg 10 , Arg 11 , Arg 12 , Arg 13 , Arg14 , Arg 15-30 , Lys7, Lys8, Lys9, Lys 10 , Lys 11 , Lys 12 , Lys 13 , Lys 14 , Lys 15-30 , His7, His8, His9, His 10 , His 11 , His 12 , His 13 , His 14 , His 15-30 , Orn7, Orn8, Orn9, Orn 10 , Orn 11 , Orn 12 , Orn 13 , Orn 14 , Orn 15-30

[0076] In a further particularly preferred embodiment, the polymeric carrier cationic or polycationic peptide or protein having the above empirical sum formula (I) and containing at least one -SH moiety or additionally modified to contain at least one -SH moiety includes, but is not limited to, at least one of the subgroups of the following formulas: The following formulas (like empirical formula (I)) do not specify any amino acid order, but are intended to reflect the empirical formula by exclusively specifying the (number of) amino acids as components of each peptide. Thus, by way of example, the empirical formula Arg (7-29) Lys1 is intended to mean that peptides corresponding to this formula contain 7-19 arginine residues and one Lys residue, in any order. If a peptide contains seven Arg residues and one Lys residue, all variants having seven Arg residues and one Lys residue are included. The Lys residue can therefore be located anywhere in the eight amino acid long sequence of, for example, seven Arg and one Lys residue. Subgroups preferably include:

[0077] Arg (4-29) Lys1, Arg (4-29) His1, Arg (4-29 Orn1, Lys(4-29) His1、Lys (4-29) Orn1、His (4-29) Orn1、Arg (3-28) Lys2、Arg (3-28) His2、Arg (3-28) Orn2、Lys (3-28) His2、Lys (3-28) Orn2、His (3-28) Orn2、Arg (2-27) Lys3、Arg (2-27) His3、Arg (2-27) Orn3、Lys (2-27) His3、Lys (2-27) Orn3、His (2-27) Orn3、Arg (1-26) Lys4、Arg (1-26) His4、Arg (1-26) Orn4、Lys (1-26) His4、Lys (1-26) Orn4、His (1-26) Orn4、

[0078] Arg (3-28) Lys1His1、Arg (3-28) Lys1Orn1、Arg (3-28) His1Orn1、Arg1Lys (3-28) His1、Arg1Lys (3-28) Orn1、Lys (3-28) His1Orn1、Arg1Lys1His (3-28) 、Arg1His (3-28) Orn1、Lys1His (3-28) Orn1、

[0079] Arg (2-27) Lys2His1、Arg (2-27) Lys1His2、Arg (2-27) Lys2Orn1、Arg (2-27) Lys1Orn2、Arg (2-27) His2Orn1、Arg (2-27) His1Orn2、Arg2Lys (2-27) His1、Arg1Lys (2-27) His2、Arg2Lys (2-27) Orn1、Arg1Lys (2-27) Orn2、Lys(2-27) His2Orn1、Lys (2-27) His1Orn2、Arg2Lys1His (2-27) 、Arg1Lys2His (2-27) 、Arg2His (2-27) Orn1、Arg1His (2-27) Orn2、Lys2His (2-27) Orn1、Lys1His (2-27) Orn2、

[0080] Arg (1-26) Lys3His1、Arg (1-26) Lys2His2、Arg (1-26) Lys1His3、Arg (1-26) Lys3Orn1、Arg (1-26) Lys2Orn2、Arg (1-26) Lys1Orn3、Arg (1-26) His3Orn1、Arg (1-26) His2Orn2、Arg (1-26) His1Orn3、Arg3Lys (1-26) His1、Arg2Lys (1-26) His2、Arg1Lys (1-26) His3、Arg3Lys (1-26) Orn1、Arg2Lys (1-26) Orn2、Arg1Lys (1-26) Orn3、Lys (1-26) His3Orn1、Lys (1-26) His2Orn2、Lys (1-26) His1Orn3、Arg3Lys1His (1-26) 、Arg2Lys2His (1-26) 、Arg1Lys3His (1-26) 、Arg3His (1-26) Orn1、Arg2His (1-26) Orn2、Arg1His (1-26) Orn3、Lys3His (1-26) Orn1、Lys2His (1-26) Orn2、Lys1His (1-26) Orn3、

[0081] Arg (2-27) Lys1His1Orn1、Arg1Lys (2-27)His1Orn1, Arg1Lys1His (2-27) Orn1, Arg1Lys1His1Orn (2-27) ,

[0082] Arg (1-26) Lys2His1Orn1, Arg (1-26) Lys1His2Orn1, Arg (1-26) Lys1His1Orn2, Arg2Lys (1-26) His1Orn1, Arg1Lys (1-26) His2Orn1, Arg1Lys (1-26) His1Orn2, Arg2Lys1His (1-26) Orn1, Arg1Lys2His (1-26) Orn1, Arg1Lys1His (1-26) Orn2, Arg2Lys1His1Orn (1-26) , Arg1Lys2His1Orn (1-26) , Arg1Lys1His2Orn (1-26)

[0083] In further particularly preferred embodiments, the polymeric carrier cationic or polycationic peptides or proteins having the above empirical sum formula (I) and containing at least one -SH moiety or additionally modified to contain at least one -SH moiety include, but are not limited to, those having the general formula Arg7 (also referred to as R7), Arg9 (also referred to as R9), and Arg 12 (R 12 (also referred to as "anti-cancer drugs").

[0084] In one further particularly preferred embodiment, the cationic or polycationic peptide or protein of the polymeric carrier has the formula {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x} (formula (I)) and containing at least one -SH moiety or further modified to contain at least one -SH moiety, is selected from, but not limited to, the following sub-formula (Ia):

[0085] {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa') x (Cys) y Formula (Ia)

[0086] where (Arg) l , (Lys) m , (His) n , (Orn) o and x are defined herein; Xaa' is selected from a native (naturally occurring) or non-native amino acid excluding Arg, Lys, His, Orn, or Cys; and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, provided that the total content of Arg (arginine), Lys (lysine), His (histidine), and Orn (ornithine) accounts for at least 10% of the total amino acids of the oligopeptide.

[0087] This embodiment can be achieved, for example, by the empirical formula (Arg) shown above. l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x (formula (I)), can be applied to situations in which the cationic or polycationic peptide or protein of the polymeric carrier is characterized in that it contains or is modified with at least one cysteine, as an -SH moiety in the above sense, so that the cationic or polycationic peptide as cationic component carries at least one cysteine ​​capable of forming a disulfide bond with another component of the polymeric carrier.

[0088] In another particularly preferred embodiment, the cationic or polycationic peptide or protein of the polymeric carrier has the formula {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x} (formula (I)), is selected from, but not limited to, the following sub-formula (Ib):

[0089] Cys 1 {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x} Cys 2 Formula (Ib)

[0090] where the empirical formula {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x} (Formula (I)) is defined herein and (semi-empirical experiments) forms the core of an amino acid sequence according to Formula (I), 1 and Cys 2 is (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x Exemplary examples may include any of the above sequences with two Cys adjacent to each other, as well as the following sequences:

[0091] CysArg7Cys Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (SEQ ID NO: 1) CysArg8Cys Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (SEQ ID NO:2) CysArg9Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:3) CysArg 10 Cys Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:4) CysArg 11 Cys Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:5) CysArg 12 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:6) CysArg 13 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:7) CysArg 14 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number: 8) CysArg 15 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:9) CysArg 16 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number:10) CysArg 17 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (sequence number: 11) CysArg 18Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (SEQ ID NO: 12) CysArg 19 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (SEQ ID NO: 13) CysArg 20 Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Cys (SEQ ID NO: 14)

[0092] This embodiment can be achieved, for example, by the empirical formula (Arg) shown above. l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x This can be applied to the situation where the cationic or polycationic peptides or proteins of the polymeric carriers that can be used to complex at least one mRNA of the vaccine of the invention, when defined according to (formula (I)), are characterized in that they are modified with at least two cysteines as -SH moieties in the above sense, so that the cationic or polycationic peptides of the polymeric carriers of the invention carry at least two (terminal) cysteines capable of forming disulfide bonds with other components of the polymeric carrier.

[0093] According to a second alternative, at least one cationic (or polycationic) component of the polymeric carrier may, for example, be selected from any (non-peptidic) cationic or polycationic polymer suitable in this context, provided that this (non-peptidic) cationic or polycationic polymer presents, or has been modified to present, at least one -SH moiety, which provides for a disulfide bond between the cationic or polycationic polymer and other components of the polymeric carrier as defined herein. Thus, the polymeric carrier may comprise the same or different cationic or polycationic polymers, also as defined herein.

[0094] In certain cases where the cationic component of the polymeric carrier comprises a (non-peptidic) cationic or polycationic polymer, the cationic character of the (non-peptidic) cationic or polycationic polymer can be determined by the amount of cationic charge compared to the overall charge of the cationic polymer component. Preferably, the amount of cationic charge on the cationic polymer at (physiological) pH is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60%, or 70%, but also at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99%, or 100%, and most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. %, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 30% to 100%, and even more preferably in the range of 50% to 100%, for example, 50%, 60%, 70%, 80%, 90% or 100%, or a range formed by any two of the aforementioned values ​​(wherein the total charge amount, for example, the positive and negative charges at (physiological) pH as defined herein in the entire cationic polymer, is taken as 100%).

[0095] Preferably, the (non-peptidic) cationic component of the polymeric carrier typically represents a cationic or polycationic polymer exhibiting a molecular weight of 0.1 kDa or 0.5 kDa, preferably from about 1 kDa to about 75 kDa, more preferably from about 5 kDa to about 50 kDa, even more preferably from about 5 kDa to about 30 kDa, or from about 10 kDa to about 50 kDa, even more preferably from about 10 kDa to about 0 kDa. In addition, the (non-peptidic) cationic or polycationic polymer typically presents at least one -SH moiety capable of forming a disulfide bond by condensation with another cationic component or other components of the polymeric carrier as defined herein.

[0096] In this context, the (non-peptidic) cationic component of the polymeric carrier that can be used to complex at least one mRNA of the vaccine of the present invention can be selected from acrylates, modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methyl acrylate)), chitosan, aziridine, or 2-ethyl-2-oxazoline (forming oligoethyleneimines or modified oligoethyleneimines), polymers obtained by reacting bisacrylates with amines to form oligo-β-aminoesters or polyamidoamines, or other polymers such as polyesters and polycarbonates. Each molecule of these (non-peptidic) cationic or polycationic polymers typically presents at least one -SH moiety, where this at least -SH may be introduced into the (non-peptidic) cationic or polycationic polymer by chemical modification, such as the introduction of iminothiolane, 3-thiopropionic acid, or an -SH moiety-containing amino acid, such as cysteine ​​or any further (modified) amino acid. Such -SH moieties are preferably as defined above.

[0097] In relation to the polymeric carrier, the cationic components forming the basis of the polymeric carrier that can be used to complex at least one mRNA of the vaccine of the invention by disulfide bridges can be the same or different from each other. It is also particularly preferred that the polymeric carrier of the invention comprises a mixture of cationic peptides, proteins or polymers and, optionally, further components as defined herein, cross-linked by disulfide bonds as described herein.

[0098] In this regard, the polymeric carriers of the present invention, which can be used to complex at least one mRNA of the vaccine of the present invention, allow for the combination of the desired properties of different (short) cationic or polycationic peptides, proteins, or polymers, or other components. The polymeric carriers allow for the efficient condensation of nucleic acids without loss of activity, for example, for the purpose of efficient transfection of nucleic acids for adjuvant therapy, gene therapy for gene knockdown, or other methods, particularly for efficient transfection of nucleic acids into different cell lines in vitro, but also for in vivo transfection. Furthermore, the polymeric carriers are not toxic to cells, provide efficient release of their nucleic acid cargo, are stable during lyophilization, and are applicable as immunostimulants or adjuvants. In this regard, the components of the polymeric carriers of the present invention can be varied in such a way that the induced cytokine pattern can be determined.

[0099] Specifically, polymeric carriers formed with disulfide-linked cationic moieties allow for significant changes in their peptide or polymer content, for example, by introducing the same or different cationic peptides or polymers as the cationic moiety into the polymeric carrier and optionally adding other moieties, thereby allowing for extremely simple and rapid adjustment of their biophysical / biochemical properties, particularly the cationic properties of the polymeric carrier. Despite being composed of very small, non-toxic monomer units, the polymeric carrier forms long cationic binding sequences that provide strong condensation of mRNA as a nucleic acid cargo and stability of the complex. Under reducing conditions in the cytosol (e.g., cytosolic GSH), the complex rapidly degrades into its (cationic) components, which are further degraded (e.g., oligopeptides). This supports the decomposition of nucleic acid cargo in the cytosol. No toxicity is observed due to degradation into small oligopeptides or polymers in the cytosol, as is known for larger oligopeptides or polymers, such as polymeric polyarginine.

[0100] Thus, the polymeric carriers that may be used to complex at least one mRNA of the vaccine of the invention may comprise different (short) cationic or polycationic peptides, proteins or polymers selected from the above-mentioned cationic or polycationic peptides, proteins or (non-peptidic) polymers, optionally together with further components as defined herein.

[0101] In addition, the polymeric carrier that can be used to complex at least one mRNA of the vaccine of the present invention, more preferably at least one different (short) cationic or polycationic peptide or (non-peptidic) polymer that forms the basis of the polymeric carrier via disulfide bridges, may preferably be modified with at least one additional component prior to disulfide bridge formation. Alternatively, such a polymeric carrier may be modified with at least one additional component. It may also optionally contain at least one additional component that typically forms the disulfide of the polymeric carrier with another (short) cationic or polycationic peptide as defined above via disulfide bridges.

[0102] To allow modification of the cationic or polycationic peptides or (non-peptidic) polymers defined above, each component of the polymeric carrier may also (preferably already before the disulfide bridge) comprise at least one further functional group, which allows the linking of further components as defined herein. Such functional groups can be, for example, by amide formation (e.g., carboxylic acids, sulfonic acids, amines, etc.), by Michael addition (e.g., maleimide moieties, α,β-unsaturated carbonyls, etc.), by click chemistry (e.g., azides or alkynes), alkene / alkyne metathesis (e.g., alkenes or alkynes), imine or hydrazone formation (aldehydes or ketones, hydrazines, hydroxylamines, amines), conjugation reactions (avidin, biotin, protein G), or S n The functional groups may be selected from compounds that permit substitution reactions of this type (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonate esters, oxyphosphonium salts) or other chemical moieties available for the attachment of further components, e.g., functional groups that permit the attachment of further components, such as those defined herein.

[0103] In a particularly preferred embodiment, a further component forming the basis of the polymeric carrier or the biophysical / biochemical properties of the polymeric carrier as defined herein is an amino acid component (AA), which may be included in the polymeric carrier and used to conjugate at least one of the vaccines of the invention or to modify different (short) cationic or polycationic peptides or (non-peptidic) polymers. According to the present invention, the amino acid component (AA) preferably comprises a number of amino acids selected from the range of about 1 to 100, preferably from the range of about 1 to 50, more preferably from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to 20, inclusive, or may be selected from the range formed by any two of the aforementioned values. In this context, the amino acids of the amino acid component (AA) can be selected independently of each other. For example, if two or more (AA) components are present in the polymeric carrier, they may be identical to each other or different from each other.

[0104] The amino acid component (AA) may contain or be adjacently (e.g., terminally) positioned -SH-containing moieties, which allow the component (AA) to be incorporated into a polymeric carrier as defined herein via a disulfide bond. When the -SH-containing moiety represents cysteine, the amino acid component (AA) may also be interpreted as -Cys-(AA)-Cys-, where Cys represents cysteine ​​and provides the -SH moiety necessary for disulfide bonding. The -SH-containing moiety may also be introduced into the amino acid component (AA) using any modification or reaction for a cationic moiety or any of its components, as described above.

[0105] Furthermore, for example, when an amino acid component (AA) is used as a linker between two additional components (e.g., as a linker between two cationic polymers), the amino acid component (AA) may be provided with two -SH groups (or more), e.g., in the form of the formula HS-(AA)-SH, to allow for linking the two functional groups via a disulfide bond. In this case, one -SH moiety is preferably protected in an initial step using a protecting group known in the art, leading to an amino acid component (AA) of the formula HS-(AA)-S-protecting group. The amino acid component (AA) may then be coupled to an additional component of the polymeric carrier via the unprotected -SH moiety to form a first disulfide bond. The protected -SH moiety is then typically deprotected and coupled to an additional free -SH moiety of the additional component of the polymeric carrier to form a second disulfide bond.

[0106] Alternatively, the amino acid component (AA) may be provided on other functional groups already mentioned above for other components of the polymeric carrier, which allows for the attachment of the amino acid component (AA) to any component of the polymeric carrier.

[0107] Thus, according to the present invention, the amino acid component (AA) may be attached to a further component of the polymeric carrier, which can be used to complex at least one mRNA of the vaccine of the present invention, with or without a disulfide bond. Attachment without a disulfide bond may be achieved by any of the reactions described above, preferably by attaching the amino acid component (AA) to another component of the polymeric carrier using amide chemistry as defined herein. If desired or necessary, the other end of the amino acid component (AA), e.g., the N-terminus or C-terminus, may be used to link another component, e.g., a ligand L. For this purpose, the other end of the amino acid component (AA) preferably contains or is modified to contain a further functional group, such as an alkyne species (see above), which can be used to attach another component, e.g., via click chemistry. If the ligand is attached via an acid-labile bond, the bond is preferably cleaved in the endosome, leaving the polymeric carrier presenting the amino acid component (AA) on its surface.

[0108] The amino acid component (AA) may be present as a further component of a polymeric carrier that can be used to complex at least one mRNA of a vaccine of the invention as defined above, e.g. as a linker between cationic components, all preferably as defined herein, e.g. as a linker between one cationic peptide and a further cationic peptide, as a linker between one cationic polymer and a further cationic polymer, or may be present as an additional component of a polymeric carrier, e.g. by binding the amino acid component (AA) to the polymeric carrier or a component thereof, e.g. via a side chain, an SH moiety, or via a further moiety as defined herein, wherein the amino acid component (AA) is preferably modified accordingly.

[0109] According to a further particularly preferred alternative, amino acid components (AA) may be used to modify the content of cationic components in the polymeric carrier, in particular in the polymeric carriers defined above.

[0110] In this context, the content of cationic components in the polymeric carrier is preferably at least 10%, 20% or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, and most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 30% to 100%, more preferably in the range of about 50% to 100%, even more preferably in the range of 70% to 100%, for example 70%, 80%, 90% or 100%, or a range formed by any two of the aforementioned values ​​(where the total amino acid content is taken as 100%).

[0111] In the context of the present invention, the amino acid components (AA) may be selected from the following options:

[0112] According to a first alternative, the amino acid moiety (AA) may be an aromatic amino acid moiety (AA). The incorporation of aromatic amino acids, or sequences of amino acids as aromatic acid moieties (AA), into the polymeric carrier of the present invention allows for a different (secondary) binding of the polymeric carrier to nucleic acids, through interaction of the aromatic amino acid with the bases of the nucleic acid cargo, as opposed to binding to its phosphate backbone through the cationic charged sequence of the polymeric carrier molecule. This interaction can occur, for example, by intercalation or by minor or major groove binding. This type of interaction is not prone to decompactation by anionic complexing partners (e.g., heparin, hyaluronic acid) found predominantly in the extracellular matrix in vivo, and is also less sensitive to salts.

[0113] For this purpose, the amino acids in the aromatic amino acid component (AA) may be selected from the same or different aromatic amino acids, for example selected from Trp, Tyr, or Phe. Alternatively, the amino acids (or all aromatic amino acid components (AA)) may be selected from the following peptide combinations: Trp-Tyr, Tyr-Trp, Trp-Trp, Tyr-Tyr, Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Trp-Trp, Tyr-Tyr-Tyr, Trp-Tyr-Trp, Tyr-Trp-Tyr-Trp, Trp-Trp-Trp, Phe-Tyr, Tyr-Phe, Phe-Phe, Phe-Ty The peptides may be selected from r-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr, Tyr-Phe-Tyr-Phe, Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe, Phe-Trp-Phe, Trp-Phe-Trp, Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, or Tyr-Tyr-Tyr-Tyr (SEQ ID NOs: 15-42). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times. These peptide combinations may also be appropriately combined with each other.

[0114] Additionally, the aromatic amino acid component (AA) may contain or be adjacent to an -SH-containing moiety, which allows the component to be incorporated via a disulfide bond into a further component of the polymeric carrier defined above, e.g., as a linker. Such an -SH-containing moiety may be any moiety as defined herein that is suitable for linking one component as defined herein to a further component as defined herein. By way of example, such an -SH-containing moiety may be cysteine. And, for example, the aromatic amino acid component (AA) may be, for example, a peptide combination Cys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys, Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys, Cys-Trp p-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys, Cy s-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys, Cys-Phe-Tyr-Cys, Cys-Tyr-Ph e-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Ph e-Phe-Phe-Cys, Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys, or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Cys, Cys-Trp-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys, Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. Each of the above Cys may also be replaced by any modified peptide or compound bearing a free -SH moiety as defined herein (SEQ ID NOs: 43 to 75).Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times. These peptide combinations may also be combined with each other as appropriate.

[0115] In addition, the aromatic amino acid component (AA) may contain or represent at least one proline, preferably two, three or more prolines, which may act as a structural breaker for long sequences of Trp, Tyr and Phe in the aromatic amino acid component (AA).

[0116] According to a second alternative, the amino acid moiety (AA) may be a hydrophilic (and preferably uncharged polar) amino acid moiety (AA). The incorporation of a sequence as a hydrophilic (and preferably uncharged polar) amino acid or amino hydrophilic (and preferably uncharged polar) acid moiety (AA) into the polymeric carrier of the present invention allows for more flexible binding to the nucleic acid cargo. This results in more effective compaction of the nucleic acid cargo and thus better protection against nucleases and unwanted decompaction. It also allows the provision of (long) polymeric carriers that, if desired or necessary, exhibit a reduced cationic charge throughout the entire carrier, with the associated improved tailored binding properties.

[0117] For this purpose, the amino acids in the hydrophilic (and preferably uncharged polar) amino acid component (AA) may be selected from the same or different hydrophilic (and preferably uncharged polar) amino acids, for example selected from Thr, Ser, Asn, or Gln. Alternatively, the amino acids (or all hydrophilic (and preferably uncharged polar) amino acids (AA)) may be selected from the following peptide combinations: Ser-Thr, Thr-Ser, Ser-Ser, Thr-Thr, Ser-Thr-Ser, Thr-Ser-Thr, Ser-Ser-Thr, Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser, Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr, Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn, Gln-Asn-Gln, Asn-Gln-Asn, Gl ... It may be selected from n-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn, Asn-Gln-Asn-Gln, Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser, Ser-Ser, Asn-Asn, Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn, Ser-Asn-Ser-Asn, Asn-Ser-Asn-Ser, Ser-Ser-Ser, or Asn-Asn-Asn-Asn, etc. (SEQ ID NOs: 76 to 111). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times. These peptide combinations may also be combined with each other as appropriate.

[0118] In addition, the hydrophilic (and preferably uncharged polar) amino acid component (AA) may contain or be adjacently positioned with an -SH-containing moiety, which allows this component to be incorporated via a disulfide bond as a further moiety, e.g., a linker, of the polymeric carrier defined above. Such an -SH-containing moiety may be any moiety as defined herein that is suitable for linking one component as defined herein to a further component as defined herein. By way of example, such an -SH-containing moiety may be cysteine.and for example, hydrophilic (and preferably uncharged polar) amino acid components (AA) can be represented by, for example, peptide combinations Cys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys, Cys-Thr-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys, Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr- Cys, Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys, Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-As n-Cys, Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys, Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-A sn-Gln-Asn-Cys, Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys, Cys-Asn-Gln-Asn-Gln-Cys, Cys -Gln-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys, Cys-Asn-Ser-Cys, Cys-Se Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys, Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Asn-Cys, Cys-Ser-Asn-Ser-Asn-Cys, Cys-Ser-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Asn-Cys, etc. Each of the above Cys may also be replaced by any modified peptide or compound carrying a free -SH moiety as defined herein. (SEQ ID NOs: 112-153) Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times.Combinations of these peptides may also be combined with each other as appropriate.

[0119] Additionally, the hydrophilic (and preferably uncharged polar) amino acid component (AA) may contain at least one proline, preferably two, three or more prolines, which may act as a structural breaker for long sequences of Ser, Thr and Asn in the hydrophilic (and preferably uncharged polar) amino acid component (AA).

[0120] According to a third alternative, the amino acid moiety (AA) may be a lipophilic amino acid moiety (AA). The incorporation of a lipophilic amino acid or amino lipophilic acid moiety (AA) sequence into the polymeric carrier of the present invention allows for stronger compaction of the nucleic acid cargo and / or polymeric carrier and / or the nucleic acid cargo when forming a complex. This is due in particular to the interaction between one or more polymer chains of the polymeric carrier, in particular the lipophilic amino acid moiety (AA), and the lipophilic portion of the nucleic acid cargo. This interaction preferably adds additional stability to the complex between the polymeric carrier and its nucleic acid cargo. This stabilization can be compared in some way to a kind of non-covalent bond between different polymer chains. Especially in an aqueous environment, this interaction is typically strong and provides a significant effect.

[0121] For this purpose, the amino acids in the lipophilic amino acid component (AA) may be selected from the same or different lipophilic amino acids, for example selected from Leu, Val, Ile, Ala, and Met. Alternatively, the amino acids (or the entire lipophilic amino acid component (AA)) may be selected from the following peptide combinations: Leu-Val, Val-Leu, Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val, Leu-Leu-Leu, Val-Val-Val, Leu-Val-Leu-Val, Val-Leu-Val-Leu, Leu-Leu-Leu-Leu, Val-Val-Val-Val, Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala, Ile-Ala-Ile, Ala-Ile-Ala, Ile ... The peptide combinations may be selected from Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Ile-Ala, Ala-Ile-Ala-Ile, Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met, Met-Met, Ala-Ala, Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala, Met-Ala-Met-Ala, Ala-Met-Ala-Met, or Met-Met-Met-Met, etc. (SEQ ID NOs: 154-188). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or more times. These peptide combinations may also be combined with each other as appropriate.

[0122] In addition, the lipophilic amino acid component (AA) may contain or be adjacent to an -SH-containing moiety, which allows this component to be incorporated via a disulfide bond as a further component, e.g., a linker, of the polymeric carrier defined above. Such an -SH-containing moiety may be any moiety as defined herein that is suitable for linking one component as defined herein to a further component as defined herein. By way of example, such an -SH-containing moiety may be cysteine.and for example, the lipophilic amino acid moiety (AA) may be any of the peptide combinations Cys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys, Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys, Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys- Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys, Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys , Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys, Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala -Ile-Ala-Cys, Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys, Cys-Ala-Ile-Ala-Ile-Cys, Cy s-Ile-Ile-Ile-Ile-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys, Cys-Met-Met- Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys, Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. Each of the above Cys may also be replaced by any modified peptide or compound bearing a free -SH moiety as defined herein (SEQ ID NOs: 189 to 229). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times.Combinations of these peptides may also be combined with each other as appropriate.

[0123] In addition, the lipophilic amino acid component (AA) may contain at least one proline, preferably two, three or more prolines, which may act as a structural breaker for long sequences of Leu, Val, Ile, Ala and Met in the lipophilic amino acid component (AA).

[0124] Finally, according to a fourth alternative, the amino acid component (AA) may be a weakly basic amino acid component (AA). The incorporation of a weakly basic amino acid or a sequence of a weakly basic amino acid component (AA) into the polymeric carrier of the present invention may function as a proton sponge and promote endosomal escape (also called endosomal release) (proton sponge effect). The incorporation of such a weakly basic amino acid component (AA) preferably improves transfection efficiency.

[0125] For this purpose, the amino acids in the weakly basic amino acid component (AA) can be selected from the same or different weak amino acids, for example, histidine or aspartate (aspartic acid). Alternatively, the weakly basic amino acids (or the entire weakly basic amino acid component (AA)) can be selected from the following peptide combinations: Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp, His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp, Asp-Asp-Asp-Asp, or His-His-His-His, etc. (SEQ ID NOs: 230-241). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or more times. Combinations of these peptides may also be combined with each other as appropriate.

[0126] In addition, the weakly basic amino acid component (AA) may contain or be adjacent to an -SH-containing moiety, which allows the component to be introduced via a disulfide bond into a further component of the polymeric carrier defined above, e.g., as a linker. Such an -SH-containing moiety may be any moiety as defined herein that is suitable for linking one component as defined herein to a further component as defined herein. By way of example, such an -SH-containing moiety may be cysteine. And, for example, the weakly basic amino acid component (AA) may be, for example, a peptide combination Cys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys, Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys, Cys-His-Asp- The amino acid sequence may be selected from His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys, Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys, Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-Cys. Each Cys may also be replaced by any modified peptide or compound bearing a free -SH moiety as defined herein (SEQ ID NOs: 242-255). Such peptide combinations may be repeated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or more times. These peptide combinations may also be combined with each other as appropriate.

[0127] Additionally, the weakly basic amino acid component (AA) may contain at least one proline, preferably two, three or more prolines, which may act as a structural breaker for long sequences of histidine or aspartate (aspartic acid) in the weakly basic amino acid component (AA).

[0128] According to a fifth alternative, the amino acid component (AA) may be a signal peptide or sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell-penetrating peptide (e.g., TAT), etc. Preferably, such an amino acid component (AA) is linked to the polymeric carrier or another component of the polymeric carrier via a (reversible) disulfide bond. In this context, the signal peptide or sequence, the localization signal or sequence, the nuclear localization signal or sequence (NLS), the antibody, the cell-penetrating peptide (e.g., TAT), etc. additionally comprises at least one -SH moiety. In this regard, a signal peptide, localization signal or sequence, or nuclear localization signal or sequence (NLS) can be used to guide the polymeric carrier cargo complex of the present invention to a specific target cell (e.g., stem cell or antigen-presenting cell), preferably a mitochondrial matrix sequence, a cell membrane sequence, a Golgi apparatus, a nucleus, a cytoplasm, a cytoskeleton sequence, etc., which allows the transport and localization of the polymeric carrier to a specific target, for example, a cell, a nucleus, an endosomal compartment, etc. Such a signal peptide, localization signal or sequence, or nuclear localization signal can be used for transporting any nucleic acid defined herein, preferably RNA or DNA, more preferably shRNA or pDNA, for example, to the nucleus. Without being limited thereto, such a signal peptide, localization signal or sequence, or nuclear localization signal can include, for example, an endoplasmic reticulum localization sequence.Specific localization signals or localization sequences, or nucleic acid localization signals include, for example, KDEL (SEQ ID NO: 256), DDEL (SEQ ID NO: 257), DEEL (SEQ ID NO: 258), QEDL (SEQ ID NO: 259), RDEL (SEQ ID NO: 260), and GQNLSTSN (SEQ ID NO: 261), PKKKRKV (SEQ ID NO: 262), PQKKIKS (SEQ ID NO: 263), QPKKP (SEQ ID NO: 264), RKKR (SEQ ID NO: 265), RKKRRQRRRAHQ. (SEQ ID NO: 266), RQARRNRRRRWRERQR (SEQ ID NO: 267), MPLTRRRPAASQALAPPTP (SEQ ID NO: 268), GAALTILV (SEQ ID NO: 269), and GAALTLLG (SEQ ID NO: 270), a nuclear localization sequence including MDDQRDLISNNEQLP (SEQ ID NO: 271), a mitochondrial localization sequence including MLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 272), and a targeting sequence for the endosomal compartment including MDDQRDLISNNEQLP (SEQ ID NO: 273). GCVCSSNP (SEQ ID NO: 273), GQTVTTPL (SEQ ID NO: 274), GQELSQHE (SEQ ID NO: 275), GNSPSYNP (SEQ ID NO: 276), GVSGSKGQ (SEQ ID NO: 277), GQTITTPL (SEQ ID NO: 278), GQTLTTPL (SEQ ID NO: 279), GQIFSRSA (SEQ ID NO: 280), GQIHGLSP (SEQ ID NO: 281), GARASVLS (SEQ ID NO: 282), and GCTLSA Examples of secretory signal peptide sequences as defined herein include, but are not limited to, signal sequences for targeting the cell membrane, such as EE (SEQ ID NO: 283), sequences for targeting the endoplasmic reticulum and nucleus, including GAQVSSQK (SEQ ID NO: 284) and GAQLSRNT (SEQ ID NO: 285), sequences for targeting the Golgi apparatus, nucleus, cytoplasm, and cytoskeleton, including GNAAAAKK (SEQ ID NO: 286), sequences for targeting the cytoplasm and cytoskeleton, including GNEASYPL (SEQ ID NO: 287), and sequences for targeting the cell membrane and cytoskeleton, including GSSKSKPK (SEQ ID NO: 288). Examples of secretory signal peptide sequences as defined herein include, but are not limited to, signal sequences of classical MHC molecules or non-classical MHC molecules (e.g., MHC I molecules and MHC II molecules, such as the MHC class I molecule HLA-A). *0201), the signal sequence of a cytokine or immunoglobulin as defined herein, the signal sequence of the invariant chain of an immunoglobulin or antibody as defined herein, the signal sequences of Lamp1, Tapasin, Erp57, calreticulin, calnexin and further membrane-associated proteins, or the signal sequences of proteins associated with the endoplasmic reticulum (ER) or endosomal-lysosomal compartments. Particularly preferred are the signal sequences of the MHC class I molecule HLA-A * A signal sequence of 0201 may be used in accordance with the present invention. Such additional components may, for example, be attached to any other component of the cationic polymer or polymeric carrier defined herein. Preferably, the signal peptide, localization signal or sequence, or nuclear localization signal or sequence (NLS) is attached to the polymeric carrier or other component of the polymeric carrier via a (reversible) disulfide bond. For this purpose, the (AA) component additionally comprises at least one -SH moiety as defined herein. Attachment to any component of the polymeric carrier may also be carried out using an acid-labile bond, preferably via any side chain of the component of the polymeric carrier that allows cleavage or release of the additional component at low pH values, e.g., physiological pH values ​​as defined herein.

[0129] Additionally, according to another alternative, the amino acid component (AA) may be a functional peptide or a functional protein that can modulate the function of the polymeric carrier accordingly. Such functional peptides or proteins as amino acid components (AA) may preferably comprise any peptide or protein defined herein, e.g., as defined below as a therapeutic protein. According to one alternative, such additional functional peptides or proteins may comprise so-called cell-penetrating peptides (CPPs) or cationic peptides for transport. Particularly preferred are CPPs that induce pH-mediated conformational changes within endosomes and insert into the lipid layer of liposomes, thereby resulting in improved release of the polymeric carrier (complexed with nucleic acid) from endosomes. These cell penetrating peptides (CPPs) or cationic peptides for delivery include, but are not limited to, protamine, nucleolin, spermine or spermidine, oligo- or poly-L-lysine (PLL), basic polypeptides, oligo- or polyarginine, cell penetrating peptides (CPPs), chimeric CPPs such as transportan or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat Examples of CPPs that may be used include HIV (Human Immunodeficiency Virus), Tat-derived peptides, such as penetratin, Antennapedia-derived peptides (particularly from Drosophila Antennapedia), members of the penetratin family, such as pAntp and pIsl, and CPPs derived from antibacterial agents, such as buforin 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, oligomers, FGF, lactoferrin, histones, VP22-derived or similar peptides, Erns of plague virus, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, and calcitonin peptides. Such amino acid moieties (AA) may also be conjugated to any polymeric carrier as defined herein. Preferably, it is bound to the polymeric carrier or to another component of the polymeric carrier via a (reversible) disulfide bond.For the above purposes, the amino acid component (AA) preferably comprises at least one -SH moiety as defined herein. Attachment to any component of the polymeric carrier may also be achieved using an SH moiety or an acid-labile bond, preferably via any side chain of the component of the polymeric carrier that allows for cleavage or release of the additional component at low pH values, e.g., physiological pH values ​​as defined herein.

[0130] According to a last alternative, the amino acid component (AA) may consist of any peptide or protein capable of performing any useful function in the cell. Particularly preferred are peptides or proteins selected from therapeutic proteins or peptides derived from antigens, such as tumor antigens, pathogenic antigens (animal antigens, viral antigens, protozoan antigens, bacterial antigens, allergenic antigens), autoimmune antigens, or further antigens derived from allergens, antibodies, immunostimulatory proteins or peptides, antigen-specific T cell receptors, or any other protein or peptide suitable for a particular (therapeutic) use as defined below for the encoding nucleic acid. Particularly preferred are peptide epitopes derived from the antigens defined herein.

[0131] The polymeric carrier that can be used to complex at least one mRNA of the vaccine of the present invention may comprise at least one of the cationic or polycationic peptides, proteins or polymers mentioned above, or additional components such as (AA), where any of the above alternatives may be combined with each other or formed by polymerizing them in a polycondensation reaction via their -SH moieties.

[0132] In another embodiment, the polymeric carrier, e.g., the above-mentioned cationic or polycationic peptide, protein, or further component (AA), which can be used to complex at least one mRNA of the vaccine of the present invention or a single component thereof, can be further modified with a ligand, preferably a carbohydrate, more preferably a sugar, and even more preferably mannose. Preferably, the ligand is attached to the polymeric carrier or a component of the polymeric carrier via a (reversible) disulfide bond or via Michael addition. When the ligand is attached by a disulfide bond, it further comprises at least one -SH- moiety. These ligands can be used to target the polymeric carrier cargo complex of the present invention to specific target cells (e.g., hepatocytes or antigen-presenting cells). In this regard, mannose is particularly preferred as a ligand, especially when dendritic cells are the target for vaccination or adjuvant purposes.

[0133] According to one particular embodiment, the entire polymeric carrier of the present invention is formed in a first step by condensation polymerization of (at least one) of the above-mentioned cationic or polycationic peptides, proteins or polymers, or further components, such as (AA), via their -SH moieties, and in a second step by complexing nucleic acids to such a polymeric carrier. The polymeric carrier may thus contain a large number of at least one or more of the same or different cationic or polycationic peptides, proteins or polymers, or further components, such as (AA), as defined above, the number of which is preferably determined within the above ranges.

[0134] In one alternative specific embodiment, the polymeric carrier of the present invention, which can be used to complex at least one mRNA of the vaccine of the present invention, is formed by polycondensation of at least one of the above-mentioned cationic or polycationic peptides, proteins or polymers, or further components, such as (AA), via their -SH moieties, and simultaneously complexing at least one mRNA encoding at least one antigen to the (in situ) polymeric carrier. Likewise, the polymeric carrier may thus comprise at least one or more of the same or different cationic or polycationic peptides, proteins or polymers, or further components, such as (AA), as defined above, the number of which is preferably determined within the ranges mentioned above.

[0135] According to a further alternative embodiment, the polymeric carrier of the present invention may be selected from polymeric carrier molecules of general formula (VI) below:

[0136] LP 1 -S-[SP 2 -S] n -SP 3 -L Formula (VI) where P 1 and P 3 are different or identical to each other and represent linear or branched hydrophilic polymer chains, and each P 1 and P 3 is the component P 2 or by condensation with (AA), (AA) x or [(AA) x ] z (Such components are P 1 and P 2 or P 3 and P 2 (AA), (AA) x , [(AA) x ] zor L), and the linear or branched hydrophilic polymer chains are independently selected from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholine, poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine), hydroxyethyl starch, or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chains exhibit a molecular weight of about 1 kDa to about 100 kDa, preferably about 2 kDa to about 25 kDa, more preferably about 2 kDa to about 10 kDa, for example, about 5 kDa to about 25 kDa or 5 kDa to about 10 kDa; P 2 is, for example, a cationic or polycationic peptide or protein as defined herein, preferably having a length of about 3 to 100 amino acids, more preferably about 3 to about 50 amino acids, even more preferably about 3 to about 25 amino acids, for example about 3 to 10 amino acids, 5 to 15 amino acids, 10 to 20 amino acids, or 15 to 25 amino acids, more preferably about 5 to 20 amino acids, and even more preferably about 10 to about 20 amino acids, or P 2 is typically a cationic or polycationic polymer as defined herein, having a molecular weight of about 0.5 kDa to about 30 kDa, including a molecular weight of about 1 kDa to about 20 kDa, more preferably about 1.5 kDa to about 10 kDa, or about 0.5 kDa to about 100 kDa, including a molecular weight of about 10 kDa to about 50 kDa, more preferably about 10 kDa to about 30 kDa, and each P 2 is a further component P 2 , or component P 1 and component P 3 and / or alternatively further components (e.g., (AA), (AA) x , or [(AA) x ] z presenting at least two -SH moieties capable of forming disulfide bonds by condensation with -SS- is a (reversible) disulfide bond (brackets omitted for readability), where S preferably represents sulfur or an -SH-bearing moiety that has formed a (reversible) disulfide bond. The (reversible) disulfide bond is preferably formed by the bonding of component P 1 and P 2 , P 2 and P 2 , or P 2 and P 3 or optionally further components as defined herein (e.g., L, (AA), (AA) x , [(AA) x ] z etc.), which may be part of the structure of these components or may be added by modifications as defined below, L are optional ligands that may or may not be present, each independently selected from RGD, transferrin, folate, a signal peptide or sequence, a localization signal or sequence, a nucleic acid localization signal or sequence (NLS), an antibody, a cell penetrating peptide (e.g., TAT, or KALA), a ligand of a receptor (e.g., a cytokine, hormone, growth factor, etc.), a small molecule (e.g., a carbohydrate such as mannose, galactose, or a synthetic ligand), a small molecule agonist, an inhibitor or antagonist of a receptor (e.g., an RGD peptidomimetic analog), etc.; n is typically an integer selected from the range of about 1 to 50, including, for example, the range of about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, the range of about 3 to 15, 4 to 15, or 10 to 15, or the range of about 6 to 11 or 7 to 10, preferably the range of about 1, 2, or 3 to 30, more preferably the range of about 1, 2, 3, 4, or 5 to 25, the range of about 1, 2, 3, 4, or 5 to 20, the range of about 1, 2, 3, 4, or 5 to 15, or the range of about 1, 2, 3, 4, or 5 to 10. Most preferably, n is in the range of about 1, 2, 3, 4, or 5 to 10, more preferably the range of about 1, 2, 3, or 4 to 9, the range of about 1, 2, 3, or 4 to 8, or the range of about 1, 2, or 3 to 7.

[0137] As defined above, ligands (L) may optionally be used in the polymeric carrier molecules of the invention of general formula (VI), for example, to target the carrier polymer of the invention and all its "cargo" (adjuvant component and / or antigen, or vaccine composition of the invention) to specific cells. They may be selected independently from one another from RGD, transferrin, folate, signal peptides or sequences, localization signals or sequences, nucleic acid localization signals or sequences (NLS), antibodies, cell penetrating peptides (CPPs) (e.g., TAT, KALA), receptor ligands (e.g., cytokines, hormones, growth factors, etc.), small molecules (e.g., carbohydrates such as mannose, galactose, or synthetic ligands), small molecule agonists, receptor inhibitors or antagonists (e.g., RGD peptidomimetic analogs), or molecules as further defined below. Particularly preferred are cell-penetrating peptides (CPPs) that induce pH-mediated structural changes within endosomes and insert into the lipid layer of liposomes, thereby resulting in improved release of the polymeric carriers of the present invention (complexed with nucleic acid) from endosomes.So-called CPPs or cationic peptides for transport include, but are not limited to, protamine, nucleolin, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, polyarginine, cell penetrating peptides (CPPs), chimeric CPPs such as transportan or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, such as penetratin, antennapedia-derived peptides (particularly from Drosophila antennapedia), members of the penetratin family such as pAntp and pIsl, and peptides such as buforin 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, proline-rich peptides, oligomers, arginine-rich peptides, calcitonin, and the like. Examples of CPPs include antibacterial agents such as calcitonin peptides, FGF, lactoferrin, poly-L-lysine, polyarginine, histones, VP22-derived or similar peptides, Erns of the plague virus, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, and calcitonin peptides. Particularly preferred in this context is mannose as a ligand, which targets antigen-presenting cells bearing mannose receptors on their cell membranes. In a further preferred aspect of the first embodiment of the present invention, galactose can be used as an optional ligand to target hepatocytes. Such ligands can be formed by reversible disulfide bonds as defined below, or by other possible chemical additions, such as amide formation (e.g., carboxylic acids, sulfonic acids, amines, etc.), Michael addition (e.g., maleimide moieties, α,β-unsaturated carbonyls, etc.), by click chemistry (e.g., azides or alkynes), imine or hydrazone formation (aldehydes or ketones, hydrazines, hydroxylamines, amines), conjugation reactions (avidin, biotin, protein G), or S. nComponent P can be linked to a compound that undergoes a substitution reaction (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonate esters, oxyphosphonium salts) or other chemical moieties that can be used to link further components. 1 and component P 3 It may be attached to at least one of the above.

[0138] In relation to formula (VI) of the present invention, P 1 and P 3 represents a linear or branched hydrophilic polymer chain containing at least one -SH moiety, and each P 1 and P 3 are independently selected from, for example, polyethylene glycol (PEG), poly-N-(2-hydroxypropyl) methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholine, poly(hydroxyalkyl L-asparagine), or poly(hydroxyalkyl L-glutamine). 1 and P 3 may be the same or different from each other. Preferably, each hydrophilic polymer P 1 and P 2 represents a molecular weight of about 1 kDa to about 100 kDa, preferably about 1 kDa to about 75 kDa, more preferably about 5 kDa to about 50 kDa, and even more preferably about 5 kDa to about 25 kDa. 1 and P 3 typically contains at least one -SH moiety, where optionally, for example, L, and (AA) or (AA) x P as defined below, together with further components such as at least one of 1 and P 2 , or P 3 and P 2 When used as a linker between moieties P, for example, when two or more -SH moieties are included, at least one -SH moiety is 2 , or component (AA) or component (AA) x Disulfide bonds can be formed by reaction with S, P 1 and P 3and any of the following sub-formulas "P" in the above general formula (VI) is as defined herein. 1 -SSP 2 " and "P 2 -SSP 3 ” (brackets omitted for readability) is typically a hydrophilic polymer P 1 and P 3 one -SH moiety of the component P of the above general formula (VI) 2 and the sulfurs of both -SH moieties form a disulfide bond -SS- as defined in formula (VI). These -SH moieties are typically attached to the respective hydrophilic polymer P via, for example, an internal cysteine ​​or any further (modified) amino acid or amino compound bearing -SH moieties. 1 and P 2 Therefore, the sub-formula "P 1 -SSP 2 " and "P 2 -SSP 3 " also refers to "P" when the -SH moiety is provided by a cysteine. 1 -Cys-Cys-P 2 " and "P 2 -Cys-Cys-P 3 ", where the expression Cys-Cys represents two cysteines linked via a disulfide bond rather than a peptide bond. In this case, the expression "-SS-" in these formulas may also be written as "-S-Cys", "-Cys-S" or "-Cys-Cys-". In this context, the expression "-Cys-Cys-" represents the bond of two cysteines that form a disulfide bond via their -SH moieties rather than a peptide bond. Therefore, the expression "-Cys-Cys-" may also generally be understood as "-(Cys-S)-(S-Cys)-", where in certain cases S refers to the sulfur of the -SH moiety of cysteine. Similarly, the expressions "-S-Cys" and "-Cys-S" refer to a disulfide bond between an -SH-containing moiety and a cysteine, which may also be written as "-S-(S-Cys)" and "-(Cys-S)-S". Alternatively, the hydrophilic polymer P1 and P 3 is the hydrophilic polymer P 1 and P 3 may be modified with an -SH moiety, preferably via chemical reaction with a compound carrying an -SH moiety, so that carries at least one such -SH moiety. Such a compound carrying an -SH moiety may for example be an (additional) cysteine ​​or any further (modified) amino acid carrying an -SH moiety. Such compounds may also be modified with a hydrophilic polymer P as defined herein. 1 and P 3 The amino acid may be a non-amino compound or moiety that contains or allows the introduction of an -SH moiety into the amino acid. Such non-amino compounds may be, for example, by coupling of 3-thiopropionic acid or thioimolane, by amide formation (e.g., carboxylic acids, sulfonic acids, amines, etc.), by Michael addition (e.g., maleimide moieties, α,β-unsaturated carbonyls, etc.), by click chemistry (e.g., azides or alkynes), alkene / alkyne metathesis (e.g., alkenes or alkynes), imine or hydrazone formation (aldehydes or ketones, hydrazines, hydroxylamines, amines), complexation reactions (avidin, biotin, protein G), or S n The hydrophilic polymer P of formula (VI) of the polymeric carrier according to the invention can be reacted or bonded to a compound that is amenable to a substitution reaction (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonic acid esters, oxyphosphonium salts) or other chemical moieties that can be used to link further components. 1 and P 3 A particularly preferred PEG derivative in this context is α-methoxy-ω-mercaptopoly(ethylene glycol). In each case, for example, cysteine ​​or any further (modified) amino acid or SH moiety of the compound may be linked to the hydrophilic polymer P 1 and P 3 As defined herein, each hydrophilic polymer P 1 and P 3typically presents at least one -SH moiety, preferably at one terminus, but may contain two or more -SH moieties, which may be linked to further moieties as defined herein, preferably further functional peptides or proteins, such as ligands, amino acid moieties (AA) or (AA) x , antibodies, cell-penetrating peptides, or enhancer peptides (e.g., TAT, KALA), etc.

[0139] According to one preferred alternative, such additional functional peptides or proteins may comprise so-called cell penetrating peptides (CPPs) or cationic peptides for transport. Particularly preferred are CPPs that induce pH-mediated structural changes within endosomes and insert into the lipid layer of liposomes, thereby resulting in improved release of the polymer carrier of the present invention (complexed with nucleic acid) from endosomes. Examples of so-called cell penetrating peptides (CPPs) or cationic peptides for transport include, but are not limited to, protamine, nucleolin, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, polyarginine, cell penetrating peptides (CPPs), chimeric CPPs such as transportan or MPG peptide, HIV-binding peptides, Tat, HIV-1, and the like. Tat (HIV), Tat-derived peptides, oligoarginines, such as penetratin, Antennapedia-derived peptides (especially from Drosophila Antennapedia), members of the penetratin family such as pAntp and pIsl, as well as peptides such as buforin 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, proline-rich peptides, oligomers, arginine-rich peptides, cal CPPs derived from antibacterial agents such as calcitonin peptides, FGF, lactoferrin, poly-L-lysine, polyarginine, histones, VP22-derived or similar peptides, Erns of the plague virus, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, and calcitonin peptides may be mentioned.

[0140] According to a further preferred aspect of the first embodiment of the present invention, each hydrophilic polymer P of formula (VI) of the polymeric carrier used in accordance with the present invention is 1 and P 3may, for example, have at least one further functional group that allows for the attachment of a further moiety as defined herein, for example, by amide formation (e.g., carboxylic acid, sulfonic acid, amine, etc.), by Michael addition (e.g., maleimide moiety, α,β-unsaturated carbonyl, etc.), by click chemistry (e.g., azide or alkyne), imine or hydrazone formation (aldehyde or ketone, hydrazine, hydroxylamine, amine), conjugation reaction (avidin, biotin, protein G), or S n The further functional moiety may comprise a ligand or functional group as defined above that allows for the attachment of further moieties by compounds that allow for substitution reactions of this type (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonate esters, oxyphosphonium salts) or other chemical moieties that can be used to attach further moieties. The further functional moiety may comprise an amino acid moiety (AA) or (AA) as defined herein. x wherein (AA) is preferably an amino acid component as defined above. In this context, x is preferably selected from the range of about 1 to 100, preferably from the range of about 1 to 50, more preferably from 1 to 30, and even more preferably from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to 30, inclusive, for example from the range of about 1 to 30, from the range of about 1 to 15, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, inclusive, or from the range formed by any two of the aforementioned values. More preferably, x is 1. In such an amino acid component (AA) or (AA) x may be contained in any part of the polymeric carrier of the present invention according to the above formula (VI), and therefore may be linked to all components of the polymeric carrier of the present invention according to formula (VI). x is a ligand or a repeating component [SP 2 -S] n It is particularly preferred that the hydroxyl group is present as part of the formula:

[0141] In relation to the overall formula (VI) of the polymeric carrier of the present invention, it can preferably be defined as follows:

[0142] LP 1 -S-[Cys-P 2 -Cys] n -SP 3 -L Formula (VI)

[0143] where L, P 1 , P 2 , P 3 and n are as defined herein, S is sulfur, and each Cys provides an -SH moiety for a disulfide bond.

[0144] According to a particular embodiment, the polymeric carrier of the invention according to formula (VI) as defined above comprises at least one amino acid component (AA) or (AA) as defined above. x Such amino acid components (AA) or (AA) may be included. x may be contained in all parts of the polymer carrier of the present invention according to the above formula (VI), and therefore may be linked to all components of the polymer carrier of the present invention according to formula (VI). x is a ligand or a repeating component [SP 2 -S] n It is particularly preferred that the amino acid component (AA) or (AA) is present as part of x is preferably connected to this component (AA) or (AA) via a disulfide bond. x The polymeric carrier according to formula (VI) as defined herein may comprise at least one -SH-containing moiety or be adjacent to (e.g., terminally) an -SH-containing moiety, which allows the incorporation of the -SH-containing moiety into the polymeric carrier according to formula (VI) as defined herein. Such -SH-containing moieties may be, for example, a cysteine ​​residue, where any -SH-containing moiety (or, of course, one sulfur of a disulfide bond) is a cysteine ​​residue. In the particular case where the -SH-containing moiety represents a cysteine, the amino acid component (AA) x Also, -Cys-(AA) x -or-Cys-(AA) x-Cys-, where Cys represents cysteine ​​and provides the -SH moiety necessary for disulfide bonding. The -SH-containing moiety can also be prepared by converting the amino acid moiety (AA) to a hydroxyl group using any of the modifications or reactions as shown above for moieties P1, P2, or P3. x Amino acid components (AA) may be introduced into x In the particular case where (AA) or (AA) are linked to two components of the polymeric carrier of the invention according to formula (VI), x Preferably, the amino acid sequence (AA) contains at least two -SH moieties, e.g., at least two cysteines, preferably at its terminus. x are the respective components [SP 2 -S] n It is particularly preferred that the aryl group is a part of (AA) or (AA) x is introduced into the polymeric carrier of the present invention according to formula (VI) as defined herein by any possible chemical addition reaction. Thus, the amino acid component (AA) or (AA) x may contain a further component as defined herein, for example a component P1 or P3, P2, L, or a further amino acid component (AA) or (AA) x etc. Such functional groups may be, for example, by amide formation (e.g., carboxylic acids, sulfonic acids, amines, etc.), by Michael addition (e.g., maleimide moieties, α,β-unsaturated carbonyls, etc.), by click chemistry (e.g., azides or alkynes), imine or hydrazone formation (aldehydes or ketones, hydrazines, hydroxylamines, amines), conjugation reactions (avidin, biotin, protein G), or S n The functional groups may be selected from compounds that permit substitution reactions of this type (e.g., halogenated alkanes, thiols, alcohols, amines, hydrazides, sulfonate esters, oxyphosphonium salts) or other chemical moieties available for the attachment of further components, e.g., functional groups that permit the attachment of further components, such as those defined herein.

[0145] The amino acid component (AA) or (AA) in the polymeric carrier of formula (VI) xalso contain mixed repeating amino acid components [(AA) x ] z where the amino acid component (AA) or (AA) x is further defined by the integer z. In this context, z may be selected from the range of about 1 to 30, preferably from the range of about 1 to 15, more preferably from the range of 1 to 10 or 1 to 5, and even more preferably from a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or from the range formed by any two of the foregoing values.

[0146] According to certain particularly preferred alternatives, the amino acid components (AA) or (AA) x is preferably S-(AA) x -S or [S-(AA) x -S] and component P 2 , in particular the repeating component [SP 2 -S] n Ingredients SP 2 In relation to the overall polymeric carrier according to formula (VI), this can be represented for example by the following formula (VIa):

[0147] LP 1 -S-{[SP 2 -S] a [S-(AA) x -S] b}-SP 3 -L formula (VIa)

[0148] where x, S, L, AA, P 1 , P 2 and P 3 is preferably as defined herein. In the above formula (VIa), the single component [SP 2 -S] and [S-(AA) x -S] is a sub-expression {[SP 2 -S] a [S-(AA) x -S] b} can occur in any order. 2 -S] a [S-(AA) x -S] b} in the single component [SP 2 -S] and [S-(AA) x The number of -S] is determined by the integers a and b, where a+b=n, where n is an integer and is defined above for formula (VI).

[0149] a is an integer and is typically selected, independently of the integer b, from a range of about 1 to 50, preferably from a range of about 1, 2, or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or from a range of about 1, 2, 3, 4, or 5 to 20, or from a range of about 1, 2, 3, 4, or 5 to 15, or from a range of about 1, 2, 3, 4, or 5 to 10, including, for example, a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or from a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or from a range of about 6 to 11, or 7 to 10. Most preferably, a is in a range of about 1, 2, 3, 4, or 5 to 10, more preferably a range of about 1, 2, 3, or 4 to 9, or a range of about 1, 2, 3, or 4 to 8, or a range of about 1, 2, or 3 to 7.

[0150] b is an integer, typically selected independently from the integer a from a range of about 0 to 50 or 1 to 50, preferably from a range of about 1, 2, or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or from a range of about 1, 2, 3, 4, or 5 to 20, or from a range of about 1, 2, 3, 4, or 5 to 15, or from a range of about 1, 2, 3, 4, or 5 to 10, including, for example, a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or from a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or from a range of about 6 to 11 or 7 to 10. Most preferably, b is in a range of about 1, 2, 3, 4, or 5 to 10, more preferably from a range of about 1, 2, 3, or 4 to 9, or from a range of about 1, 2, 3, or 4 to 8, or from a range of about 1, 2, or 3 to 7.

[0151] According to a preferred aspect, the mRNA of the vaccine of the invention, encoding at least one antigen as defined above, may be formulated with a cationic or polycationic compound and / or a polymeric carrier, preferably as defined herein.

[0152] According to a further preferred embodiment, the mRNA of the vaccine of the invention encoding at least one antigen as defined above may be formulated with an (adjuvant) component. According to a particularly preferred embodiment, the mRNA of the vaccine of the invention encoding at least one antigen as defined above is formulated to comprise a) an (adjuvant) component comprising or consisting of at least one immunostimulatory nucleic acid complexed with a cationic or polycationic compound and / or a polymeric carrier as defined herein, and b) at least one free mRNA, preferably encoding an antigen as defined herein for the vaccine of the invention.

[0153] In this regard, the cationic or polycationic compounds and / or polymeric carriers used to complex at least one immunostimulatory nucleic acid in the adjuvant component may be selected from the cationic or polycationic compounds and / or polymeric carriers described above.

[0154] Furthermore, the immunostimulatory nucleic acid as defined above for the adjuvant component may preferably be selected from the mRNA as defined herein for the vaccine of the invention, encoding at least one antigen. Alternatively, such immunostimulatory nucleic acid may be selected from the immunostimulatory nucleic acids as defined herein, preferably from the immunostimulatory RNA (isRNA) as defined herein.

[0155] In this context, the immunostimulatory nucleic acid used herein is preferably selected from immunostimulatory nucleic acids known to bind to TLR receptors. Such immunostimulatory nucleic acids are preferably in the form of (immunostimulatory) CpG nucleic acids that induce an innate immune response, particularly CpG-RNA or CpG-DNA. The CpG-RNA or CpG-DNA used in the present invention can be single-stranded CpG-DNA (ssCpG-DNA), double-stranded CpG-DNA (dsDNA), single-stranded CpG-RNA (ssCpG-RNA), or double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid used in the present invention is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ssCpG-RNA). Also preferably, such CpG nucleic acids have the lengths described above. Preferably, the CpG motifs are unmethylated.

[0156] Furthermore, the immunostimulatory nucleic acid used herein is preferably selected from immunostimulatory RNA (isRNA), which preferably induces an innate immune response. Preferably, the immunostimulatory RNA is single-stranded, double-stranded, or partially double-stranded RNA, more preferably single-stranded RNA and / or circular or linear RNA, more preferably linear RNA. More preferably, the immunostimulatory RNA may be a (linear) single-stranded RNA. Even more preferably, the immunostimulatory RNA may be a (long) (linear) (single-stranded) non-coding RNA. In this regard, it is particularly preferred that the isRNA, when transcribed in vivo, carries a triphosphate at its 5' end. The immunostimulatory RNA may be present as a short RNA oligonucleotide as defined herein. The immunostimulatory RNA used herein may further be selected from any class of RNA molecules found in nature or synthetically produced, which can induce an innate immune response and support an adaptive immune response induced by an antigen. In this regard, the immune response can be generated in various ways. A substantial factor for an appropriate (adaptive) immune response is the stimulation of different T cell subpopulations. T lymphocytes are typically divided into two subpopulations, T helper 1 (Th1) cells and T helper 2 (Th2) cells, which allow the immune system to destroy intracellular (Th1) and extracellular (Th2) pathogens (e.g., antigens). The two Th cell populations differ in the patterns of effector proteins (cytokines) they produce. Thus, Th cells support the cellular immune response by activating macrophages and cytotoxic T cells. Th2 cells, on the other hand, promote the humoral immune response by stimulating the transformation of B cells into plasma cells and the formation of antibodies (e.g., against antigens). The Th1 / Th2 ratio is therefore crucial for the induction and maintenance of an adaptive immune response. In the context of the present invention, the Th1 / Th2 ratio of the (adaptive) immune response is preferably shifted toward a cellular response (Th1 response), thereby inducing a cellular immune response. By way of example, the innate immune system, which can support an adaptive immune response, can be activated by ligands of Toll-like receptors (TLRs).TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMPs) and play an important role in innate immunity in mammals. Currently, at least 13 family members, designated TLR1 through TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13), have been identified. Furthermore, numerous specific TLR ligands have been identified. For example, unmethylated bacterial DNA and its synthetic analog (CpG DNA) have been found to be ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl. Acad. Sci. USA 98, 9237-42). Furthermore, ligands for specific TLRs include specific nucleic acid molecules, and specific types of RNAs have been reported to be immunostimulatory in a sequence-independent or sequence-dependent manner, where these various immunostimulatory RNAs can stimulate intracellular receptors such as, for example, TLR3, TLR7, or TLR8, or RIG-I and MDA-5.

[0157] Preferably, immunostimulatory nucleic acids, preferably immunostimulatory RNA (isRNA) as used herein, may comprise any RNA known to be immunostimulatory, including, but not limited to, a ligand for a TLR, preferably selected from human family members TLR1-TLR10 or murine family members TLR1-TLR10, more preferably selected from (human) family members TLR1-TLR10, even more preferably TLR7 and TLR8, an RNA sequence that represents or encodes a ligand for an intracellular receptor for RNA (such as RIG-I or MDA-5), or both (see, e.g., Meylan, E., Tschopp, J. (2006) Toll-like receptors and RNA helicases: two parallel ways of triggering antiviral responses Mol. Cell 22, 561-569), or any other immunostimulatory RNA sequence. Furthermore, immunostimulatory RNA molecules (classes) used as additional compounds in the vaccines of the present invention may also comprise any other RNA capable of eliciting an immune response. Such immunostimulatory RNAs may include, but are not limited to, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such immunostimulatory RNAs may comprise a length of 1000 to 5000, 500 to 5000, 5 to 5000, 5 to 1000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30 amino acids.

[0158] In particularly preferred embodiments, the immunostimulatory nucleic acid sequence, particularly the isRNA used herein, may consist of or comprise amino acids of formula (I) or formula (II):

[0159] G l X m G n Formula (I) wherein G is guanosine, uracil, or a guanosine analog or a uracil analog; X is guanosine, uracil, adenosine, thymidine, cytosine, or an analog of the above nucleotides; and 1 is an integer from 1 to 40; where: When l=1, G is guanosine or an analog thereof; when l>1, at least 50% of the nucleotides are guanosine or an analog thereof; where: When m=3, X is uracil or an analogue thereof; When m>3, at least three consecutive uracils or uracil analogs occur; n is an integer from 1 to 40, where: When n=1, G is guanosine or an analog thereof; If n>1, then at least 50% of the nucleotides are guanosine or an analog thereof.

[0160] C l X m C n Formula (II) where: C is cytosine, uracil, or an analog of cytosine or an analog of uracil; X is guanosine, uracil, adenosine, thymidine, cytosine, or an analog of the above nucleotides; 1 is an integer from 1 to 40; where: When l=1, C is cytosine or an analog thereof; when l>1, at least 50% of the nucleotides are cytosine or an analogue thereof; m is an integer and is at least 3; where: When m=3, X is uracil or an analogue thereof; When m>3, at least three consecutive uracils or uracil analogs occur; n is an integer from 1 to 40, where: When n=1, C is cytosine or an analog thereof; If n>1, then at least 50% of the nucleotides are cytosine or an analogue thereof.

[0161] Nucleic acids of Formula (I) or Formula (II), particularly isRNA, that can be used as immunostimulatory nucleic acid sequences may be relatively short nucleic acid molecules with typical lengths of about 5 to 100 (although in certain embodiments, they may be longer than 100 nucleotides, e.g., 200 nucleotides), 5 to 90 nucleotides, or 5 to 80 nucleotides, preferably about 5 to 70 nucleotides, more preferably about 8 to 60 nucleotides, more preferably about 15 to 60 nucleotides, more preferably 20 to 60 nucleotides, and most preferably 30 to 60 nucleotides. When a nucleic acid of Formula (I) or Formula (II) has a maximum length of, for example, 100 nucleotides, m will typically be 98 or less. The number of nucleotides, G, in a nucleic acid of Formula (I) is defined by l or n, which are independently integers of 1 to 40, respectively, where when l or n is 1, G is guanosine or an analog thereof, and when l or n is greater than 1, at least 50% of the nucleotides are guanosine or an analog thereof. For example, and without implying any limitation, when l or n is 4, G l or G n can be, for example, GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG, or GGGG, etc., and when l or n is 5, G l or G n can be, for example, GGGUU, GGUGU, GUGGU, UGGGU, UGGUG, UUGGG, UUGGG, GUGUG, GGGGU, GGUGG, GGUGG, GGGGG, UGGGG, or GGGGG, etc. X in the nucleic acid of formula (I) according to the present invention mThe nucleotide adjacent to is preferably not uracil. Similarly, the number of nucleotides C in the nucleic acid of formula (II) according to the present invention is defined by l or n. l and n are each independently an integer of 1 to 40, where when l or n is 1, C is cytosine or an analog thereof, and when l or n is greater than 1, at least 50% of the nucleotides are cytosine or an analog thereof. For example, without any limitation, when l or n is 4, C l or C n can be, for example, CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC, UCCC, or CCCC, and when l or n is 5, C l or C n can be, for example, CCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UUCCC, UUCCC, CCCUC, CCCCU, CCUCC, CCUCC, UCCCC, or CCCCC, etc. X in the nucleic acid of formula (II) according to the present invention m The nucleotides adjacent to the flanking sequences G1 and G2 are preferably not uracil. Preferably, for formula (I), when l or n is greater than 1, at least 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosine or an analogue thereof as defined above. n In at least one of the above, up to 100% of the remaining nucleotides (when guanosine constitutes less than 100% of the nucleotides) are uracil or an analog thereof as defined herein. Also preferably, l and n are each independently an integer of 2 to 30, more preferably an integer of 2 to 20, and even more preferably an integer of 2 to 15. The lower limit of l or n can be varied as necessary, and is at least 1, preferably at least 2, and more preferably at least 3, 4, 5, 6, 7, 8, 9, or 10. This definition also applies to formula (II).

[0162] In a particularly preferred embodiment, the nucleic acid according to either formula (I) or formula (II) above, which may be used as an immunostimulatory nucleic acid sequence, in particular an isRNA, is

[0163] GGUUUUUUUUUUUUUUUGGG (SEQ ID NO: 289), GGGGGUUUUUUUUUUGGGGG (SEQ ID NO: 290), GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG (SEQ ID NO: 291), GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU (SEQ ID NO: 292), GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU (SEQ ID NO: 293), GGGGGGGGGUUUGGGGGGGG (SEQ ID NO: 294), GGGGGGGGUUUUGGGGGGGG (SEQ ID NO: 295), GGGGGGGUUUUUUGGGGGGG (SEQ ID NO: 296), GGGGGGGUUUUUUUGGGGGG (SEQ ID NO: 297), GGGGGGUUUUUUUUGGGGGG (SEQ ID NO: 298), GGGGGGUUUUUUUUUGGGGG (SEQ ID NO: 299), GGGGGGUUUUUUUUUUUGGGG (SEQ ID NO: 300), GGGGGUUUUUUUUUUUUGGGG (SEQ ID NO: 301), GGGGGUUUUUUUUUUUUGGG (SEQ ID NO: 302), GGGGUUUUUUUUUUUUUGGG (SEQ ID NO: 303), GGGGUUUUUUUUUUUUUUGG (SEQ ID NO: 304), GGUUUUUUUUUUUUUUUUGG (SEQ ID NO: 305), GUUUUUUUUUUUUUUUUUUG (SEQ ID NO: 306), GGGGGGGGGGUUUGGGGGGGGG (SEQ ID NO: 307), GGGGGGGGGUUUUGGGGGGGGG (SEQ ID NO: 308), GGGGGGGGUUUUUUGGGGGGGG (SEQ ID NO: 309), GGGGGGGGUUUUUUUGGGGGGG (SEQ ID NO: 310), GGGGGGGUUUUUUUUGGGGGGG (SEQ ID NO: 311), GGGGGGGUUUUUUUUUGGGGGG (SEQ ID NO: 312), GGGGGGGUUUUUUUUUUUGGGGG (SEQ ID NO: 313), GGGGGGUUUUUUUUUUUUGGGGG (SEQ ID NO: 314), GGGGGGUUUUUUUUUUUUUGGGG (SEQ ID NO: 315), GGGGGUUUUUUUUUUUUUUGGGG (SEQ ID NO: 316), GGGGGUUUUUUUUUUUUUUUGGG (SEQ ID NO: 317), GGGUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 318), GGUUUUUUUUUUUUUUUUUUGG (SEQ ID NO: 319), GGGGGGGGGGGUUUGGGGGGGGGG (SEQ ID NO: 320), GGGGGGGGGGUUUUGGGGGGGGGG (SEQ ID NO: 321), GGGGGGGGGUUUUUUGGGGGGGGG (SEQ ID NO: 322), GGGGGGGGGUUUUUUUGGGGGGGG (SEQ ID NO: 323), GGGGGGGGUUUUUUUUGGGGGGGG (SEQ ID NO: 324), GGGGGGGGUUUUUUUUUGGGGGGG (SEQ ID NO: 325), GGGGGGGGUUUUUUUUUUUGGGGGG (SEQ ID NO: 326), GGGGGGGUUUUUUUUUUUUGGGGGG (SEQ ID NO: 327), GGGGGGGUUUUUUUUUUUUUGGGGG (SEQ ID NO: 328), GGGGGGUUUUUUUUUUUUUUGGGGG (SEQ ID NO: 329), GGGGGGUUUUUUUUUUUUUUUGGGG (SEQ ID NO: 330), GGGGUUUUUUUUUUUUUUUUUGGGG (SEQ ID NO: 331), GGGUUUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 332), GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG (SEQ ID NO: 333), GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG (SEQ ID NO: 334), GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 335), GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 336), GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG (SEQ ID NO: 337), GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG (SEQ ID NO: 338), GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG (SEQ ID NO: 339), GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG (SEQ ID NO: 340), GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG (SEQ ID NO: 341), GGUUUGG (SEQ ID NO: 342), GGUUUUGG (SEQ ID NO: 343), GGUUUUUGG (SEQ ID NO: 344), GGUUUUUUGG (SEQ ID NO: 345), GGUUUUUUUGG (SEQ ID NO: 346), GGUUUUUUUUGG (SEQ ID NO: 347), GGUUUUUUUUUGG (SEQ ID NO: 348), GGUUUUUUUUUUGG (SEQ ID NO: 349), GGUUUUUUUUUUUGG (SEQ ID NO: 350), GGUUUUUUUUUUUUGG (SEQ ID NO: 351), GGUUUUUUUUUUUUUGG (SEQ ID NO: 352), GGUUUUUUUUUUUUUUGG (SEQ ID NO: 353), GGUUUUUUUUUUUUUUUGG (SEQ ID NO: 354), GGGUUUGGG (SEQ ID NO: 355), GGGUUUUGGG (SEQ ID NO: 356), GGGUUUUUGGG (SEQ ID NO: 357), GGGUUUUUUGGG (SEQ ID NO: 358), GGGUUUUUUUGGG (SEQ ID NO: 359), GGGUUUUUUUUGGG (SEQ ID NO: 360), GGGUUUUUUUUUGGG (SEQ ID NO: 361), GGGUUUUUUUUUUGGG (SEQ ID NO: 362), GGGUUUUUUUUUUUGGG (SEQ ID NO: 363), GGGUUUUUUUUUUUUGGG (SEQ ID NO: 364), GGGUUUUUUUUUUUUUGGG (SEQ ID NO: 365), GGGUUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 366), GGGUUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUUUGGG (SEQ ID NO: 367), GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGG (SEQ ID NO: 368), GGUUUUUUUUUUUUUUUGGG (short, GU-rich, SEQ ID NO: 369), or

[0164] CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUUCCC (SEQ ID NO: 370), CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCC (SEQ ID NO: 371), CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCC (SEQ ID NO: 372), The sequence may be selected from a sequence consisting of or including any of the sequences:

[0165] Alternatively, it may be selected from sequences having at least 60%, 70%, 80%, 90% or even 95% sequence identity with any of these sequences.

[0166] In further particularly preferred embodiments, the immunostimulatory nucleic acid sequence, particularly the isRNA used herein, may consist of or comprise a nucleic acid of formula (III) or formula (IV).

[0167] (N u G l X m G n N v ) a Formula (III)

[0168] where: G is guanosine (guanine), uridine (uracil), or an analog of guanosine (guanine) or uridine (uracil), preferably guanosine (guanine) or an analog thereof; X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or a nucleotide (nucleoside) analog thereof, preferably uridine (uracil) or an analog thereof; N is a nucleic acid sequence having a length of about 4 to 50, preferably about 4 to 40, more preferably about 4 to 30 or 4 to 20 nucleic acids, and each N is independently selected from guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or a nucleotide (nucleoside) analog thereof; a is an integer of 1 to 20, preferably 1 to 15, and most preferably 1 to 10; l is an integer from 1 to 40, where: When l=1, G is guanosine (guanine) or an analog thereof; If l>1, then at least 50% of these nucleotides (nucleosides) are guanosine (guanine) or an analog thereof; m is an integer and is at least 3; where: When m=3, X is uridine (uracil) or an analog thereof; When m>3, there are at least 3 consecutive uridine (uracil) or uridine (uracil) analogs; n is an integer from 1 to 40, where: When n=1, G is guanosine (guanine) or an analog thereof; when n>1, at least 50% of these nucleotides (nucleosides) are guanosine (guanine) or an analog thereof; u and v are each independently an integer of 0 to 50; Preferably, where u=0 and v≧1, or v=0 and u≧1, Here, the nucleic acid molecule of formula (III) has a length of at least 50 nucleotides, preferably at least 100 nucleotides, more preferably at least 150 nucleotides, even more preferably 200 nucleotides, and most preferably at least 250 nucleotides.

[0169] (N u C l X m C n N v ) a Formula (IV)

[0170] where: C is cytidine (cytosine), uridine (uracil), or an analog of cytidine (cytosine) or an analog of uridine (uracil), preferably cytidine (cytosine) or an analog thereof; X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or an analog of the above-mentioned nucleotides (nucleosides), preferably uridine (uracil) or an analog thereof; N's are each, independently of one another, a nucleic acid sequence having a length of about 4 to 50, preferably about 4 to 40, more preferably about 4 to 30 or 4 to 20 nucleic acids, and each N is independently selected from guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or a nucleotide (nucleoside) analog thereof; a is an integer of 1 to 20, preferably 1 to 15, and most preferably 1 to 10; l is an integer from 1 to 40, where: When l=1, C is cytidine (cytosine) or an analog thereof; If l>1, then at least 50% of these nucleotides (nucleosides) are cytidine (cytosine) or an analog thereof; m is an integer and is at least 3; where: When m=3, X is uridine (uracil) or an analog thereof; When m>3, there are at least 3 consecutive uridine (uracil) or uridine (uracil) analogs; n is an integer from 1 to 40, where: When n=1, C is cytidine (cytosine) or an analog thereof; if n>1, at least 50% of these nucleotides (nucleosides) are cytidine (cytosine) or an analog thereof; u and v are each independently an integer of 0 to 50; Preferably, where u=0 and v≧1, or v=0 and u≧1, Here, the nucleic acid molecule of formula (IV) according to the present invention has a length of at least 50 nucleotides, preferably at least 100 nucleotides, more preferably at least 150 nucleotides, even more preferably 200 nucleotides, and most preferably at least 250 nucleotides.

[0171] In any of formulas (I) and (II), the above definitions, for example, element N (i.e., N u and N v ) and X(X m ), in particular the definitions for the integers a, l, m, n, u and v as well as for the core structure defined above apply correspondingly to the elements of formula (III) and formula (IV). u and N v is defined as N in formula (IV). u and N v is the same as the above definition for

[0172] According to a very particularly preferred embodiment, the nucleic acid molecule of the invention according to formula (IV) which may be used as an immunostimulatory nucleic acid sequence, in particular as an isRNA, may for example be selected from any of the following sequences:

[0173] UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG (SEQ ID NO: 373)

[0174] UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG AUCGCUUCGA GAACCUGGAUCCAAAAAAAAAAAAAAACCCACGCAAGGAUCUUCAUGUGC (SEQ ID NO: 374)

[0175] GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUC (SEQ ID NO: 375)

[0176] GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAG (SEQ ID NO: 376)

[0177]

[0178] GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUC (SEQ ID NO: 378)

[0179] (R 722 SEQ ID NO: 379)

[0180]

[0181] According to another very particularly preferred embodiment, the nucleic acid molecule according to formula (V) may, for example, be selected from any of the following sequences:

[0182] UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACG (SEQ ID NO: 381), or

[0183] UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUUCCCUGCGUUCCUA GAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAAAAAAAAAGGGACGCAAGGAUCUUCAUGUGC (SEQ ID NO: 382)

[0184] Alternatively, it may be selected from sequences having at least 60%, 70%, 80%, 90%, or even 95% sequence identity with any of these sequences.

[0185] Finally, the so-called "(adjuvant) component" that can be used together with the mRNA in the vaccine of the present invention is preferably prepared in a first step by complexing at least one (m)RNA of the (adjuvant) component with a cationic or polycationic compound, preferably as defined herein, and / or a polymeric carrier in a specific ratio to form a stable complex. In this regard, it is highly preferred that, after complexing the (m)RNA, no or negligibly small amounts of free cationic or polycationic compounds or polymeric carriers remain in the (adjuvant) component. Therefore, the ratio of (m)RNA to cationic or polycationic compounds and / or polymeric carriers in the (adjuvant) component is typically selected within a range in which the (m)RNA is completely complexed and no or negligibly small amounts of free cationic or polycationic compounds or polymeric carriers remain in the composition. Preferably, the ratio of the (adjuvant) component, i.e., the ratio of (m)RNA to at least one of a cationic or polycationic compound, preferably as defined herein, and a polymeric carrier, is selected from the range of about 6:1 (w / w) to about 0.25:1 (w / w), more preferably about 5:1 (w / w) to about 0.5:1 (w / w), even more preferably about 4:1 (w / w) to about 1:1 (w / w), or about 3:1 (w / w) to about 1:1 (w / w), and most preferably about 3:1 (w / w) to about 2:1 (w / w). Alternatively, the ratio of (m)RNA to at least one of a cationic or polycationic compound, preferably as defined herein, and a polymeric carrier in the (adjuvant) component can also be calculated based on the nitrogen / phosphate ratio (N / P ratio) of the entire complex of the (adjuvant) component.In the context of the present invention, preferably, the cationic or polycationic compound in the complex is cationic or polycationic, or, when the cationic or polycationic protein or peptide and / or polymeric carrier are as defined above, the N / P ratio, with respect to the ratio of the cationic or polycationic compound, preferably as defined herein, and / or polymeric carrier in the complex to the (m)RNA ((m)RNA:cationic or polycationic compound, preferably as defined herein, and / or polymeric carrier in the complex), is preferably in the range of about 0.1 to 10, preferably in the range of about 0.3-4, most preferably in the range of about 0.5 to 2 or 0.7 to 2, and most preferably in the range of about 0.7 to 1.5. Such ratios, in particular the weight ratio and / or N / P ratio, may also apply to the ratio of at least one mRNA encoding at least one antigen as defined herein to the cationic or polycationic polymer or polymeric carrier as defined herein used to complex the at least one mRNA.

[0186] According to a further preferred embodiment, the mRNA of the vaccine of the present invention encoding at least one antigen as defined above may be formulated with an (adjuvant) component as defined above, wherein the vaccine of the present invention may comprise: a) at least one (m)RNA complexed with a cationic or polycationic compound and / or a polymeric carrier, preferably as defined herein; and b) at least one free mRNA encoding the antigen, preferably as defined herein. This formulation is preferably as defined above. Furthermore, the entire formulation of a) and b) may additionally be packaged with a carrier molecule to allow combined packaging of the (adjuvant) component and the antigen. Such a carrier molecule may be selected from any polymer suitable for packaging and delivery of the entire formulation of a) and b) to, preferably, cells, tissues, etc. of a patient as defined herein, for example, from the cationic or polycationic polymers as defined herein, or any further polymer suitable for this purpose, such as the polymeric carriers described above.

[0187] The ratio of all components of the overall vaccine composition of the present invention, as defined above, preferably the cationic or polycationic compound, at least one mRNA encoding at least one antigen, and / or the adjuvant comprising or consisting of at least one immunostimulatory nucleic acid sequence complexed with a carrier molecule and formulated into the vaccine of the present invention, can be calculated based on the nitrogen / phosphate ratio (N / P ratio) of all these components. In the context of the present invention, the N / P ratio, with respect to the ratio of nucleic acid to cationic or polycationic peptide contained in the vaccine of the present invention (nucleic acid:cationic or polycationic peptide contained in the vaccine of the present invention), is preferably in the range of about 0.01-4, 0.01-2, 0.1-2, or 0.1-1.5, most preferably in the range of about 0.1-1. Such an N / P ratio is preferably designed to provide good in vivo transfection properties and transport to and across cell membranes. Preferably, for this purpose, the cationic or polycationic compound and / or polymeric carrier used herein are based on peptide sequences.

[0188] In a further preferred embodiment of the present invention, the vaccine of the present invention may comprise a pharmaceutically acceptable carrier and / or excipient. In the context of the present invention, a pharmaceutically acceptable carrier typically comprises a liquid or non-liquid basis for a composition comprising the components of the vaccine of the present invention. When the composition is provided in liquid form, the carrier is typically pyrogen-free water, isotonic saline, or a buffered (aqueous) solution, such as a solution buffered with phosphate or citrate. The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium. For example, the buffer may have a higher, equal, or lower salt content relative to the reference medium. Preferably, such concentrations of the aforementioned salts are used, which do not lead to cell damage due to osmotic or other concentration effects. The reference medium may be a liquid occurring in an "in vivo" method, such as blood, lymph, cytoplasmic fluid, or other body fluid, or a liquid that can be used as a reference medium in an "in vitro" method, such as a common buffer or liquid. Such common buffers or liquids are known to those skilled in the art. Lactated Ringer's solution is particularly suitable as a liquid base.

[0189] However, one or more compatible solid or liquid fillers or diluents, or encapsulating compounds suitable for administration to a patient to be treated, may likewise be used in the vaccines of the present invention. As used herein, the term "compatible" means that these components of the vaccines of the present invention are capable of being mixed with the components of the vaccines of the present invention such that there is substantially no interaction that would substantially reduce the pharmaceutical efficacy of the vaccines of the present invention under typical conditions of use.

[0190] According to a particular embodiment, the vaccine of the present invention may comprise an adjuvant. In this context, an adjuvant may be understood as any compound suitable for initiating or enhancing an immune response of the innate immune system, i.e., a non-specific immune response. In other words, when administered, the vaccine induces an innate immune response, preferably due to the adjuvant optionally contained therein. Preferably, such adjuvants may be selected from adjuvants known to those skilled in the art and suitable in the present case, i.e., adjuvants that support the induction of an innate immune response in mammals, such as the adjuvant proteins defined above or the adjuvants defined below.

[0191] According to one embodiment, such adjuvants may be selected from the (adjuvant) components defined above.

[0192] According to a further aspect, such adjuvants may be selected from those known to those skilled in the art and suitable in the present invention, i.e., adjuvants that support the induction of an innate immune response in mammals and / or adjuvants suitable for storage and delivery of the components of the vaccine of the present invention. Suitable adjuvants for storage and delivery are cationic or polycationic compounds as defined above. Similarly, adjuvants include, but are not limited to, cationic or polycationic compounds as defined above, chitosan, TDM, MDP, muramyl dipeptide, Pluronic®, alum solution, aluminum hydroxide, ADJUMER® (polyphosphazene), aluminum phosphate gel, algae-derived glucan, algammulin, aluminum hydroxide gel (alum), high protein-adsorbing aluminum hydroxide gel, low viscosity aluminum hydroxide gel, AF or SPT (an emulsion of squalene (5%), Tween 80 (0.2%), Pluronic® L121 (1.25%), and phosphate buffered saline at pH 7.4), AVRIDINE® (propanediamine), BAY R1005® ((N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyldodecanoylamide hydroacetate), CALCITRIOL® (1α,25-dihydroxyvitamin D3), calcium phosphate gel, CAP® (calcium phosphate nanoparticles), cholera holotoxin, cholera toxin A1-protein AD fragment fusion protein, B subunit of cholera toxin, CRL 1005 (block copolymer P1205), cytokine-containing liposomes, DDA (dimethyldioctadecylammonium bromide), DHEA (dehydroepiandrosterone), DMPC (dimyristoylphosphatidylcholine), DMPG (dimyristoylphosphatidylglycerol), DOC / alum complex (deoxycholic acid sodium salt), Freund's complete adjuvant, Freund's incomplete adjuvant, gamma inulin, Gerbu adjuvant (a mixture of the following: i) N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc L-proline salt complex (ZnPro-8), GM-CSF, GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine), imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine), ImmTher® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate), DRVs (immunoliposomes prepared from dehydrated-rehydrated vesicles), interferon-γ, interleukin-1β, interleukin-2, interleukin-7, interleukin-12, ISCOMS®, ISCOPREP 7.0.3.®, liposomes, LOXORIBINE® (7-allyl-8-oxoguanosine), LT5 oral adjuvant (E. coli labile endotoxin protoxin), microspheres and microparticles of any composition, MF59®, (squalene water emulsion), MONTANIDE ISA 51® (purified incomplete Freund's adjuvant), MONTANIDE ISA 720® (metabolizable oil adjuvant), MPL® (3-Q-desacyl-4'-monophosphoryl lipid A), MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-Dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)-ethylamide, monosodium salt), MURAMETIDE® (Nac-Mur-L-Ala-D-Gln-OCH3), MURAPALMITINE® and DMURAPALMITINE® (Nac-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl), NAGO (neuraminidase-galactose oxidase), nanospheres or nanoparticles of any composition, NISVs (nonionic surfactant vesicles), PLEURAN® (β-glucan), PLGA, PGA and PLA (homopolymers and copolymers of lactic and glycolic acid, microspheres / nanospheres), PLURONIC® L121, PMMA (polymethyl methacrylate), PODDS® (proteinoid microspheres), polyethylene carbamate derivatives, poly rA:poly rU (polyadenylic acid-polyuridylic acid complex), polysorbate 80 (Tween 80), Protein Cochleates (Avanti Polar Lipids, Inc., Alabaster, Alabama), STIMULON® (QS-21), Quil-A (Quil-A saponin), S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol), SAF-1® (Syntex adjuvant formulation), Sendai proteoliposomes and Sendai-containing lipid matrices, Span-85 (sorbitan trioleate), Specol (Marcol 52, Span 85 and Tween 85 emulsion), squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane), stearyl tyrosine (octadecyl tyrosine hydrochloride), Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide), threonyl-MDP (Termurtide® or [thr1]-MDP, N-acetylmuramyl-L-threonyl-D-isoglutamine), Ty particles (Ty-VLPs or virus-like particles), Walter-Reed tyrosine containing Pam3Cys Liposomes (liposomes containing lipid A adsorbed to aluminum hydroxide), especially aluminum salts such as Adju-phos, Alhydrogel, and Rehydragel; emulsions including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, and Vaxfectin; copolymers including Optivax (CRL1005), L121, and Poloxamer 4010; liposomes containing Stealth; cochlear structures including BIORAL, QS21, and Quil A, plant-derived adjuvants including Iscomatrix and ISCOM, adjuvants suitable for costimulation including Tomatine, biopolymers including PLG, PMM and Inulin, Romurtide, DETOX, MPL, CWS, mannose, CpG nucleic acid sequences, CpG7909, ligands for human TLR1-10, ligands for mouse TLR1-13, ISS-1018, 35 IC31, imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchors, LNFPIII / Lewis X, antimicrobial peptides, UC-1V150, RSV fusion proteins, and peptides derived from microorganisms including cdiGMP, and adjuvants suitable as antagonists including the CGRP neuropeptide.

[0193] Particularly preferably, the adjuvant may be selected from adjuvants that support the induction of a Th1-type immune response or maturation of naive T cells, such as GM-CSF, IL-12, IFNγ, immunostimulatory RNA sequences as defined herein, and CpG DNA.

[0194] The vaccine of the present invention may further comprise an additional immunotherapeutic agent selected from immunoglobulins, preferably IgG, monoclonal or polyclonal antibodies, polyclonal sera or serum, etc. Preferably, such additional immunotherapeutic agent may be provided as a peptide / protein or may be encoded by a nucleic acid, preferably DNA or RNA, more preferably mRNA. Such an immunostimulant allows the provision of an inactive vaccine in addition to the active vaccination caused by the mRNA-encoded antigen of the composition or vaccine composition of the present invention.

[0195] The vaccine of the present invention may further contain one or more auxiliary substances to enhance its immunogenicity or immunostimulatory capacity, if necessary. The interaction between the vaccine of the present invention and the auxiliary substances, which may optionally be included in the vaccine or formulated with inhibitors, is preferably achieved thereby. Depending on the type of auxiliary substance, various mechanisms can be considered in this regard. For example, compounds that allow dendritic cell (DC) maturation, such as lipopolysaccharide, TNF-α, or CD40 ligand, form a first class of suitable auxiliary substances. Generally, any agent that affects the immune system in the form of "danger signals" (e.g., LPS, GP96) or cytokines, such as GM-CFS, can be used as an auxiliary substance, allowing the immune response to be enhanced and influenced in a targeted manner. Particularly preferred supplemental substances are cytokines such as monokines, lymphokines, interleukins or chemokines that further enhance the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFNα, IFNβ, IFNγ, GM-CSF, G-CSF, M-CSF, LT-β or TNF-α, and growth factors such as hGH.

[0196] The vaccines of the invention may also comprise any further compounds known to be immunostimulatory due to their binding affinity (as ligands) to the human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 or to the murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, ligands of NOD-like receptors, or ligands of RIG-I-like receptors.

[0197] In this regard, the vaccine of the invention may also further comprise an immunostimulatory nucleic acid, preferably an immunostimulatory RNA (isRNA) as defined above.

[0198] The vaccine of the present invention as defined in accordance with the first embodiment of the present invention may further comprise further additives or additional compounds. Further additives that may be included in the vaccine of the present invention are emulsifiers, such as Tween®, humectants, such as sodium lauryl sulfate, colorants, pharmaceutical carriers, taste enhancers, pharmaceutical carriers, tableting agents, stabilizers, antioxidants, and preservatives.

[0199] One further additive that may be included in the vaccine of the present invention may be an antibacterial agent. In this regard, any antibacterial agent known to those skilled in the art may be used in combination with the components of the vaccine of the present invention as defined herein. Non-limiting examples of antibacterial agents include amikacin, amoxicillin, amoxicillin-clavulanic acid, amphotericin B, ampicillin, ampicillin-sulbactam, apramycin, azithromycin, aztreonam, bacitracin, benzylpenicillin, caspofungin, cefaclor, cefadroxil, cephalexin, cephalothin, cefazolin, cefdinir, cefepime, cefixime, cefmenoxime, cefoperazone, cefoperazone-sulbactam, cefotaxime, cefoxitin, cefotaxime ... Cefbrozil, Cefquinome, Ceftazidime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxone, Cefuroxime, Chloramphenicol, Florfenicol, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazole (Trimethoprim / Sulfamethoxazole) oxazole), dalbavancin, dalfopristin / quinopristin, daptomycin, dibekacin, dicloxacillin, doripenem, doxycycline, enrofloxacin, ertapenem, erythromycin, flucloxacillin, fluconazole, flucytosine, fosfomycin, fusidic acid, garenoxacin, gatifloxacin, gemifloxacin, gentamicin, imipenem, itraconazole, kanamycin, ketoconazole, levofloxacin, lincomycin Isin, linezolid, loracarbef, mecillinam (amdinocillin), meropenem, metronidazole, mediocillin, mezlocillin-sulbactam, minocycline, moxifloxacin, mupirocin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin, pefloxacin, penicillin V, piperacillin, piperacillin-sulbactam, piperacillin-tazobactam, rifampicin, roxithromycin,These include sparfloxacin, spectinomycin, spiramycin, streptomycin, sulbactam, sulfamethoxazole, teicoplanin, telavancin, telithromycin, temocillin, tetracycline, ticarcillin, ticarcillin-clavulanate, tigecycline, tobramycin, trimethoprim, trovafloxacin, tylosin, vancomycin, virginiamycin, and voriconazole.

[0200] Another additive that may be included in the vaccines of the present invention may be an antiviral agent, preferably, but not limited to, nucleoside analogues (e.g., zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, peramivir, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferon and other interferons, AZT, t-705, zanamivir (Relenza®), and oseltamivir (Tamiflu®). Other antiviral agents include influenza virus vaccines, such as Fluarix® (GlaxoSmithKline), FluMist® (MedImmune Vaccines), Fluvirin® (Chiron Corporation), Flulaval® (GlaxoSmithKline), Afluria® (CSL Biotherapies Inc.), Agriflu® (Novartis), or Fluzone® (Aventis Pasteur).

[0201] Vaccines of the present invention typically contain a "safe and effective amount" of a component of a vaccine of the present invention as defined herein. As used herein, "safe and effective amount" preferably means an amount of a component, preferably at least one mRNA, sufficient to significantly induce a positive modification of a disease or disorder as defined herein. However, at the same time, a "safe and effective amount" is small enough to avoid serious side effects and to allow a reasonable relationship between benefit and risk. Determining these limits is typically within the scope of reasonable medical judgment.

[0202] As defined in the first embodiment, the vaccine of the present invention, comprising at least one mRNA encoding at least one antigen, may be used for the prevention and treatment of diseases in at least one of newborns and infants, preferably aged 3 years or less or 2 years or less, preferably 1 year (12 months) or less, more preferably 9 months or less, 6 months or less, or 3 months or less. The treatment preferably includes vaccinating the newborn or infant to induce an immune response in the newborn or infant. Preferably, the newborn or infant is a mammal (patient), preferably a human (patient), typically aged 3 years or less or usually 2 years or less, preferably 1.5 years or less, more preferably 1 year (12 months) or less, even more preferably 9 months or less, 6 months or less, or even 3 months or less. Accordingly, the newborn or infant may be approximately 0 to 3 years old, or usually 0 to 2 years old, preferably 0 to 1.5 years old, more preferably 0 to 1 year (0 months to 12 months), even more preferably 0 to 9 months or less, 0 to 6 months or even 0 to 3 months or less. A newborn or infant may be further distinguished as a newborn, typically referring to an age of 1 year (12 months) or less, preferably 9 months or less, 6 months or less, or even 3 months or less. Thus, a newborn or infant may be approximately 0 to 1 year (0 months to 12 months), preferably 0 months to 9 months or less, 0 months to 6 months or less, or even 0 months to 3 months or less. A newborn or infant may be further distinguished as a newborn, typically referring to an age greater than 3 months, preferably 6 months, more preferably greater than 9 months, but also referring to an age less than 3 years or usually less than 2 years, preferably 1.5 years or less, more preferably 1 year (12 months) or less, even more preferably 9 months or less, or even 6 months or less. Thus, the newborn or infant may be about 3 months to about 3 years, about 3 months to about 2 years, about 3 months to about 1.5 years, or about 3 months to about 1 year (12 months), about 6 months to about 3 years, about 6 months to about 2 years, about 6 months to about 1.5 years, or about 6 months to about 1 year (12 months), about 9 months to about 3 years, about 9 months to about 2 years, or about 9 months to about 1.5 years, about 12 months to about 3 years, about 12 months to about 2 years, or about 12 months to about 1.5 years. The newborn or infant may be male or female.

[0203] As further defined in the first embodiment of the present invention, treatment includes vaccination of a patient to induce an immune response in said patient. In this context, vaccination is typically carried out via administration of a vaccine of the present invention. Administration may be parenteral, oral, nasal, pulmonary, by inhalation (e.g., via aerosol or spray), topical, rectal, buccal, vaginal, or via an implanted reservoir. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, and sublingual injection or infusion techniques. Preferably, the vaccine of the present invention may be administered intradermally to reach APCs in the dermis. Also preferably, the vaccine of the present invention as defined herein may be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. Also preferably, the vaccine of the present invention may be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including, for example, diseases of the skin or other accessible epithelial tissues. Suitable topical formulations are readily prepared for each of these areas or organs. For topical application, the vaccine of the present invention may be formulated into a suitable ointment containing the vaccine of the present invention and, optionally, further components as defined herein, suspended or dissolved in one or more carriers. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and formulating with an aerosolizing agent for use as a spray.

[0204] The vaccines of the present invention may be used in combination with other therapies, preferably therapies for diseases as defined herein, or other therapies. As defined herein, the term "in combination" refers to the use of more than one therapy, preferably two or more therapies, in reference to the administration of two or more therapies to a newborn or infant as defined herein. The use of the term "in combination" does not limit the order in which the therapies are administered to a newborn or infant as defined herein. For example, a first therapy (e.g., a first prophylactic or therapeutic agent) may be administered concurrently with the second therapy (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before) the second therapy. The first or second therapy may be administered to a neonate or infant as defined herein any time after the first or second therapy (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later). In some embodiments, the one or more other therapies are surgery, immunotherapy, gene therapy, pain therapy, anti-fever therapy, therapy to relieve or assist breathing, other (active or passive) vaccination / immunization, anti-viral therapy, anti-bacterial therapy, anti-fungal therapy, anti-parasitic therapy, anti-allergy therapy, conventional tumor therapy, and chemotherapy, or include prophylactic use in or for any of the diseases described herein, preferably for rabies, RSV viral infection, and the like.

[0205] In certain embodiments, treatments are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about They may be administered 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. In certain embodiments, two or more therapies are administered during the same patient visit.

[0206] Typical doses of mRNA encoding at least one antigen as defined herein may range, but are not limited to, from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 μg, or 30 μg to 300 μg of mRNA per patient. Preferably, the vaccines of the present invention are formulated accordingly to include one, two, three or more doses.

[0207] According to certain embodiments, the vaccine of the present invention may be administered to a newborn or infant as a single dose. In certain embodiments, the vaccine of the present invention may be administered to a newborn or infant as a single dose followed by a second administration, and optionally even a third, fourth (or more) administrations. According to this embodiment, a booster vaccination of the vaccine of the present invention may be administered to a newborn or infant after the second (or third, fourth, etc.) administration, preferably at specific time intervals as defined below. In certain embodiments, such a booster vaccination of the vaccine of the present invention may utilize an additional compound or component as defined for the vaccine of the present invention as defined herein. In some embodiments, administrations and / or booster administrations of the same vaccine of the present invention may be repeated, such administrations being repeated for at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, e.g., 1-5 days, 1-10 days, 5-15 days, 10-20 days, 15-25 days, 20-30 days, 25-35 days, 30-5 days, The intervals may be 0 days, 40 to 60 days, 50 to 70 days, 1 to 75 days, or 1 month, 2 months, 3 months, 4 months, 5 months, or at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 30 months, 36 months, 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, or more. In certain embodiments, the vaccines of the present invention may be administered to a subject as a single dose once per year.

[0208] In certain embodiments, the vaccines of the present invention may be administered to newborns or infants in the fall or winter, i.e., before or during the influenza season in each hemisphere. In one embodiment, the newborn or infant receives his / her first dose early in the season, for example, in late September or early October, so that the second dose (if needed) can be given before the peak of the influenza season.

[0209] In certain embodiments, the vaccines of the present invention may be administered at least once, and preferably more than once, to a newborn or infant prior to treatment for a disease as defined herein, preferably at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, e.g., 1 to 5 days, 1 to 10 days, 5 to 15 days, 10 to 20 days, 15 to 25 days, 20 to 30 days, 25 to 35 days, 30 to 50 days, 40 to 60 days, 50 to 70 days, 1 to 75 days, or 1 month, 2 months, 3 months, 4 months, 5 months, or at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months prior to treatment for a disease as defined herein. The second or further dose may then be administered prior to, simultaneously with, or directly after treatment.

[0210] Furthermore, the disease defined according to the first embodiment of the present invention is any disease selected from an infectious disease, preferably a (viral, bacterial or protozoal) infectious disease, an autoimmune disease, an allergy, an allergic disease, or a cancer or tumor disease.

[0211] Such diseases include cancer or tumor diseases, preferably melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, blastoma, renal cancer, gastrointestinal tumors, glioma, prostate tumor, bladder cancer, rectal tumor, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, breast cancer (= breast cancer), uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), leukemia, liver cancer, various virus-induced tumors, such as papillomavirus-induced cancers (e.g. cervical carcinoma = cervical cancer), cancer), adenocarcinoma, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1-induced lymphoma and HTLV-2-induced lymphoma, acoustic neuroma, lung carcinoma (= lung cancer) cancer)=bronchial cancer), small cell lung cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumor, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, pituitary tumor, mycosis fungoides, carcinoid, neurofibromatosis, spinal cell carcinoma, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, uterine cancer, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP syndrome, head / neck tumor, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, esophageal cancer (=oesophageal cancer), wart lesions, small intestine tumor, craniopharyngioma, ovarian cancer, reproductive organ tumor, ovarian cancer (=ovarian cancer), pancreatic cancer carcinoma (=pancreatic cancer), endometrial cancer, liver metastasis, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumor, and prostate cancer (=prostate tumor), etc.

[0212] According to one further particular aspect, a disease as defined herein comprises an infectious disease, preferably a (viral, bacterial or protozoal) infectious disease. Such an infectious disease, preferably a viral, bacterial or protozoal infection, is typically an influenza, preferably influenza A, B, C or Thogotovirus, more preferably e.g. hemagglutinin subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14 or H15 and / or neuraminidase subtype N1, N2, N3, N4, N5, N6, N7, N8 or N9, or preferably influenza A subtype. Combinations of types H1N1, H1N2, H2N2, H2N3, H3N1, H3N2, H3N3, H5N1, H5N2, H7N7, or H9N2, malaria, severe acute respiratory syndrome (SARS), respiratory syncytial virus infection, yellow fever, AIDS, Lyme borreliosis, leishmaniasis, anthrax, meningitis, condyloma acuminata, hollow warts, dengue fever, tertian fever, Ebola virus, the common cold, early summer meningoencephalitis (FSME), shingles, hepatitis, herpes simplex type I, herpes simplex type II, herpes zosterzoster), Japanese encephalitis, arenavirus-related diseases (Lassa fever infection), Marburg virus, measles, foot-and-mouth disease, infectious mononucleosis (Pfeiffer's glandular fever), mumps, Norwalk virus infection, smallpox, polio (infantile lameness), pseudocroup, erythema infectiosum (fifth disease), rabies, warts, West Nile fever, chickenpox, and viral infections such as cytomegalovirus (CMV), abortion (prostatitis), anthrax, appendicitis, borreliosis, botulism Toxicosis, Campylobacter, Chlamydia trachomatis (inflammation of the urethra, conjunctivitis), cholera, diphtheria, granuloma venereum (donavanosis), epiglottitis, typhus, gas gangrene, gonorrhea, tularemia, Helicobacter pylori, whooping cough, climatic bubo, osteomyelitis, Legionnaires' disease, leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis, anthrax, otitis media, Mycoplasma hominis, neonatal sepsis (chorioamnionitis), rhinoplasty, paratyphoid fever, Bacterial infections such as plague, Reiter's syndrome, Rocky Mountain spotted fever, Salmonella paratyphoid, Salmonella typhoid, scarlet fever, syphilis, tetanus, Triple flu, scrub typhus, tuberculosis, typhoid, vaginitis (colpitis), and chancroid, as well as amebiasis, schistosomiasis, Chagas' disease, echinococcus, diphyllobothrix, fish poisoning (ciguatera), fox tapeworm, athlete's foot, dog tapeworm, candidiasis, yeast spots, scabies, skin Selected from infections caused by parasites, protozoa or fungi such as leishmaniasis, lambliasis (giardia), lice, malaria, microscopy, onchocerciasis (river blindness), fungal diseases, bovine tapeworm, schistosomiasis, pig tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral leishmaniasis, nappy / diaper dermatitis or small tapeworms.

[0213] According to another particular embodiment, the diseases defined herein include autoimmune diseases as defined below. Autoimmune diseases can be broadly divided into systemic and organ-specific or localized autoimmune diseases, depending on the predominant clinicopathological features of each disease. Autoimmune diseases may be categorized as systemic syndromes, including systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, rheumatoid arthritis, and polymyositis, or as localized syndromes, which may be endocrinological (type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, etc.), cutaneous (pemphigus vulgaris), hematological (autoimmune hemolytic anemia), and neurological (multiple sclerosis), or may involve a localized mass of virtually any body tissue. The autoimmune disease to be treated may be selected from the group consisting of type I, type II, type III, or type IV autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes (type 1 diabetes), chronic polyarthritis, Graves' disease, chronic hepatitis, ulcerative colitis, type I allergic disease, type II allergic disease, type III allergic disease, type IV allergic disease, fibromyalgia, alopecia, Bechterew's disease, Crohn's disease, myasthenia gravis, neurodermatitis, polymyalgia rheumatica, progressive systemic sclerosis (PSS), Reiter's syndrome, rheumatoid arthritis, psoriasis, and vasculitis, or type II diabetes. The exact mechanism by which the immune system elicits an immune response against self-antigens has not yet been elucidated, but some research has provided insight into the pathogenesis. Therefore, autoreactions may be due to T cell bypass. A normal immune system requires the activation of B cells by T cells before the former can produce large amounts of antibodies. This requirement for T cells can be bypassed in rare cases, such as infection with organisms that produce superantigens that can initiate polyclonal activation of B cells, or even T cells, by directly binding to the β-subunit of the T cell receptor in a nonspecific manner. Another explanation posits autoimmune disease from "molecular mimicry," where a foreign antigen may share structural similarity with a particular host antigen, and therefore any antibody produced against this antigen (mimicking the self-antigen) would theoretically also bind to the host antigen and amplify the immune response.Autoimmune diseases based on molecular mimicry are known to those skilled in the art involving various viral and bacterial antigens. The most striking form of molecular mimicry is observed in group A beta-hemolytic streptococci, which share antigens with human myocardium and are the cardiac cause of rheumatic fever.

[0214] Thus, according to a further specific embodiment, the diseases defined herein include allergies or allergic disorders, i.e., disorders related to allergies. Allergies are typically conditions associated with abnormal adaptive immune hypersensitivity to specific foreign antigens or allergens, such as the allergens defined herein. Such allergens or allergens may be selected from the allergens defined herein, which are antigens derived from different sources, such as animals, plants, fungi, and bacteria. Related allergens include, for example, dander, grass, pollen, mold, drugs, or numerous environmental factors. Allergies typically trigger a local or systemic inflammatory response to these antigens or allergens, resulting in the body's internal immunity to these allergens. Without being bound by theory, several different disease mechanisms are believed to be involved in the development of allergies. According to the classification system by P. Gell and R. Coombs, the term "allergy" is restricted to type I hypersensitivity, which is triggered by the classical IgE mechanism. Type I hypersensitivity is characterized by excessive activation of mast cells and basophils by IgE antibodies, which causes a systemic inflammatory response that can range from benign symptoms such as a runny nose to life-threatening anaphylactic shock and death. Well-known types of allergies include, but are not limited to, asthma, allergic asthma (resulting in swelling of the nasal mucosa), allergic conjunctivitis (resulting in redness and itching of the conjunctiva), allergic rhinitis ("hay fever"), anaphylaxis, angioedema, atopy, atopic dermatitis (eczema), urticaria (hives), eosinophilia, respiratory allergies, allergies to insect bites, skin allergies (resulting in or including various rashes such as eczema, urticaria, and (contact) dermatitis), food allergies, and allergies to drugs. Treatment of such allergic disorders or diseases can occur, preferably by desensitizing the immune response that triggers a specific immune response. Such desensitization can be achieved by administering an effective amount of an allergen or allergen encoded by a nucleic acid as defined herein, preferably when formulated as a pharmaceutical composition, to induce a slight immune response.The amount of allergen or allergy antigen may be increased stepwise with subsequent administrations until the immune system of the patient being treated can tolerate a particular amount of the allergen or allergy antigen.

[0215] Diseases relevant to the present invention also include type II hypersensitivity reactions (cytotoxic, antibody-dependent), including, for example, autoimmune hemolytic anemia, thrombocytopenia, erythroblastosis fetalis, Goodpasture's syndrome, Graves' disease, and myasthenia gravis; type III hypersensitivity reactions (immune complex diseases), including, for example, serum sickness, Arthus reaction, and systemic lupus erythematosus (SLE); type IV hypersensitivity reactions (delayed-type hypersensitivity (DTH), cell-mediated immune memory response, antibody-independent), including, for example, contact dermatitis, tuberculin reaction, chronic graft rejection, and multiple sclerosis; and type V hypersensitivity reactions (receptor-mediated autoimmune diseases), including, for example, Graves' disease and myasthenia gravis.

[0216] In a further preferred embodiment, the vaccine of the present invention may be formulated as a kit, preferably as a kit of parts. Accordingly, the present invention also provides kits, particularly kits of parts, comprising the components of the vaccine of the present invention, alone or in combination with other components as defined above, and optionally technical instructions with information on the administration and dosage of the vaccine of the present invention. The components of the vaccine of the present invention may be included in the kit alone or in combination with further components as defined above, as part of the kit or as separate parts of the kit, such as, for example, at least one mRNA encoding at least one antigen as defined above in one part of the kit, and preferably, further components mixed with at least one mRNA encoding at least one antigen as defined above or separate in the further part of the kit. Such kits, preferably kits of parts, can be applied, for example, to any of the applications or uses described above.

[0217] In the present invention, unless otherwise stated, different features of alternatives and embodiments may be combined with each other where suitable. Furthermore, unless specifically stated, the term "comprising" is not to be interpreted as meaning "consisting of". However, in the context of the present invention, the term "comprising" may be replaced with the term "consisting of" where suitable.

[0218] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0219] The following drawings are intended to further illustrate the invention, but are not intended to limit the subject matter of the invention thereto. [Brief explanation of the drawings]

[0220] [Figure 1A] Figure 1A shows the weight progression of mice during the experiment. As a result, neonatal mice vaccinated with mRNA encoding PR8 H1 hemagglutinin showed significantly better survival rates (all mice survived) than those challenged with influenza infection along with control mRNA alone (all mice vaccinated with control mRNA encoding chicken ovalbumin on the first day of week 8 died approximately 5 days after vaccination with control mRNA, and all mice vaccinated with control mRNA at week 8 died approximately 6 days after vaccination with control mRNA). Remarkably, the survival rates were comparable to those of adult mice. [Figure 1B]FIG. 1B shows the coding sequence of the mRNA encoding PR8 H1 HA (hemagglutinin of influenza virus A / Puerto Rico / 8 / 1934) (SEQ ID NO: 384) used for vaccination of newborn and 8-week-old mice (see FIG. 1A). [Figure 1C] FIG. 1C shows the coding sequence of the mRNA encoding chicken ovalbumin (control mRNA) (SEQ ID NO: 385) used as a control for vaccination of newborn and 8-week-old mice (see FIG. 1A). [Example]

[0221] The following examples are intended to further illustrate the invention without, however, intending to limit the subject matter of the invention thereto.

[0222] Example 1 - Preparation of mRNA constructs For this example, DNA sequences encoding PR8 H1 HA (hemagglutinin of influenza virus A / Puerto Rico / 8 / 1934) (SEQ ID NO: 384) and chicken ovalbumin (control mRNA) (SEQ ID NO: 385) as a control were prepared and used in the subsequent in vivo transcription reaction.

[0223] In the first preparation, a DNA sequence designated PR8 H1 HA (hemagglutinin of influenza virus A / Puerto Rico / 8 / 1934) (SEQ ID NO: 384) (see Figure 1B) was prepared by modifying a wild-type hemagglutinin-encoding DNA sequence by introducing a GC-optimized sequence for better codon usage and stabilization. The corresponding mRNA sequence is shown in SEQ ID NO: 384 (see Figure 1B). This sequence was further introduced into the pCV19 vector and modified to include a stabilizing sequence derived from α-globin-3'-UTR (muag (mutated α-globin-3'-UTR)), a stretch of 70 adenosines at the 3' end (poly-A tail), and a stretch of 30 cytosines at the 3' end (poly-C tail). The sequence of the final DNA construct was designated "PR8 HA H1."

[0224] In the second preparation, a DNA sequence designated as control chicken ovalbumin (control mRNA) (SEQ ID NO: 385) (see Figure 1C) was prepared by modifying the wild-type chicken ovalbumin-encoding DNA sequence by introducing a GC-optimized sequence for better codon usage and stabilization. The corresponding mRNA sequence is shown in SEQ ID NO: 385 (see Figure 1C). This sequence was further introduced into the pCV19 vector and modified to include a stabilizing sequence derived from α-globin-3'-UTR (muag (mutated α-globin-3'-UTR)), a stretch of 70 adenosines at the 3' end (poly-A tail), and a stretch of 30 cytosines at the 3' end (poly-C tail). The sequence of the final DNA construct was designated "chicken ovalbumin."

[0225] In a further step, each of the DNA plasmids prepared above was transcribed in vitro into mRNA using T7 polymerase, and the resulting mRNA was then purified using PureMessenger® (CureVac, Tübingen, Germany).

[0226] All mRNAs used herein were further complexed with protamine before use. The RNA complex consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine (mRNA:protamine = 2:1 (weight ratio)). First, mRNA was complexed with protamine by slowly adding protamine-lactated Ringer's solution to the mRNA. As soon as the complex was stable, free mRNA was added, briefly stirred, and the final vaccine concentration was adjusted with lactated Ringer's solution.

[0227] Example 2 - Vaccination of newborn and 8-week-old mice In this experiment, newborn or 8-week-old mice were inoculated intradermally twice with 80 μg of mRNA encoding PR8 H1 HA (influenza virus A / Puerto Rico / 8 / 1934 hemagglutinin; Figure 1B) or chicken ovalbumin (control mRNA; Figure 1C). The first injection was administered within 24 hours of birth and at 8 weeks of age. Five weeks after the final vaccination, mice were challenged with 10 times the median lethal dose (LD50) of PR8 virus (10 LD50). Mouse weights were monitored over a two-week period and sacrificed when they lost more than 25% of their original weight. The results are shown in Figure 1A. Figure 1A shows the progression of mouse weight over the course of the experiment. As a result, mice vaccinated with mRNA encoding PR8 H1 hemagglutinin showed significantly better survival (all mice survived) against influenza challenge with control mRNA alone (all mice in the control experiment died approximately 5 days after vaccination with control mRNA when vaccinated with control mRNA encoding chicken ovalbumin on day 1 and approximately 6 days after vaccination with control mRNA when vaccinated with control mRNA at 8 weeks). All vaccinated newborn mice survived challenge with PR8 H1 hemagglutinin, in contrast to controls.

Claims

1. A composition comprising at least one mRNA encoding at least one viral antigen selected from antigens derived from respiratory syncytial virus (RSV) for use in preventing viral infections of respiratory syncytial virus (RSV) in at least one of newborns and infants aged 2 years or younger, wherein the prevention comprises administering the composition to the newborn or infant, thereby inducing an immune response in the newborn or infant.

2. 2. The composition for use according to claim 1, wherein the induction of an immune response in a newborn or infant comprises induction of a Th1 immune response.

3. 3. The composition for use according to claim 1 or 2, wherein the newborn or infant is 1) male or female, mammal, or human, or 2) aged 2 years or less, 1.5 years or less, 1 year (12 months) or less, 9 months or less, 6 months or less, or 3 months or less, or both 1) and 2).

4. 4. A composition for use according to any one of claims 1 to 3, wherein the viral antigen is an RSV fusion (F) protein.

5. 5. A composition for use according to any one of claims 1 to 4, wherein the G / C content of the coding region of at least one mRNA is increased by at least 15% compared to the G / C content of the coding region of the wild-type mRNA.

6. 6. The composition for use according to any one of claims 1 to 5, wherein at least one mRNA is associated with a cationic compound selected from the group consisting of cationic peptides or proteins, cationic polysaccharides, cationic polymers, and cationic lipids.

7. The composition for use according to claim 6, wherein the cationic compound is a cationic lipid.

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