RNA vaccine against SARS-CoV-2 variants

Abstract

The present invention is directed to nucleic acids suitable for use in the treatment or prevention of infection with a coronavirus, preferably the coronavirus SARS-CoV-2, or a disorder associated with such an infection, preferably COVID-19. The present invention is also directed to compositions, polypeptides, and vaccines. The compositions and vaccines preferably comprise at least one of said nucleic acid sequences, preferably a nucleic acid sequence associated with a lipid nanoparticle (LNP). The present invention is also directed to first and second medical uses of the nucleic acids, compositions, polypeptides, combinations, vaccines, and kits, as well as methods for treating or preventing a coronavirus infection, preferably a Coronavirus infection.

Description

[Technical Field]

[0001] This application claims priority based on U.S. Provisional Application No. 63 / 129,395 filed on 22 December 2020; PCT Application No. PCT / EP2021 / 052455 filed on 3 February 2021; PCT Application No. PCT / EP2021 / 069626 filed on 14 July 2021; and PCT Application No. PCT / EP2021 / 069632 filed on 14 July 2021. Each of the aforementioned applications is incorporated herein by reference. [Background technology]

[0002] The present invention is not limited in any way to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 The present invention relates to RNAs suitable for use in treating or preventing infection with SARS-CoV-2 variants, or disorders associated with such infection, including (gamma, Brazil), B.1.427 / B.1.429 (epsilon, California, USA), B.1.525 (eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (copper, India), B.1.617.2 (delta, India), P.2 (zeta, Brazil), C37.1 (lambda, Peru), P.3 (theta, Philippines), and / or B.1.621 (mu, Colombia). The present invention also relates to compositions, polypeptides, and vaccines. The compositions and vaccines preferably comprise at least one of the RNA sequences, preferably RNA associated with lipid nanoparticles (LNPs).

[0003] list The present invention also includes, but is not limited to, the first and second medical uses of RNA, compositions, vaccines, and kits, as well as C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), This covers methods for treating or preventing SARS-CoV-2 infection caused by SARS-CoV-2 variants including B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0004] Coronavirus is a highly contagious, enveloped, positive-positive, single-stranded zoonotic RNA virus belonging to the Coronaviridae family.

[0005] Coronaviruses are genetically highly variable, and individual virus strains have the potential to infect several host species by overcoming species barriers. In late 2019, an outbreak of respiratory illness caused by a novel coronavirus strain was reported in Wuhan, Hubei Province, China. The novel coronavirus was named "Severe Acute Respiratory Syndrome Coronavirus 2" (SARS-CoV-2). Typical symptoms of the viral infection caused by SARS-CoV-2, also known as COVID-19 disease, include fever, cough, shortness of breath, and pneumonia, and are associated with a high mortality rate in the elderly population. In March 2020, the WHO declared the SARS-CoV-2 outbreak a pandemic. Furthermore, some individuals suffer from the effects of COVID-19 infection for several weeks to several months after infection. This group is referred to as "longcovid." General signs and symptoms that persist over time include fatigue, shortness of breath or difficulty breathing, cough, joint pain, chest pain, memory, concentration or sleep problems, muscle pain or headache, rapid or severe palpitations, loss of smell or taste, depression or anxiety, fever, dizziness upon standing, and symptoms that worsen after physical or mental activity.

[0006] During the pandemic, new SARS-CoV-2 variants often emerged that were more transmissible or pathogenic than the original SARS-CoV-2 strain. These newly emerging SARS-CoV-2 strains could potentially lead to reduced efficacy of first-generation vaccines developed against the original SARS-CoV-2 strain. Furthermore, it remains unclear whether boost vaccination with vaccines specifically designed against newly emerging SARS-CoV-2 strains in a population vaccinated against the original SARS-CoV-2 strain would induce a protective immune response against these newer strains. [Overview of the Initiative]

[0007] Therefore, one of the fundamental objectives of the present invention is to provide an RNA-based vaccine for SARS-CoV-2 infection, particularly SARS-CoV-2 infection caused by newly emerging SARS-CoV-2 variant strains. These newly emerging strains are not limited to, but include C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), and B.1.3 This includes 51 (Beta, South Africa), B.1.1.7 (Alpha, United Kingdom), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia). RNA-based vaccination represents one of the most promising technologies for new vaccines against the newly emerging SARS-CoV-2 virus. RNA can be genetically modified to suit newly emerging SARS-CoV-2 strains and administered to human subjects, where the transfected cells directly produce RNA-encoded antigens that elicit an immunological response.

[0008] As further defined in the claims and fundamental description, these objectives include, in particular, but are not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, UK) This is resolved by providing RNA containing at least one coding sequence encoding at least one antigenic peptide or protein derived from SARS-CoV-2, including at least one mutation derived from SARS-CoV-2 strains including B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0009] In preferred embodiments of the present invention, RNA and RNA-based vaccines include, for example, but are not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), The SARS-CoV-2 spike protein comprises RNA encoding at least one antigenic peptide derived from SARS-CoV-2 strains, including the spike protein derived from SARS-CoV-2 strains including P.1 (gamma, Brazil), B.1.427 / B.1.429 (epsilon, California, USA), B.1.525 (eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (copper, India), B.1.617.2 (delta, India), P.2 (zeta, Brazil), C37.1 (lambda, Peru), P.3 (theta, Philippines), and / or B.1.621 (mu, Colombia). [Brief explanation of the drawing]

[0010] [Figure 1-1]In groups vaccinated with mRNA vaccines CV2CoV and CV2CoV.351, both formulated within LNPs, significant IgG1 and IgG2a binding antibody responses to the receptor-binding domain (RBD) of ancestral SARS-CoV-2 and the RBD of the B.1.351 variant are shown on day 14 (Figure 1A-D) and day 21 (Figure 1E-H). On day 14, Figure 1A shows comparable IgG1 responses for all groups (ancestral SARS-CoV-2 receptor-binding domain (RBD) protein coating), and Figure 1B shows comparable IgG2a titers for all vaccine designs (ancestral RBD protein coating). On day 14, Figure 1C shows a comparable IgG1 response for all vaccination designs (RBD B.1.351 variant K417N, E484K, N501Y protein coating), and Figure 1D shows comparable IgG2a titers for all vaccination designs (RBD B.1.351 variant K417N, E484K, N501Y protein coating). On day 21, Figure 1E shows a comparable IgG1 response for all vaccination designs (ancestral SARS-CoV-2 receptor-binding domain (RBD) protein coating), and Figure 1F shows comparable IgG2a titers for all vaccination designs (ancestral SARS-CoV-2 receptor-binding domain (RBD) protein coating). On day 21, Figure 1G shows comparable IgG1 responses for all vaccine designs (RBD B.1.351 variant K417N, E484K, N501Y protein coating), and Figure 1H shows comparable IgG2a titers for all vaccine designs (RBD B.1.351 variant K417N, E484K, N501Y protein coating). [Figure 1-2] Continuation of Figure 1-1. [Figure 1-3] Continuation of Figure 1-2. [Figure 1-4] Continuation of Figure 1-3. [Figure 2-1]Figure 2A shows a significant induction of viral neutralizing titer (VNT) as assessed in a cytopathic effect (CPE)-based assay using ancestral SARS-CoV-2. Figure 2B and Figure 2A show increased VNT for all groups B-H on day 14 and day 21, respectively. Co-delivery of mRNA vaccine CV2CoV and CV2CoV.351 to the same leg (groups F and G) or different legs (group H) can produce responses to both vaccine variants on day 14 and day 21. Figure 2C shows increased VNT levels for all groups (groups B-H) on day 42. Co-delivery of both vaccine variants to the same leg (groups F and G) or different legs (group H) can produce responses to both variants on day 42. [Figure 2-2] Continuation of Figure 2-1. [Figure 3-1] Figure 3A shows a significant induction of VNT in a CPE-based assay using the B.1.351 variant of SARS-CoV-2, and Figure 3B shows an increase in VNT for all groups B-H on day 14 and day 21, respectively. Co-delivery of mRNA vaccines CV2CoV and CV2CoV.351 to the same leg (groups F and G) or different legs (group H) can produce a response to both vaccine variants on day 14 and day 21. Figure 3C shows an increase in VNT levels for all groups (groups B-H) on day 42. Co-delivery of both vaccine variants to the same leg (groups F and G) or different legs (group H) can produce a response to both variants on day 42. [Figure 3-2] Continuation of Figure 3-1. [Figure 4]Figure 4B shows significant induction of VNT as assessed in a CPE-based assay using the B.1.1.7 variant SARS-CoV-2 (Figure 4A) or P.1 (B.1.1.28) (Figure 4B). Figure 4A shows the increase in VNT levels using the B.1.1.7 variant for all groups (groups B-H) on day 42. Co-delivery of both vaccine variants to the same leg (groups F and G) or different legs (group H) can produce a response to both variants on day 42. Figure 4B shows the increase in VNT levels using the B.1.1.28 P.1 variant for all groups (groups B-H) on day 42. Co-delivery of both vaccine variants to the same leg (groups F and G) or different legs (group H) can produce a response to both variants on day 42. [Figure 5-1]In the groups vaccinated with CV2CoV and CV2CoV.351, significant IgG1 and IgG2a binding antibody responses were observed on day 14 (Figures 5A-D) and day 21 (Figures 5E-H). On day 14, Figures 5A (IgG1 titer) and 5B (IgG2a titer) showed dose-dependent levels of binding antibody titer using doses of 0.5 μg, 2 μg, 8 μg, and 40 μg, reaching saturation in the group vaccinated with 40 μg (ancestral SARS-CoV-2 receptor-binding domain (RBD) protein coating). On day 14, Figures 5C (IgG1 titer) and 5D (IgG2a titer) also showed dose-dependent levels of binding antibody titer using doses of 0.5 μg, 2 μg, 8 μg, and 40 μg, reaching saturation in the group vaccinated with 40 μg (RBD B.1.351 variant K417N, E484K, N501Y protein coating). On day 21, Figures 5E (IgG1 titer) and 5F (IgG2a titer) show the dose-dependent levels of conjugated antibody titers, saturated IgG1 and IgG2a titers, and saturation at doses >8 μg (Group I) for CV2CoV (ancestral SARS-CoV-2 receptor-binding domain (RBD) protein coating) for all doses of CV2CoV.351 vaccination. On day 21, Figures 5G (IgG1 titer) and 5H (IgG2a titer) show the dose-dependent levels of conjugated antibody titers, saturated IgG1 and IgG2a titers, and saturation at doses >8 μg (Group I) for mRNA vaccine CV2CoV (RBD B.1.351 variant K417N, E484K, N501Y protein coating) for all doses of CV2CoV.351 vaccination. [Figure 5-2] Continuation of Figure 5-1. [Figure 5-3] Continuation of Figure 5-2. [Figure 5-4] Continuation of Figure 5-3. [Figure 6-1]Figure 6 shows significant induction of VNT assessed in CPE-based assays using ancestral SARS-CoV-2 (CV2CoV, Figures 6A, 6C, and 6E) or B.1.351 variant SARS-CoV-2 (CV2CoV.351, Figures 6B, 6D, and 6F) on days 14, 21, and 42. Figure 6 also shows significant induction of VNT assessed in CPE-based assays using B.1.1.7 variant SARS-CoV-2 (Figure 6G) or B.1.1.28 P.1 variant (Figure 6H) on day 42. Figure 6A shows that the B.1.351 variant vaccine CV2CoV.351 induces dose-dependent VNT (heterogeneous response) to ancestral SARS-CoV-2 on day 14 in all dose groups. Compared to the response to vaccination with CV2CoV (allogeneic response), VNT in the group vaccinated with CV2CoV.351 decreased by approximately half on day 14. Figure 6B shows that CV2CoV.351 induces dose-dependent VNT against B.1.351 SARS-CoV-2 (allogeneic response) on day 14 in all dose groups. CV2CoV.351 vaccination induced high levels of VNT against the allogeneic virus, increasing 45-fold on day 14 compared to heterologous VNT against the ancestral virus (mean difference across all dose groups). Compared to vaccination with CV2CoV, VNT induced by CV2CoV.351 increased 41-fold on day 14 (mean difference across all dose groups). Figure 6C shows that the B.1.351 variant vaccine CV2CoV.351 induces dose-dependent VNT against ancestral SARS-CoV-2 (heterogeneous response) on day 21 in all dose groups. Compared to the response to vaccination with CV2CoV (allogeneic response), VNT in the group vaccinated with CV2CoV.351 was reduced by approximately half on day 21. Figure 6D shows that CV2CoV.351 induced a slightly dose-dependent VNT against B.1.351 SARS-CoV-2 (allogeneic response) on day 21 in all dose groups. CV2CoV.351 vaccination induced high levels of VNT against the allogeneic virus, which increased 35-fold on day 21 compared to heterologous VNT against the ancestral virus (mean difference across all dose groups).Compared with vaccination with CV2CoV, the VNT induced by CV2CoV.351 increased 42-fold on day 21 (mean difference across all dose groups). Figure 6E shows that the B.1.351 variant vaccine CV2CoV.351 induced a dose-dependent VNT against ancestral SARS-CoV-2 (heterologous response) on day 42 in all dose groups. Except for all vaccinations with 0.5 μg (group F), a slightly higher response was shown upon vaccination with CV2CoV (homologous response). Figure 6F shows that CV2CoV.351 induced a dose-dependent VNT against B.1.351 SARS-CoV-2 (homologous response) on day 42 in all dose groups. The VNT induced by CV2CoV.351 increased on day 42 compared with vaccination with CV2CoV. Figure 6G shows that the B.1.351 variant vaccine CV2CoV.351 induced a dose-dependent VNT against B.1.1.7 variant SARS-CoV-2 (heterologous response) on day 42 in all dose groups. Except for vaccination with 0.5 μg (group F), a similar response for group H was shown upon vaccination with CV2CoV (homologous response). Figure 6H shows that CV2CoV.351 induced a dose-dependent VNT against B.1.1.28 P.1 variant SARS-CoV-2 (homologous response) on day 42 in all dose groups. A lower response was observed upon vaccination with CV2CoV (homologous response). [Figure 6-2] Continuation of Figure 6-1. [Figure 6-3] Continuation of Figure 6-2. [Figure 6-4] Continuation of Figure 6-3. [Figure 7-1]Figures 7A-D show significant IgG1 and IgG2a binding antibody responses on day 14 in groups vaccinated with the bivalent mRNA vaccine composition CV2CoV+CV2CoV.351 formulated in LNP. On day 14, Figures 7A (IgG1 titer) and 7B (IgG2a titer) show dose-dependent levels of binding antibody titer using doses of 0.5 μg, 2 μg, and 8 μg (SARS-CoV-2 ancestral receptor-binding domain (RBD) protein coating). On day 14, Figures 7C (IgG1 titer) and 7D (IgG2a titer) show dose-dependent levels of binding antibody titer using doses of 0.5 μg, 2 μg, and 8 μg (B.1.351 RBD variant K417N, E484K, N501Y protein coating). Figures 7K and 7L also show significant induction of VNT as assessed in CPE-based assays at days 14, 21, and 42, respectively, using either ancestral SARS-CoV-2 (Figures 7E, 7F, and 7I) or B.1.351 variant SARS-CoV-2 (Figures 7G, ​​7H, and 7J). [Figure 7-2] Continuation of Figure 7-1. [Figure 7-3] Continuation of Figure 7-2. [Figure 8A]VNTs evaluated in a CPE-based assay using ancestral SARS-CoV-2 (Figure 8A) or the B.1.351 variant of SARS-CoV-2 (Figure 8B) are shown. Boosting with CV2CoV or the B.1.351 variant vaccine CV2CoV.351 showed strong boosting ability against ancestral SARS-CoV-2 and the B.1.351 variant of SARS-CoV-2 for both homologous and heterologous responses. Homologous responses are shown in Figure 8A for groups B, D, and F and in Figure 8B for groups C, E, and G. Heterologous responses are shown in Figure 8A for groups C, E, and G and in Figure 8B for groups B, D, and F. Viral neutralization responses against ancestral SARS-CoV-2 and the SARS-CoV-2 B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) variants were tested 14 days after boosting, on day 119 (Figures 8C–8F) (VNTs against ancestral SARS-CoV-2 (Figure 8C), SARS-CoV-2 B.1.351 (Figure 8D), SARS-CoV-2 B.1.1.7 (Figure 8E), and P.1 (Figure 8F)). [Figure 8B] Continuation of Figure 8A. [Figure 8C] Continuation of Figure 8B. [Figure 8D] Continuation of Figure 8C. [Figure 8E] Continuation of Figure 8D. [Figure 8F] Continuation of Figure 8E. [Figure 9-1]All groups show a significant total IgG spike-binding antibody response to ancestral SARS-CoV-2 RBD (Figure 9A) and B.1.351 RBD variants K417N, E484K, and N501Y (Figure 9B) at day 14. Time-dependent induction of VNTs to different SARS-CoV-2 variants is shown in Figures 9C-F (Figure 9C: Ancestral; Figure 9D: B.1.1.7; Figure 9E: B.1.351; Figure F: P1). Figures 9G-J show the cellular immune response of CD8 (Figures 9G and 9I) and CD4 (Figures 9H and 9J) positive T cells in mice stimulated with a mixture of ancestral SARS-CoV-2 peptide libraries (Figures 9G and H) or a mixture of B.1.351 SARS-CoV-2 peptide libraries (Figures 9I and J) using intracellular cytokine staining assays. [Figure 9-2] Continuation of Figure 9-1. [Figure 9-3] Continuation of Figure 9-2. [Figure 9-4] Continuation of Figure 9-3. [Figure 9-5] Continuation of Figure 9-4. [Figure 10] This shows the cellular immune response of CD8 (Figure 10B) and CD4 (Figure 10A)-positive T cells in mice stimulated with a mixture of ancestral SARS-CoV-2 peptide libraries, using an intracellular cytokine staining assay. [Figure 11-1] The time course of VNT in rats up to 133 days after the first vaccination, following prime and boost vaccinations with CVnCoV or CV2CoV, and a third vaccination with a bivalent CV2CoV+CV2CoV.351 vaccine composition, is shown (Figure 11A: VNT against ancestral SARS-CoV-2, Figure 11B: VNT against SARS-CoV-2 B.1.351). A strongly elevated VNT at day 119 was induced not only against ancestral and B.1.351 SARS-CoV-2, but also against B.1.1.7 and P.1 SARS-CoV-2 variants (Figure 11C: ancestral, Figure 11D: B.1.351, Figure 11E: B.1.1.7, Figure 11F: P.1). [Figure 11-2] Continuation of Figure 11-1. [Figure 12-1] The antibody response to vaccination in rats with vaccine compositions encoding different mRNA formats of the stabilized spike (S_stab pp) of the delta variant SARS-CoV-2 B1.617.2 is shown (Figures 12A and 12B: spike-binding antibodies detected by ELISA against delta B.1.617.2 variant RBD on days 14 and 42, respectively; Figures 12C, 12D, and 12E: VNT against SARS-CoV-2 B.1.617.2 on days 14, 21, and 42, respectively). Strong VNT was induced not only against the allogeneic SARS-CoV-2 variant (B.1.617.2) but also against heterologous SARS-CoV-2 ancestors and SARS-CoV-2 variants B.1.351 and P.1 (Figure 12F: ancestor, Figure G: B.1.351, Figure 12H: P.1). [Figure 12-2] Continuation of Figure 12-1. [Figure 12-3] Continuation of Figure 12-2. [Figure 13] This shows the initial antibody response (total IgG) at day 14 after vaccination with vaccine compositions encoding different mRNA constructs that encode different variant SARS-CoV-2 S_stab pp in rats. Furthermore, a bivalent approach compares chemically modified mRNA with unmodified mRNA (Figure 13A: ancestral RBD; Figure 13B: delta RBD (L452R, T478K); Figure 13C: beta RBD (K417N, E484K, N501Y)). [Figure 14-1]This study demonstrates vaccine efficacy by challenging mice with either SARS-CoV-2 variant B.1.351 or SARS-CoV-2 variant B.1.627.2. Survival of challenged mice is shown in Figure 14A (challenge using B.1.351) and Figure 14B (challenge using B.1.617.2). Mean percentage body weight change is shown in Figure 14C (challenge using B.1.351) and Figure 14D (challenge using B.1.617.2). Salivary viral RNA levels are shown in Figure 14E (B.1.351 challenge group) and Figure 14F (B.1.617.2 challenge group). The viral load in the upper respiratory tract (URT) (turbinates) is shown in Figure 14G for the B.1.351 challenge group and Figure 14H for the B.617.2 challenge group, while the viral load in the lower respiratory tract (LRT) (lungs) is shown in Figure 14I (challenge using B.1.351) and J (challenge using B.1.617.2). The viral load in the brain is shown in Figures 14K to N (Figures 14K and L for the cerebellum, and Figures 14M and N for the cerebrum (for challenge group B.1.351: Figures 14K and M, for B.1.617.2: Figures 14L and N)). The induction of total anti-RBD immunoglobulin is shown in Figure 14O: Challenge group B.1.351 and Figure 14P: Challenge group B.1.617.2, and VNT is shown in Figure 14Q: Post-challenge group B.1.351, Figure 14R: Pre-challenge group B.1.617.2, and Figure 14S: Post-challenge group B.1.617.2. [Figure 14-2] Continuation of Figure 14-1. [Figure 14-3] Continuation of Figure 14-2. [Figure 14-4] Continuation of Figure 14-3. [Figure 14-5] Continuation of Figure 14-4. [Figure 15-1] This figure shows the vaccine efficacy in hamsters challenged with SARS-CoV-2 variant B.1.351. Figure 15A shows the percentage change in body weight over the number of days after challenge infection. Figure 15B shows the amount of viral RNA in saliva, and Figure 15C shows the amount of viral RNA in lung tissue. Figure 15D shows the induction of anti-RBD total immunoglobulin (Ig), and Figure 15E shows VNT. [Figure 15-2] Continuation of Figure 15-1. [Figure 15-3] Continuation of Figure 15-2. [Modes for carrying out the invention]

[0011] definition For clarity and readability, the following definitions are provided. Any technical features described in these definitions can be read in any embodiment of the invention. Additional definitions and explanations may be provided specifically in the context of these embodiments.

[0012] In a numerical context, percentages should be understood as relative to the total number of each item. Otherwise, unless otherwise indicated by the context, percentages should be understood as weight percentages (wt.-%).

[0013] Approximately: The term "approximately" is used when a determinant or value does not need to be identical, i.e., 100% the same. Thus, "approximately" means that a determinant or value can deviate from any point within the range of 0.1% to 20%, or 0.1% to 10%; for example, including 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. A person skilled in the art will know, for example, that a particular parameter or determinant can vary slightly depending on how the parameter is determined. For example, if a particular determinant or value is defined herein as having, for example, a length of "about 1000 nucleotides," then the length may be any point within these ranges, such as 0.1% to 20%, or 0.1% to 10%; for example, it may be far from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Therefore, those skilled in the art will know that in certain examples the length may deviate from 1 to 200 nucleotides, or from 1 to 100 nucleotides, particularly 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides, or any integer value within the range.

[0014] Adaptive Immune Response: As used herein, the term “adaptive immune response” is intended to be recognized and understood by those skilled in the art, for example, to refer to an antigen-specific response of the immune system (adaptive immune system). Antigen specificity results in a response tailored to a particular pathogen or to cells infected with that pathogen. The ability to initiate these tailored responses is typically maintained in the body by “memory cells” (B cells). In the context of this invention, the antigen is SARS-CoV-2, for example, but not limited to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (A This is brought about by at least one antigenic peptide or protein-coding RNA derived from SARS-CoV-2 strains including Rufa (UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).Preferably, the antigens are, for example, but not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B It is brought about by RNA encoding at least one antigenic peptide derived from SARS-CoV-2 spike protein, including the spike protein derived from SARS-CoV-2 strains including .1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0015] Antigen: As used herein, the term “antigen” is intended to mean a substance that is recognized and understood by those skilled in the art and can be recognized, for example, by the immune system, preferably by the adaptive immune system, and which has the ability to induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or may contain, a peptide or protein that can be presented to T cells by MHC. Also, for example, the SARS-CoV-2 spike protein (S), for example, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 ( Peptides or protein fragments, variants, and derivatives derived from the spike protein (S) of SARS-CoV-2 strains, including Alpha (UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia), are understood as antigens in the context of the present invention. In the context of the present invention, the antigen may also be the product of translation of the RNA provided as identified herein.

[0016] Antigenic peptides or proteins: The terms “antigenic peptides or proteins” or “immunogenic peptides or proteins” are intended to be recognized and understood by those skilled in the art to refer, for example, peptides or proteins derived from (antigenic or immunogenic) proteins that stimulate the body’s adaptive immune system, thereby eliciting an adaptive immune response. Therefore, antigenic / immunogenic peptides or proteins are derived from (e.g., the spike protein (S) of SARS-CoV-2), for example, but not limited to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (B The product contains at least one epitope or antigen of a protein derived from the spike protein (S) of SARS-CoV-2 strains, including B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0017] Cationicity: Unless a different meaning is evident from the specific context, the term "cationicity" means that each structure is positively charged, either constitutively or non-constitutively, but in response to certain conditions, such as pH. Therefore, the term "cationicity" covers both "constitutive cationicity" and "cationicizable."

[0018] Cationizable: As used herein, the term “cationizable” means that a compound, group, or atom is positively charged at lower pH and uncharged at higher pH in that environment. Furthermore, in non-aqueous environments where the pH value cannot be determined, cationizable compounds, groups, or atoms are positively charged at high hydrogen ion concentrations and uncharged at low concentrations or active hydrogen ions. Whether charged or uncharged depends on the individual properties of the cationizable or polycationizable compound at pH or hydrogen ion concentration, particularly the pKa of each cationizable group or atom. In a diluted aqueous environment, the proportion of positively charged cationizable compounds, groups, or atoms can be estimated using the so-called Henderson-Hasselbalch formula, which is well known to those skilled in the art. For example, in some embodiments, if the compound or part is cationizable, it is preferable to be positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even more preferably 9 or less, 8 or less, or 7 or less, most preferably at a physiological pH, for example, about 7.3 to 7.4, i.e., under physiological conditions, particularly under physiological salt conditions of cells in vivo. In other embodiments, the cationizable compound or part is predominantly neutral at a physiological pH, for example, about 7.0 to 7.4, but is preferably positively charged at lower pH values. In some embodiments, the preferred range of pKa of the cationizable compound or part is about 5 to about 7.

[0019] Code Sequence / Code Region: As used herein, the terms “code sequence” or “code region” and the corresponding abbreviation “cds” are intended to refer to a sequence of several nucleotide triplets that are recognized and understood by those skilled in the art and can be translated, for example, into peptides or proteins. In the context of the present invention, a code sequence may also be an RNA sequence consisting of a number of nucleotides divisible by 3, which begins with a start codon and preferably ends with a stop codon.

[0020] Derived from: When used throughout this specification in the context of nucleic acids, i.e., nucleic acids "derived from" another nucleic acid, the term "derived from" means that a nucleic acid derived from another nucleic acid shares, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. Those skilled in the art will understand that sequence identity is typically calculated for the same type of nucleic acid, i.e., DNA sequences or RNA sequences. Therefore, when DNA is "derived from" RNA, or when RNA is "derived from" DNA, it is understood that in the first step, the RNA sequence is converted to the corresponding DNA sequence (in particular by replacing uracil (U) with thymidine (T) throughout the sequence), or vice versa, the DNA sequence is converted to the corresponding RNA sequence (in particular by replacing T with U throughout the sequence). Subsequently, the sequence identity of the DNA sequence or the sequence identity of the RNA sequence is determined. Preferably, nucleic acids "derived from" also refer to nucleic acids that are modified compared to nucleic acids that are induced, for example, to further and / or longer increase RNA stability and / or increase protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term “derived from” means that an amino acid sequence derived from another amino acid sequence shares at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.

[0021] Epitope: As used herein, the term “epitope” (also known in the art as “antigenic determinant”) is intended to be recognized and understood by those skilled in the art, for example, to refer to T cell epitopes and B cell epitopes. T cell epitopes or portions of antigenic peptides or proteins may preferably include fragments having about 6 to about 20 or more amino acid lengths, for example, preferably fragments having about 8 to about 10 amino acid lengths, for example, 8, 9, or 10 (or even 11 or 12 amino acids), which are treated and presented by MHC class I molecules, or preferably fragments having about 13 to about 20 or more amino acid lengths, which are treated and presented by MHC class I molecules. These fragments are typically recognized by T cells in the form of complexes consisting of peptide fragments and MHC molecules, i.e., the fragments are typically not recognized in their native forms. B cell epitopes are typically fragments located on the outer surface of (natural) protein or peptide antigens, preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids, i.e., recognizable by antibodies in their natural form. Such epitopes of proteins or peptides may further be selected from any of the variants of such proteins or peptides described herein. In this context, an epitope can be a three-dimensional structure or a discontinuous epitope composed of segments of a protein or peptide as defined herein, which are discontinuous in the amino acid sequence of the protein or peptide as defined herein but come together as a single polypeptide chain, or as a continuous or linear epitope.

[0022] Fragment: As used herein in the context of nucleic acid sequences (e.g., RNA or DNA) or amino acid sequences, the term “fragment” typically refers to a shorter portion of the full-length sequence of a nucleic acid or amino acid sequence, for example, a portion that still retains its intended function. Thus, a fragment typically consists of a sequence that is identical to a corresponding compartment in the full-length sequence. Preferred fragments of a sequence in the context of the present invention consist of nucleotides or amino acids corresponding to a contiguous compartment of an entity, for example, a contiguous compartment of an entity in the molecule from which the fragment is derived, which constitute at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and 95% of the total (i.e., full-length) molecule from which the fragment is derived. , corresponding to 96%, 97%, 98%, 99%, and 99.5% (for example, SARS-CoV-2 spike protein (S), for example, but not limited to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Camel 0.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (A The spike protein (S) of SARS-CoV-2 strains including B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).When used herein in the context of proteins or peptides, the term “fragment” typically includes a sequence of a protein or peptide as defined herein, which is cleaved at the N-terminus and / or C-terminus with respect to its amino acid sequence compared to the amino acid sequence of the original protein. Thus, such truncation can occur at either the amino acid level or, correspondingly, at the nucleic acid level. Therefore, sequence identity with respect to such a fragment as defined herein may preferably refer to the entire protein or peptide as defined herein, or the entire (coding) nucleic acid molecule of such a protein or peptide. A protein or peptide fragment may include at least one epitope of these proteins or peptides.

[0023] Heterogeneous: As used throughout this specification in the context of nucleic acid sequences or amino acid sequences, the term “heterogeneous” or “heterogeneous sequence” must be understood as a sequence (e.g., RNA, DNA, amino acids) that is derived from another gene, another allele, or, for example, another species or virus. Two sequences are typically understood to be “heterogeneous” if they are not derived from the same gene or the same allele. That is, heterogeneous sequences may be derived from essentially the same organism or virus, but they do not arise from the same nucleic acid or protein.

[0024] Humoral Immune Response: The term “humoral immunity” or “humoral immune response” is intended to be recognized and understood by those skilled in the art to refer, for example, to B cell-mediated antibody production, and possibly to the secondary processes associated with antibody production. Humoral immune responses can typically be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell generation. Humoral immunity may also refer to the effector function of antibodies, including pathogen and toxin neutralization, classical complement activation, and opsonin enhancement of phagocytosis and pathogen elimination.

[0025] Identity (of sequences): As used herein in the context of nucleic acid sequences or amino acid sequences, the term “identity” is intended to mean the percentage of identity that two sequences are, as recognized and understood by those skilled in the art, identical over their entire length or over a particular designated portion, region or domain. For example, identity of at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% over their entire length or over a particular designated portion, region or domain. To determine the percentage of identical sequences between two sequences, for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, preferably the aa sequences encoded by the nucleic acid sequences as defined herein, or the aa sequences themselves, the sequences can be aligned and then compared with each other. For example, a position in the first sequence may be compared with a corresponding position in the second sequence. If a position in the first sequence is occupied by the same residue as a position in the second sequence, the two sequences are identical at that position. Otherwise, the sequences are different at that position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted into the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted into the second sequence to allow for further alignment. The percentage of identical sequences is then a function of the number of identical positions, divided by the total number of positions, including those positions that are occupied in only one sequence. The percentage of two sequences that are identical can be determined using an algorithm, such as one integrated into the BLAST program.

[0026] Immunogen, Immunogenicity: The terms “immunogen” or “immunogenicity” are intended to be recognized and understood by those skilled in the art and to refer to compounds that can, for example, stimulate / induce an immune response. Preferably, the immunogen may be a peptide, polypeptide, or protein. In the sense of the present invention, an immunogen is a protein derived from the SARS-CoV-2 spike protein, for example, but not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5) as defined herein, C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 The immunogen is the translation product of a provided RNA containing at least one coding sequence encoding at least one antigenic peptide, which is a protein derived from the spike protein of SARS-CoV-2 strains, including (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia). Typically, the immunogen elicits an adaptive immune response.

[0027] Immune response: The term “immune response” is intended to be recognized and understood by those skilled in the art to refer, for example, to a specific response of the adaptive immune system to a particular antigen (so-called specific or adaptive immune response) or a nonspecific response of the innate immune system (so-called nonspecific or innate immune response), or a combination thereof. A suitable vaccine induces an efficient immune response in a normal, healthy recipient to whom the vaccine is administered. A single dose of vaccination with an efficient immune response results in a viral neutralizing antibody titer. Furthermore, or instead, an efficient immune response triggers an adaptive immune response. In some embodiments, an efficient immune response reduces coronavirus infection by at least 50% compared to the neutralizing antibody titer of an unvaccinated control. In some embodiments, an efficient immune response is one in which the neutralizing antibody titer and / or T-cell immune response is sufficient to reduce the rate of asymptomatic viral infection compared to the neutralizing antibody titer of an unvaccinated control. An efficient immune response may also be one in which the neutralizing antibody titer and / or T-cell immune response is sufficient to prevent the viral incubation period in the subject, and / or the neutralizing antibody titer is sufficient to block the fusion of the virus with the epithelial cells of the subject. In some embodiments, an efficient immune response is one in which administration of a therapeutically effective amount of nucleic acid, composition, polypeptide, or vaccine to a subject induces a T-cell immune response against coronavirus in the subject. In preferred embodiments, the T-cell immune response includes a CD4+ T-cell immune response and / or a CD8+ T-cell immune response. In further embodiments, an efficient immune response is one in which the immune response protects the subject from severe COVID-19 disease for at least about six months and / or reduces the frequency of hospitalization compared to an unvaccinated person. An efficient immune response may also reduce viral transmission compared to transmission from an unvaccinated person infected with the virus. An efficient immune response may also be considered to provide some protection against variants resulting from a heterologous immune response.

[0028] Immune System: The term “immune system” is intended to be recognized and understood by those skilled in the art and to refer to, for example, the biological system capable of protecting an organism from infection. When a pathogen succeeds in crossing the physical barriers of an organism and invades it, the innate immune system provides an immediate but nonspecific response. If the pathogen evades this innate response, vertebrates have an adaptive immune system, which is a second layer of defense. Here, the immune system adapts its response during infection to improve its recognition of the pathogen. This improved response is then retained in the form of immunological memory after the pathogen has been eliminated, allowing the adaptive immune system to launch a faster and stronger attack each time the pathogen is encountered. Thus, the immune system includes the innate and adaptive immune systems. Each of these two parts typically contains so-called humoral and cellular components.

[0029] Innate Immune System: The term “innate immune system” (also known as non-specific or non-specific immune system) is intended to be recognized and understood by those skilled in the art and to refer to a system that includes cells and mechanisms that, typically, defend a host from infection by other organisms in a non-specific manner. This means that cells of the innate system may recognize and respond to pathogens in a general manner, but unlike the adaptive immune system, it does not confer persistent or protective immunity to the host. The innate immune system can be activated by ligands such as pattern recognition receptors, e.g., Toll-like receptors, NOD-like receptors, or RIG-I-like receptors.

[0030] Lipidoid compounds: Lipidoid compounds, also called lipidoids, are lipid-like compounds, that is, amphiphilic compounds that have lipid-like physical properties. In the context of this invention, the term lipid is considered to encompass lipidoid compounds.

[0031] Constitutive Cationicity: As used herein, the term “constitutive cationicity” means, as recognized and understood by those skilled in the art, that each compound, or group, or atom, is positively charged at any pH value or hydrogen ion activity in its environment. Typically, the positive charge arises from the presence of a quaternary nitrogen atom. If a compound has multiple such positive charges, it may be referred to as constitutive polycationic.

[0032] RNA sequence: The term "RNA sequence" is recognized and understood by those skilled in the art, and refers, for example, to the specific sequence of its ribonucleotides and their individual order.

[0033] Stabilized RNA: The term "stabilized RNA" refers to RNA that has been modified to be more stable than unmodified RNA, for example, by environmental factors or enzymatic digestion, such as by exonuclease or endonuclease degradation. Preferably, stabilized RNA in the context of the present invention is stabilized in cells, for example, prokaryotic or eukaryotic cells, preferably mammalian cells, for example, human cells. The stabilizing effect may also be exerted outside the cell, for example in a buffer, for preservation of compositions containing stabilized RNA.

[0034] T-cell response: As used herein, the terms “cellular immunity,” “cellular immune response,” or “cellular T-cell response” are intended to be recognized and understood by those skilled in the art, and to refer, for example, to the activation of macrophages, natural killer cells (NKs), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. More generally, cellular immunity is based on the activation of cells in the immune system, but not on antibodies. Typically, a cellular immune response may be characterized, for example, by activating antigen-specific cytotoxic T lymphocytes that can induce apoptosis in specific immune cells, such as cells, e.g., dendritic cells or other cells, by presenting epitopes of foreign antigens on their surfaces.

[0035] UTR: The term “untranslated region” or “UTR” or “UTR element” is intended to be recognized and understood by those skilled in the art and to refer to a portion of a nucleic acid molecule typically located at 5' or 3' of a coding sequence. UTRs are not translated into proteins. UTRs may be portions of nucleic acids, such as DNA or RNA. UTRs may include elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, and the like.

[0036] 3'UTR: The terms “3' untranslated region,” “3'UTR,” or “3'UTR element” are intended to be recognized and understood by those skilled in the art to refer to, for example, a portion of a nucleic acid molecule located 3' (i.e., downstream) of a coding sequence that is not translated into a protein. The 3'UTR may also be a portion of RNA located between a coding sequence and an (optional) poly(A) sequence. The 3'UTR may include elements for controlling gene expression, also called regulatory elements. Such regulatory elements may include, for example, ribosome binding sites, miRNA binding sites, and the like.

[0037] 5'UTR: The terms “5' untranslated region,” “5'UTR,” or “5'UTR element” are intended to be recognized and understood by those skilled in the art and refer to, for example, a portion of a nucleic acid molecule located 5' (i.e., upstream) of a coding sequence that is not translated into a protein. The 5'UTR may also be a portion of RNA located between the coding sequence and (any) 5' cap. The 5'UTR may include elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc.

[0038] Variant (of a sequence): As used herein in the context of nucleic acid sequences, the term “variant” is intended to mean, as recognized and understood by those skilled in the art, a variant of a nucleic acid sequence derived, for example, from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and / or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may be at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identical to the nucleic acid sequence from which the variant is derived. A variant is a functional variant in the sense that it retains at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the functionality of the sequence from which it is induced. In one embodiment, a "variant" of a nucleic acid sequence may have at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% nucleotide identity across at least 10, 20, 30, 50, 75, or 100 nucleotide segments of such nucleic acid sequence.

[0039] When used throughout this specification in the context of proteins or peptides, the term “variant” is intended to mean a protein or peptide variant having an amino acid sequence different from the original sequence, for example, by one or more mutations / substitutions, e.g., one or more substitutions, insertions, and / or deletions of amino acids. For example, in some embodiments, insertions in a protein sequence include insertions of 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids. Preferably, these fragments and / or variants may have the same or equivalent specific antigenic properties (immunogenic variant, antigenic variant). Insertions and substitutions are possible, in particular, at sequence locations that do not cause modification to the three-dimensional structure or affect the binding region. Modification to the three-dimensional structure by insertions or deletions can be readily determined, for example, using CD spectroscopy (circular dichroism spectrum). A "variant" of a protein or peptide may have at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid identity across a parcel of at least 10, 20, 30, 50, 75, 75, 80%, 85%, 90%, 95%, or 98% of the length of such protein or peptide. Preferably, a protein variant may include a functional variant of the protein, which, in the context of the present invention, means that the variant is essentially the same as the protein from which it is derived, or exhibits at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% immunogenicity.

[0040] Brief description of the invention This invention relates to the efficient expression of RNA encoding a spike protein derived from SARS-CoV-2 variants in human cells, and to different SARS-CoV-2 variants, for example, but not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 This is partly based on the finding that antibody responses can be induced in animals that broadly neutralize SARS-CoV-2 strains, including (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, United Kingdom), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia). Furthermore, mixtures of RNAs encoding different SARS-CoV-2 spike protein variants have also been shown to be effective in producing neutralizing antibodies against a wide range of SARS-CoV-2 variants. These findings provide a basis for novel RNA-based coronavirus vaccines.

[0041] The RNA sequences, compositions, or vaccines described herein have at least some of the following advantageous features: • Translation of RNA at the injection / vaccination site (e.g., muscle) • Highly efficient induction of antigen-specific immune responses against the encoded SARS-CoV-2 protein with very low doses and drug regimens. • Suitability of vaccination for infants and / or newborns or the elderly, especially the elderly. • Suitability of composition / vaccine for intramuscular administration • Induction of a specific and functional humoral immune response to SARS-CoV-2 variants. • Induction of a broad functional cellular T cell response to SARS-CoV-2 variants. • Induction of specific B-cell memory for SARS-CoV-2 variants • Induction of functional antibodies capable of effectively neutralizing SARS-CoV-2 viral variants. • Induction of functional antibodies that can effectively neutralize the original SARS-CoV-2 virus. • Induction of mucosal IgA immunity through the induction of mucosal IgA antibodies. • Induction of balanced B cell and T cell responses • Induction of protective immunity against SARS-CoV-2 variants • Early initiation of immune protection against SARS-CoV-2 variants • Longevity of the induced immune response to SARS-CoV-2 variants • No enhancement of SARS-CoV-2 infection due to vaccination or immunopathological effects. • No antibody-dependent enhancement (ADE) caused by RNA-based SARS-CoV-2 vaccines. • No excessive induction of systemic cytokine or chemokine responses after vaccine administration, which can lead to undesirable high reactivity during vaccination. • The vaccine is well-tolerated, has no side effects, and is non-toxic. • Advantageous stability characteristics of RNA-based vaccines • Speed, adaptability, simplicity, and scalability of SARS-CoV-2 variant vaccine production • Advantageous vaccination regimens that require only one or two doses of vaccine for adequate protection • Advantageous vaccination regimens that require only low doses of vaccine for adequate protection • A favorable vaccination regimen that requires only low-dose compositions / vaccines for sufficient protection, enabling combinations of different antigens that yield RNA for multivalent vaccines. Preferably, the ability to boost existing immunity to SARS-CoV-2, which induces a further immune response to SARS-CoV-2 variants. • Induction of different SARS-CoV-2 strain-specific immune responses in subjects exposed to different strains or vaccinated with vaccines against different strains. • Induction of a broad immune response across various SARS-CoV-2 variants

[0042] In a first aspect, the present invention provides RNA encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein has the sequence H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439;V445;G446;L4 55;F456;K458;A475;G476;T478;E484;G485, F486;N487;Y489;F490;Q493;S494;P499;T500;N501;V503 ;G504;Y505;Q506;Y144;A570;P681;T716;S982;D1118;L18;D80;D215;L242;A243;L244;R246;A701;T2 0;P26;D138;R190;H655;T1027;S13;W152;L452;R346;P384;G447;G502;T748;A522;V1176;T859;S247; Y248;L249;T250;P251;G252;G75;T76;D950;E154;G769;S254;Q613;F157;R158;Q957;D253;T95;F888; The RNA includes at least one amino acid substitution, deletion, or insertion at a position corresponding to Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187;W258;T19;V126;H245;S12;A899;G142;E156;K558; and / or Q52, and the RNA includes at least one heterologous untranslated region.In certain embodiments, the RNA is a SARS-CoV-2 variant spike protein (e.g., but not limited to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1. It encodes a SARS-CoV-2 spike protein that includes at least one amino acid substitution, deletion, or insertion at the position of SARS-CoV-2 strains, including 7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0043] In a second embodiment, the present invention provides a composition, preferably an immunogenic composition, comprising at least one RNA of the first embodiment. Preferably, the composition comprises at least one RNA of the first embodiment formulated in a lipid-based carrier, preferably lipid nanoparticles (LNPs). In a preferred embodiment, the second embodiment is a polyvalent composition, for example, with different amino acid coding sequences (e.g., one or more SARS-CoV-2 variant strains, for example, but not limited to C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA. (Including 1_v4 and BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alf The present invention relates to a composition comprising RNA encoding a SARS-CoV-2 spike protein, which contains one or more SARS-CoV-2 strain-derived spike proteins, including A (United Kingdom), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0044] In a third embodiment, the present invention provides a SARS-CoV-2 variant vaccine comprising at least one RNA of the first embodiment or at least one composition of the second embodiment. In a preferred embodiment, the second embodiment relates to a polyvalent SARS-CoV-2 vaccine. In a preferred embodiment, the third embodiment relates to a SARS-CoV-2 variant booster vaccine. SARS-CoV-2 variant booster vaccines include, but are not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa) It may also be for one or more SARS-CoV-2 strains including B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0045] In a fourth aspect, the present invention provides a kit or kit of parts comprising at least one RNA of the first aspect and / or at least one composition of the second aspect and / or at least one SARS-CoV-2 variant vaccine of the third aspect.

[0046] In a fifth aspect, the present invention provides a combination comprising at least two distinct components, the at least two distinct components being an RNA species of the first aspect and / or a composition of the second aspect and / or a SARS-CoV-2 variant vaccine of the third aspect, that is, each component being an RNA species, composition and / or SARS-CoV-2 variant vaccine targeting a different SARS-CoV-2, the two distinct components being, but not limited to, C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.17 6 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1. This may include SARS-CoV-2 variants including 258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0047] Further aspects of the present invention relate to methods for treating or preventing SARS-CoV-2 infection in subjects, as well as first and second medical uses of nucleic acids, compositions, and vaccines. Methods for producing nucleic acids, compositions, or vaccines are also provided.

[0048] Detailed description of the invention This application is filed together with an electrically formatted sequence listing (WIPO Standard ST.25), which is part of the description of this application. The information contained in the sequence listing is incorporated herein by reference in its entirety. Where “sequence number” is referred herein, it refers to the corresponding nucleic acid sequence or amino acid (aa) sequence in the sequence listing having the respective identifier. For many sequences, the sequence listing also provides further detailed information, for example, certain structural features, sequence optimizations, GenBank (NCBI) or GISAID (epi) identifiers, or further detailed information regarding their coding capabilities. In particular, such information is related to the numerical identifiers in the WIPO Standard ST.25 sequence listing. <223> Provided below. Therefore, the numerical identifier <223> The information provided below is expressly included herein in whole and should be understood as an integral part of the description of the fundamental invention.

[0049] RNA suitable for SARS-CoV-2 variant vaccines: In a first aspect, the present invention relates to RNA suitable for SARS-CoV-2 variant vaccines.

[0050] It should be noted that the first aspect of the present invention, i.e., the specific features and embodiments described in the context of the RNA of the present invention, are similarly applicable to the second aspect (composition of the present invention), the third aspect (vaccine of the present invention), the fourth aspect (kit or kit of parts of the present invention), the fifth aspect (combination of the present invention), or further aspects including medical use and treatment methods.

[0051] RNA in the first embodiment forms the basis of an RNA-based composition or vaccine. Generally, protein-based vaccines or live attenuated vaccines are suboptimal for use in developing countries due to their high production costs. Furthermore, protein-based vaccines or live attenuated vaccines require long development times and are not suitable for rapid responses to pandemic virus outbreaks, such as the 2019 / 2020 SARS-CoV-2 outbreak. In contrast, RNA-based vaccines according to the present invention enable very rapid and cost-effective production. Therefore, compared to known vaccines, RNA-based vaccines of the present invention can be produced significantly cheaper and more quickly, which is particularly advantageous for use in developing countries. One further advantage of RNA-based vaccines may be their temperature stability compared to protein or peptide-based vaccines.

[0052] In a particularly preferred embodiment, a first aspect of the present invention relates to RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the RNA comprises at least one heterologous untranslated region (UTR), and the SARS-CoV-2 spike protein comprises at least one amino acid substitution selected from SARS-CoV-2 variants and optionally from stabilizing mutations of SARS-Co-2 strains.

[0053] The term “antigenic peptide or protein derived from SARS-CoV-2 spike protein” means, in this specification, (i) an antigen which is a SARS-CoV-2 spike protein having an amino acid sequence of an antigenic peptide or protein (or fragment thereof) that is identical to that of a SARS-CoV-2 variant protein (or fragment thereof), or (ii) an antigen derived from a SARS-CoV-2 spike protein having an amino acid sequence of an antigenic peptide or protein (or fragment thereof) that is not identical to that of a corresponding SARS-CoV-2 variant protein (or fragment thereof). For example, each SARS-CoV-2 spike protein may contain at least one amino acid substitution, insertion, or deletion selected from the SARS-CoV-2 variant and / or at least one pre-fusion stabilizing mutation.

[0054] The terms “immunogenic fragment” or “immunogenic variant” mean, as used herein, any fragment / variant of the corresponding SARS-CoV-2 antigen that has the ability to induce an immune response in a subject. Preferably, intramuscular or intradermal administration of the RNA of the first embodiment results in the expression of the SARS-CoV-2 spike protein encoded in the subject.

[0055] As used herein, the term “expression” refers to the production of the SARS-CoV-2 spike protein, which is provided by the coding sequence of RNA in the first embodiment. For example, “expression” of RNA refers to the production of a polypeptide of RNA, e.g., SARS-CoV-2 coronavirus, or a protein derived from SARS-CoV-2 coronavirus, via translation into a peptide or protein (e.g., after administration of the RNA to a cell or subject). The terms “expression” and “production” may be used interchangeably herein. Furthermore, the term “expression” preferably relates to the production of a particular peptide or protein upon administration of RNA to a cell or organism.

[0056] In preferred embodiments, the RNA of the present invention is suitable for SARS-CoV-2 variant vaccines.

[0057] The SARS-CoV-2 spike protein is a type I viral fusion protein that exists as a trimer on the viral surface, along with its respective monomers, consisting of a head (S1) and a stem (S2). Each precursor S polypeptide forms a homotrimer, undergoes glycosylation in the Golgi apparatus and processing to remove signal peptides, and is cleaved by cellular proteolytic enzymes to produce separate S1 and S2 polypeptide chains, which remain associated as the S1 / S2 promoter within the homotrimer, thus forming a heterodimeric trimer. The S1 domain of the spike glycoprotein contains a receptor-binding domain (RBD) that engages with the angiotensin-converting enzyme 2 receptor (most likely) and mediates viral fusion to host cells, an N-terminal domain that can first contact target cells, and two subdomains, all of which are sensitive to neutralizing antibodies. The S2 domain consists of a six-helix bundle fusion core involved in membrane fusion with the host endosomal membrane and is also a target of neutralization. The S2 subunit further comprises two 7-amino acid repeat sequences (HR1 and HR2), as well as a central helix, a transmembrane domain, and a cytosolic terminal domain, which are typical of fusion glycoproteins.

[0058] In the context of the present invention, any spike protein selected from or derived from SARS-CoV-2 variants and comprising at least one amino acid substitution, deletion, or insertion compared to SEQ ID NO: 1 may be used and may be appropriately encoded by the coding sequence or RNA of the first embodiment. Furthermore, it is within the fundamental scope of the present invention that at least one antigenic peptide or protein comprises or may consist of a synthetically produced or artificial SARS-CoV-2 spike protein. The terms “synthetically produced” SARS-CoV-2 spike protein, “artificial SARS-CoV-2 spike protein,” or “recombinant” SARS-CoV-2 spike protein refer to proteins that do not occur naturally. Therefore, “artificial SARS-CoV-2 spike protein” or “synthetically produced SARS-CoV-2 spike protein” or the term “recombinant” SARS-CoV-2 spike protein may differ from, for example, naturally occurring SARS-CoV-2 spike protein by at least one amino acid (e.g., containing one or more heterologous / introduced amino acids compared to naturally occurring SARS-CoV-2 spike protein), and / or contain further heterologous peptides or protein elements, and / or be elongated or cleaved at the N-terminus or C-terminus.

[0059] The preferred antigenic peptide or protein sequences induced by the RNA of the present invention are described in detail below.

[0060] When referring to amino acid (aa) residues and their positions in the SARS-CoV-2 spike protein (S), it should be noted that any numbering used herein, unless otherwise specified, refers to the position of each amino acid residue in the corresponding spike protein (S) of the original SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1). Throughout this disclosure, each amino acid position is shown exemplarily for the spike protein (S) of the original SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1).

[0061] As used herein, protein annotations relate to Sequence ID No. 1 as the reference protein. The full-length spike protein (S) of the original SARS-CoV-2 coronavirus reference protein has 1273 amino acid residues and includes the following elements: - Secretory signal peptide: Amino acid positions aa1~aa15 (see SEQ ID NO: 28) - Spike protein fragment S1: Amino acid positions aa1~aa681 (see SEQ ID NO: 27) - S1-N-terminal domain (S1-NTD): Amino acid positions aa13~aa303 (see SEQ ID NO: 26992) - Receptor-binding domain (RBD): Amino acid positions aa319~aa541 (see SEQ ID NO: 13243) - Important neutralizing domain (CND): Amino acid positions aa329~aa529 (see SEQ ID NO: 13310) - Spike protein fragment S2: Amino acid positions aa682~aa1273 (see SEQ ID NO: 30) - Transmembrane domain (TM): Amino acid positions aa1212~aa1273 (see SEQ ID NO: 49) - Transmembrane domain (TMflex): Amino acid positions aa1148~aa1273 (see SEQ ID NO: 13176) - Furin cleavage site region (S1 / S2): Amino acid positions aa681~aa685 (see SEQ ID NO: 26994)

[0062] It should be noted that variations in amino acid levels occur spontaneously between spike proteins derived from different SARS-CoV-2 isolates or variants. In the context of the present invention, such amino acid variations can be applied to antigenic peptides or proteins derived from the spike proteins described herein. Preferably, the amino acid variations or mutations are selected in such a way that 1) induce an immune response to the SARS-CoV-2 viral variant in which the substitution / mutation was induced, and / or 2) produce an antigenic peptide or protein (e.g., an antigenic peptide or protein derived from the spike protein in its pre-fusion form) that is desirable for inducing an immune response.

[0063] Therefore, in a particularly preferred embodiment, the RNA of the present invention comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein, or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion, or insertion selected from SARS-CoV-2 variants.

[0064] In that context, the term “at least one amino acid substitution, deletion, or insertion selected from a SARS-CoV-2 variant” means, as herein, at least one amino acid position in a SARS-CoV-2 spike protein (or a fragment thereof) that is different from the original SARS-CoV-2 spike protein (as per the reference strain of SEQ ID NO: 1).

[0065] In a preferred embodiment, the SARS-CoV-2 variants include the following SARS-CoV-2 lineages: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1 Selected from or derived from .351 (Beta, South Africa), B.1.1.7 (Alpha, United Kingdom), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0066] In a particularly preferred embodiment, the SARS-CoV-2 variant is selected from or derived from the following SARS-CoV-2 lineages: B.1.351 (South Africa), P.1 (Brazil), B.1.617.1 (India), B.1.617.2 (India), B.1.617.3 (India), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).

[0067] Accordingly, each spike protein provided herein and intended as a suitable antigen in the context of the present invention may have one or more of the following amino acid variations or mutations (amino acid positions according to reference sequence number 1) provided in List 1. The variations or mutations provided below may be derived from newly emerging SARS-CoV-2 virus variants and incorporated into the spike proteins encoded by the RNA of the present invention.

[0068] List 1A: Amino acid positions of substitutions, deletions, and / or insertions H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439;V445;G446;L455;F456;K458;A475;G476;T478;E484;G 485;F486;N487;Y489;F490;Q493;S494;P499;T500;N501;V503;G504;Y505;Q506;Y144;A570;P681;T716;S982;D 1118;L18;D80;D215;L242;A243;L244;R246;A701;T20;P26;D138;R190;H655;T1027;S13;W152;L452;R346;P38 4;G447;G502;T748;A522;V1176;T859;S247;Y248;L249;T250;P251;G252;G75;T76;D950;E154;G769;S254;Q613 ;F157;R158;Q957;D253;T95;F888;Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187;W 258;T19;V126;H245;S12;A899;G142;E156;K558;G339;P9;C136;Y449;L24;P25;P26;A27;V213;S371;T376;D405 ;A701;I210;D936;S939;R357;R682;R683;A684;R685;V143;Y144;Y145;N211;L212;R214;E241;G339;S371;S373;S375;N440;G496;Q498;Y505;T547;D614;N679;P681;N764;D796;N856;Q954;N969;L981 or Q52 (for the sequence of sequence number 1).

[0069] List 1B: Amino acid substitution, deletion, or insertion H69del;V70del;A222V;Y453F;S477N;I692V;R403K;K417N;N437S;N439K;V445A;V445I;V445F;G446V;G446S;G446A;L455F;F456L;K458N;A475V;G476S;G476A;S477I;S477R;S477G;S477T;T478I;T478K;T478R;T478A;E484Q;E484K;E484A;E484D;G485R;G485S;F486L;N487I;Y489H;F490S;F490L;Q493L;Q493K;S494P;S494L;P499L;T500I;N501Y;N501T;N501S;V503F;V503I;G504D;Y505W;Q506K;Q506H;Y144del;A570D;P681H;T716I;S982A;D1118H;L18F;D80A;D215G;L242del;A243del;L244del;L242del;A243del;L244del;R246I;A701V;T20N;P26S;D138Y;R190S;H655Y;T1027I;S13I;W152C;L452R;R346T;P384L;L452M;F456A;F456K;F456V;E484P;K417T;G447V;L452Q;A475S;F486I;F490Y;Q493R;S494A;P499H;P499S;G502V;T748K;A522S;V1176F;T859N;S247del;Y248del;L249del;T250del;P251del;G252del;R246del;S247del;Y248del;L249del;T250del;P251del;G252del;G75V;T76I;G75V;T76I;D950N;P681R;E154K;G769V;S254F;Q613H;F157L;F157del;R158del;Q957R;D253G;T95I;F888L;Q677H;A67V;Q414K;N450K;V483A;G669S;T732A;Q949R;Q1071H;E1092K;H1101Y;N1187D;W258L;V70F;T19R;T19I;Y144T;Y145S;ins145N;R346K;R346S;V126A;H245Y;ins214TDR;S12F;W152R;A899S;G142D;E156G;K558N;P9L;C136F;Y449H;L24del;P25del;P26del;A27S;V213G;S371F;T376A;D405N;D 253N;Y144S;I210T;D936N;S939F;W152L;T20I;R357K;D796H;Y145H;R682del;R683del;A684del;R685del;A701V;V1 43del;Y144del;Y145del;Y145N;N211del;L212del;L212I;ins214EPE;E241del;G339D;S371L;S373P;S375F;N440K;G496S;Q498R;Y505H;T547K;D614G;N679K;P681H;N764K;D796Y;N856K;Q954H;N969K;L981F or Q52R (for the sequence of sequence number 1).

[0070] In a preferred embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein is H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439; relative to the sequence of SEQ ID NO: 1 V445;G446;L455;F456;K458;A475;G476;T478;E484;G485;F486;N487;Y489;F490;Q493;S494;P499;T500 ;N501;V503;G504;Y505;Q506;Y144;A570;P681;T716;S982;D1118;L18;D80;D215;L242;A243;L244;R246 ;A701;T20;P26;D138;R190;H655;T1027;S13;W152;L452;R346;P384;G447;G502;T748;A522;V1176;T859 ;S247;Y248;L249;T250;P251;G252;G75;T76;D950;E154;G769;S254;Q613;F157;R158;Q957;D253;T95;F The RNA includes at least one amino acid substitution, deletion, or insertion at a position corresponding to 888;Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187;W258;T19;V126;H245;S12;A899;G142;E156;K558; and / or Q52, and the RNA includes at least one heterologous untranslated region. In certain embodiments, the RNA does not include a 3'UTR containing the sequence of SEQ ID NO: 268. In certain embodiments, the RNA includes a 3'UTR containing the sequence of SEQ ID NO: 268.

[0071] In a particularly preferred embodiment, the SARS-CoV-2 spike protein is H69del;V70del;A222V;Y453F;S477N;I692V;R403K;K417N;N437S;N439K;V445A;V445I;V445F;G446V;G446S;G446A;L455F;F456L;K458N;A475V;G476S;G476A;S477I;S477R;S477G;S477T;T478I;T478K;T478R;T478A;E484Q;E484K;E484A;E484 D;G485R;G485S;F486L;N487I;Y489H;F490S;F490L;Q493L;Q493K;S494P; S494L;P499L;T500I;N501Y;N501T;N501S;V503F;V503I;G504D;Y505W;Q50 6K;Q506H;Y144del;A570D;P681H;T716I;S982A;D1118H;L18F;D80A;D215G;L242del;A243del;L244del;L242del;A243del;L244del;R246I;A701V;T 20N;P26S;D138Y;R190S;H655Y;T1027I;S13I;W152C;L452R;R346T;P384L ;L452M;F456A;F456K;F456V;E484P;K417T;G447V;L452Q;A475S;F486I;F4 90Y;Q493R;S494A;P499H;P499S;G502V;T748K;A522S;V1176F;T859N;S247del;Y248del;L249del;T250del;P251del;G252del;R246del;S247del;Y2 48del;L249del;T250del;P251del;G252del;G75V;T76I;G75V;T76I;D950N;P681R;E154K;G769V;S254F;Q613H;F157L;F157del;R158del;Q957R;D25 3G;T95I;F888L;Q677H;A67V;Q414K;N450K;V483A;G669S;T732A;Q949R;Q 1071H;E1092K;H1101Y;N1187D;W258L;V70F;T19R;Y144T;Y145S;ins145N;Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to R346K;R346S;V126A;H245Y;ins214TDR;S12F;W152R;A899S;G142D;E156G;K558N; and / or Q52R.

[0072] In a particular embodiment, an RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion at positions corresponding to H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439;V445;G446;L455;F456;K458;A475;G476;T478;E484;G485,F486;N487;Y489;F490;Q493;S494;P499;T500;N501;V503;G504;Y505; and / or Q506 with respect to the sequence of Sequence ID No. 1. Therefore, in some embodiments, the SARS-CoV-2 spike protein is H69del;V70del;A222V;Y453F;S477N;I692V;R403K;K417N;N437S;N439K;V445A;V445I;V445F;G446V;G446S;G446A;L455F;F456L;K458N;A475V;G476S;G476A;S477I;S477R;S477G;S477T;T478I; Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to T478K;T478R;T478A;E484Q;E484K;E484A;E484D;G485R;G485S;F486L;N487I;Y489H;F490S;F490L;Q493L;Q493K;S494P;S494L;P499L;T500I;N501Y;N501T;N501S;V503F;V503I;G504D;Y505W;Q506K; and / or Q506H.

[0073] In a further embodiment, RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein is H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439;V445;G446;L455;F456;K458;A475;G476;T478;E484;G485;F486;N487;Y The molecule contains at least one amino acid substitution, deletion, or insertion at a position corresponding to 489;F490;Q493;S494;P499;T500;N501;V503;G504;Y505;Q506;Y144;A570;P681;T716;S982;D1118;L18;D80;D215;L242;A243;L244;R246;A701;T20;P26;D138;R190;H655;T1027;S13;W152;L452;R346;P384;G447;G502;T748;A522; or V1176. Therefore, in some embodiments, the SARS-CoV-2 spike protein is H69del;V70del;A222V;Y453F;S477N;I692V;R403K;K417N;N437S;N439K;V445A;V445I;V445F;G446V;G446S;G446A;L455F;F456L;K458N;A475V;G476S;G476A;S477I;S477R;S477G;S477T;T478I;T478K;T478R;T478A;E484Q;E484K;E484A;E484D;G485R; G485S;F486L;N487I;Y489H;F490S;F490L;Q493L;Q493K;S494P;S494L;P499L ;T500I;N501Y;N501T;N501S;V503F;V503I;G504D;Y505W;Q506K;Q506H;Y144 del;A570D;P681H;T716I;S982A;D1118H;L18F;D80A;D215G;L242del;A243del;L244del;L242del;A243del;L244del;R246I;A701V;T20N;P26S;D138Y;R190 Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to S;H655Y;T1027I;S13I;W152C;L452R;R346T;P384L;L452M;F456A;F456K;F456V;E484P;K417T;G447V;L452Q;A475S;F486I;F490Y;Q493R;S494A;P499H;P499S;G502V;T748K;A522S; and / or V1176F.

[0074] In a further preferred embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein is T859;R246;S247;Y248;L249;T250;P251;G252;G75;T76;D950;E154;G It includes at least one amino acid substitution, deletion, or insertion at the position corresponding to 769;S254;Q613;F157;Q957;D253;T95;F888;Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187;F157;R158;W258;T19;H245;S12;A899;G142;E156;K558 and / or Q52. Therefore, in some embodiments, the SARS-CoV-2 spike protein is T859N;S247del;Y248del;L249del;T250del;P251del;G252del;R246del;S247del;Y248del;L249del;T250del;P251del;G252del;G75V;T76I;G75V;T76I;D950N;P681R;E154K;G769V;S254F;Q613H;F157L;Q957R;D253G;T95I;F888 Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to L;Q677H;A67V;Q414K;N450K;V483A;G669S;T732A;Q949R;Q1071H;E1092K;H1101Y;N1187D;F157del;R158del;W258L;V70F;T19R;Y144T;Y145S;ins145N;R346K;R346S;V126A;H245Y;ins214TDR;S12F;W152R;A899S;G142D;E156G;K558N and / or Q52R.

[0075] In further embodiments, RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion at positions corresponding to D614;H49;V367;P1263;V483;S939;S943;L5;L8;S940;C1254;Q239;M153;V1040;A845;Y145;A831; and / or M1229 relative to the sequence of SEQ ID NO: 1. Therefore, in some embodiments, the SARS-CoV-2 spike protein includes at least one amino acid substitution, deletion, or insertion at a position corresponding to D614G;H49Y;V367F;P1263L;V483A;S939F;S943P;L5F;L8V;S940F;C1254F;Q239K;M153T;V1040F;A845S;Y145H;A831V; and / or M1229I relative to the sequence of Sequence ID No. 1.

[0076] In further embodiments, RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein is H69;V70;A222;Y453;S477;I692;R403;K417;N437;N439;V445;G446;L455;F456;K458;A475;G476;T478;E484;G485;F486;N487;Y489;F490;Q493;S494;P499;T500;N501;V503;G504;Y505;Q506;Y144;A570;P681;T716;S982 ;D1118;L18;D80;D215;L242;A243;L244;R246;A701;T20;P26;D138;R190;H655;T1027;S13;W 152;L452;R346;P384;G447;G502;T748;A522;V1176;T859;S247;Y248;L249;T250;P251;G252; It contains at least one amino acid substitution or deletion at the position corresponding to G75;T76;D950;E154;G769;S254;Q613;F157;Q957;D253;T95;F888;Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187 and / or Q52. Therefore, in some embodiments, the SARS-CoV-2 spike protein is H69del;V70del;A222V;Y453F;S477N;I692V;R403K;K417N;N437S;N439K;V445A;V445I;V445F;G446V;G446S;G446A;L455F;F456L;K458N;A475V;G476S;G476A;S477I;S477 R;S477G;S477T;T478I;T478K;T478R;T478A;E484Q;E484K;E484A;E484D;G485R;G485S;F486L;N487I;Y489H ;F490S;F490L;Q493L;Q493K;S494P;S494L;P499L;T500I;N501Y;N501T;N501S;V503F;V503I;G504D;Y505W;Q506K;Q506H;Y144del;A570D;P681H;T716I;S982A;D1118H;L18F;D80A;D215G;L242del;A243del;L244del ;L242del;A243del;L244del;R246I;A701V;T20N;P26S;D138Y;R190S;H655Y;T1027I;S13I;W152C;L452R;R 346T;P384L;L452M;F456A;F456K;F456V;E484P;K417T;G447V;L452Q;A475S;F486I;F490Y;Q493R;S494A;P 499H;P499S;G502V;T748K;A522S;V1176F;T859N;S247del;Y248del;L249del;T250del;P251del;G252del; R246del;S247del;Y248del;L249del;T250del;P251del;G252del;G75V;T76I;G75V;T76I;D950N;P681R;E1 54K;G769V;S254F;Q613H;F157L;F157del;R158del;Q957R;D253G;T95I;F888L;Q677H;A67V;Q414K;N450K; V483A;G669S;T732A;Q949R;Q1071H;E1092K;H1101Y;N1187D;W258L;V70F;T19R;Y144T;Y145S;R346K;R346S;V126A;H245Y;S12F;W152R;A899S;G142D;E156G;K558N; and / or containing at least one amino acid substitution or deletion at the position corresponding to Q52R.

[0077] In a preferred embodiment, an RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein is T19I;L24del;P25del;P26del;A27S;A67V;H69del;V70del;T95I;G142D;V143del;Y144del;Y145del;N211del;L212I;V Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to 213G;ins214EPE;G339D;S371L;S371F;S373P;S375F;T376A;D405N;K417N;N440K;G446S;S477N;T478K;E484A;Q493R;G496S;Q498R;N501Y;Y505H;T547K;D614G;H655Y;N679K;P681H;A701V;N764K;D796Y;N856K;Q954H;N969K;L981F. Therefore, in some embodiments, the SARS-CoV-2 spike protein is T19I;L24del;P25del;P26del;A27S;A67V;H69del;V70del;T95I;G142D;V143del;Y144del;Y145del;N211del;L212I;V213G;ins214EPE;G339D;S371L;S371F;S373P;S Includes at least one amino acid substitution, deletion, or insertion at the position corresponding to 375F;T376A;D405N;K417N;N440K;G446S;S477N;T478K;E484A;Q493R;G496S;Q498R;N501Y;Y505H;T547K;D614G;H655Y;N679K;P681H;A701V;N764K;D796Y;N856K;Q954H;N969K;L981F.In a particular embodiment, the SARS-CoV-2 spike protein is 90% identical to the amino acid sequence of SEQ ID NO: 10, and is T19I;L24del;P25del;P26del;A27S;A67V;H69del;V70del;T95I;G142D;V143del;Y144del;Y145del;N211del;L212I;V213G;ins214EPE;G339D;S371L;S371F;S373P;S375F;T376A;D405N;K417N;N440K;G446S;S477N;T478K It contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions, deletions, or insertions selected from the group consisting of ;E484A;Q493R;G496S;Q498R;N501Y;Y505H;T547K;D614G;H655Y;N679K;P681H;A701V;N764K;D796Y;N856K;Q954H;N969K;L981F.

[0078] In further embodiments, RNA is provided that includes at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein the SARS-CoV-2 spike protein is A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, in It includes at least one amino acid substitution, insertion, or deletion corresponding to s214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0079] In preferred embodiments, the SARS-CoV-2 spike protein includes amino acid substitutions at positions located in the RBD domain (amino acid positions aa319-aa541; amino acid positions according to reference SEQ ID NO: 1) or the CND domain (amino acid positions aa329-aa529; amino acid positions according to reference SEQ ID NO: 1). Without being constrained by theory, amino acid substitutions or mutations in the CND domain may help newly emerging SARS-CoV-2 variants evade antibody detection of certain types of antibodies induced in subjects vaccinated with first-generation vaccines (designed against the original SARS-CoV-2 strain) or in subjects after infection with the original SARS-CoV-2 strain.

[0080] Accordingly, in preferred embodiments, a first aspect of the present invention relates to RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from the SARS-CoV-2 spike protein or its immunogenic fragment or immunogenic variant, wherein the RNA comprises at least one heterologous untranslated region (UTR), and the SARS-CoV-2 spike protein comprises at least one amino acid substitution at a position located in the RBD domain (amino acid positions aa319~aa541; amino acid position according to reference sequence number 1) or the CND domain (amino acid positions aa329~aa529, amino acid position according to reference sequence number 1).

[0081] In a particular preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution, insertion, or deletion at at least one of the following positions: R346;V367, P384;R403;K417;N437;N439;V445;G446;G447;N450;L452;Y453;L455;F456;A475;G476;S477;T478;E484;G485;F486;N487;Y489;F490;Q493;S494;P499;T500;N501;G502;V503;G504;Y505;Q506;A522 (amino acid position according to reference number 1).

[0082] Accordingly, in certain preferred embodiments, a first aspect of the present invention relates to RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from the SARS-CoV-2 spike protein or its immunogenic fragment or immunogenic variant, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution at a position selected from K417;L452;T478;E484;N501 and / or P681 (amino acid positions according to reference sequence number 1), and the RNA comprises at least one heterologous untranslated region (UTR).

[0083] Without wanting to be constrained by theory, an amino acid substitution at position E484 may help the SARS-CoV-2 viral variant evade antibody detection by several types of antibodies, either induced in subjects vaccinated with first-generation vaccines (designed against the original SARS-CoV-2 strain) or in subjects after infection with the original SARS-CoV-2 strain. A mutation / substitution at N501 occurs near the peak of the coronavirus spike, where it may alter the shape of the protein and help evade several types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2 E484 variants throughout the present invention and includes, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.617 (India), P.1 (Brazil), or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and BA.1_v5).

[0084] Therefore, in some embodiments, it may be advantageous that the RNA of the present invention provides a SARS-CoV-2 spike protein comprising a substitution at position E484 to enable the induction of an efficient immune response against the SARS-CoV-2 E484 variant.

[0085] In preferred embodiments, the SARS-CoV-2 spike protein includes an amino acid substitution at position E484, where amino acid E484 is substituted with K, P, Q, A, or D (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or induced from the SARS-CoV-2 spike protein include the E484K, E484P, E484Q, E484A, and E484D amino acid substitutions.

[0086] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position E484, where amino acid E484 is substituted with K or Q (amino acid position according to reference number 1). Thus, antigenic peptides or proteins selected from or derived from the SARS-CoV-2 spike protein include the E484K or E484Q amino acid substitution. In a particular preferred embodiment, the SARS-CoV-2 spike protein includes the E484K amino acid substitution.

[0087] In a preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position N501, where amino acid N501 is substituted with a different amino acid (amino acid position according to reference SEQ ID NO: 1).

[0088] Without being bound by theory, an amino acid substitution at position N501 may help the SARS-CoV-2 viral variant evade antibody detection by certain types of antibodies, either induced in subjects vaccinated with first-generation vaccines (designed against the original SARS-CoV-2 strain) or in subjects after infection with the original SARS-CoV-2 strain. The mutation / substitution at N501 occurs near the peak of the coronavirus spike, where it may alter the shape of the protein and help evade certain types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2 N501 variant throughout the present invention and includes, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.1.7 (United Kingdom), P.1 (Brazil), or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and BA.1_v5).

[0089] Therefore, it may be advantageous that the RNA of the present invention provides a SARS-CoV-2 spike protein comprising a substitution at position N501 to enable the induction of an efficient immune response against the SARS-CoV-2 N501 variant.

[0090] In preferred embodiments, the SARS-CoV-2 spike protein contains an amino acid substitution at position N501, where amino acid N501 is substituted with Y, T, or S (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or induced from the SARS-CoV-2 spike protein contain the N501Y, N501T, and N501S amino acid substitutions.

[0091] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position N501, where amino acid N501 is substituted with Y (amino acid position according to reference sequence number 1). Therefore, antigenic peptides or proteins selected from or derived from the SARS-CoV-2 spike protein include the N501Y amino acid substitution.

[0092] In a preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position K417, where amino acid K417 is substituted with a different amino acid (amino acid position according to reference number 1).

[0093] Without wanting to be constrained by theory, an amino acid substitution at position K417 may help the SARS-CoV-2 viral variant evade antibody detection by several types of antibodies, either induced in subjects vaccinated with a vaccine designed against the original SARS-CoV-2 strain using SEQ ID NO: 1, or in subjects after infection with the original SARS-CoV-2 strain containing SEQ ID NO: 1. The mutation / substitution at K417 occurs near the peak of the coronavirus spike, where it may alter the shape of the protein and help evade several types of coronavirus antibodies. Such SARS-CoV-2 strains are referred to as SARS-CoV-2 K417 variants throughout this invention and include, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.1.7 (United Kingdom), P.1 (Brazil), AY.1 / AY.2, or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and BA.1_v5).

[0094] Therefore, it may be advantageous that the RNA of the present invention provides a SARS-CoV-2 spike protein comprising a substitution at position K417 to enable the induction of an efficient immune response against the SARS-CoV-2 K417 variant.

[0095] In preferred embodiments, the SARS-CoV-2 spike protein includes an amino acid substitution at position K417, where amino acid N501 is substituted with S, T, Q, or N (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or induced from the SARS-CoV-2 spike protein include the K417S, K417T, K417Q, or K417N amino acid substitution.

[0096] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position N501, where amino acid K417 is substituted with T or N (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or derived from the SARS-CoV-2 spike protein include the K417T or K417N amino acid substitution. In a particular preferred embodiment, antigenic peptides or proteins selected from or derived from the SARS-CoV-2 spike protein include the K417N amino acid substitution.

[0097] In a preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position L452, where amino acid L452 is substituted with a different amino acid (amino acid position according to reference sequence number 1).

[0098] Without being constrained by theory, an amino acid substitution at position L452 may help SARS-CoV-2 viral variants evade antibody detection by certain types of antibodies, either induced in subjects vaccinated with a vaccine designed against the original SARS-CoV-2 strain based on SEQ ID NO: 1, or in subjects after infection with the original SARS-CoV-2 strain having SEQ ID NO: 1. The mutation / substitution at L452 occurs near the peak of the coronavirus spike, where it may alter the shape of the protein and help evade certain types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2 L452 variant throughout this invention and includes, for example, SARS-CoV-2 B.1.617.1 (India), SARS-CoV-2 B.1.617.2 (India), or SARS-CoV-2 B.1.617.3 (India).

[0099] Therefore, it may be advantageous that the RNA of the present invention provides a SARS-CoV-2 spike protein comprising a substitution at position L452 to enable the induction of an efficient immune response against the SARS-CoV-2 L452 variant.

[0100] In preferred embodiments, the SARS-CoV-2 spike protein includes an amino acid substitution at position L452, where amino acid L452 is substituted with R or Q (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or induced from the SARS-CoV-2 spike protein include the L452R or L452Q amino acid substitution.

[0101] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position L452, where amino acid L452 is substituted with R (amino acid position according to reference sequence number 1). Therefore, antigenic peptides or proteins selected from or derived from the SARS-CoV-2 spike protein include the L452R amino acid substitution.

[0102] In a preferred embodiment, the SARS-CoV-2 spike protein contains amino acid substitutions at the furin cleavage site (amino acid positions aa681-685; amino acid positions according to reference SEQ ID NO: 1). This sequence segment (PRRAR in SEQ ID NO: 1) is thought to act as a recognition site for furin cleavage. Without being bound by theory, amino acid substitutions or mutations at the furin cleavage site may help newly emerging SARS-CoV-2 variants have increased membrane fusion and therefore cause increased infectivity.

[0103] In a preferred embodiment, the SARS-CoV-2 spike protein contains an amino acid substitution at position P681 in the furin cleavage site. Preferably, amino acid P681 is substituted with a different amino acid (amino acid position according to reference number 1), preferably an amino acid that improves furin cleavage. Such SARS-CoV-2 strains are referred to as SARS-CoV-2 P681 variants throughout this invention and include, for example, SARS-CoV-2 B.1.617.1 (India), SARS-CoV-2 B.1.617.2 (India), or SARS-CoV-2 B.1.617.3 (India), SARS-CoV-2 B.1.1.7 (United Kingdom), SARS-CoV-2 A.23.1 (Uganda), or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and BA.1_v5).

[0104] Therefore, it may be advantageous that the RNA of the present invention provides a SARS-CoV-2 spike protein comprising a substitution at position P681 to enable the induction of an efficient immune response against the SARS-CoV-2 P681 variant.

[0105] In preferred embodiments, the SARS-CoV-2 spike protein includes an amino acid substitution at position P681, where amino acid P681 is substituted with R or H (amino acid position according to reference number 1). Therefore, antigenic peptides or proteins selected from or induced from the SARS-CoV-2 spike protein include the P681R or P681H amino acid substitution.

[0106] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes an amino acid substitution at position P681, where amino acid P681 is substituted with R (amino acid position according to reference sequence number 1). Therefore, an antigenic peptide or protein selected from or induced from the SARS-CoV-2 spike protein includes the P681R amino acid substitution.

[0107] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position P681 as defined herein, preferably P681R (amino acid positions according to reference SEQ ID NO: 1).

[0108] In another particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position P681 as defined herein, preferably P681R (amino acid position according to reference SEQ ID NO: 1). In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, an amino acid substitution at position P681 as defined herein, preferably P681R, and at position D614 as defined herein, preferably D614G (amino acid position according to reference SEQ ID NO: 1).

[0109] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention includes an amino acid substitution at position N501 as defined herein, preferably N501Y, and an amino acid substitution at position E484 as defined herein, preferably E484K (amino acid position according to reference SEQ ID NO: 1).

[0110] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention includes an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position E484 as defined herein, preferably E484Q (amino acid position according to reference SEQ ID NO: 1).

[0111] In preferred embodiments, the SARS-CoV-2 spike protein includes, in addition to the substitutions defined above (at positions E484, N501, L452 and optionally P681), at least one further amino acid substitution, insertion, or deletion selected from List 1A or List 1B, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0112] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention includes an amino acid substitution or deletion at position H69 as defined herein, preferably H69del, and an amino acid substitution or deletion at position V70 as defined herein, preferably V70del (amino acid positions according to reference SEQ ID NO: 1). In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention includes deletions at both H69 and V70.

[0113] In preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is found in the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), It comprises at least one further amino acid substitution or deletion selected from B.1.351 (beta, South Africa), B.1.1.7 (alpha, UK), P.1 (gamma, Brazil), B.1.427 / B.1.429 (epsilon, California, USA), B.1.525 (eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (copper, India), B.1.617.2 (delta, India), P.2 (zeta, Brazil), C37.1 (lambda, Peru), P.3 (theta, Philippines), and / or B.1.621 (mu, Colombia).

[0114] In a preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is (relative to SEQ ID NO: 1): ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N , T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y , N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F(SA, BA.1_v1); ·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145d el、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、K417N、N440K、G4 46S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H 655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F(SA、BA.1_v0); ·K986P、V987P、A67V、T95I、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、D796Y、N856K、Q954H、N969K、L981F(SA、B.1.1.529); K986P、V987P、T19I、L24del、P25del、P26del、A27S、G142D、V213G、G339D、S371F、S373P、S375F、T376A、D405N、 S477N、T478K、E484A、Q493R、Q498R、N501Y、Y505H、D614 G、H655Y、N679K、P681H、D796Y、Q954H、N969K(SA、BA.2); ·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y1 45del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、N440K、S4 77N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F(SA, BA.1_v2); · K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v3); · K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v4); · K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v5); · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; (SA; B.1.351) · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V; (SA; B.1.351) E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I; (Brazil; P1) E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F; (Brazil P1) L452R, P681R, and D614G; (B.1.617.1; India) L452R, E484Q, P681R, E154K, D614G, and Q1071H; (B.1.617.2; India) L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N; (B.1.617.2; India) T19R, L452R, E484Q, D614G, P681R and D950N; (B.1.617.3; India) G75V, T76I, S247del, Y248del, L249del, T250del, P251del, G252del, D253del, L452Q, F490S, D614G, and T859N; (C.37.1; Peru) T95I, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N; (B.1.1.621) T95I, Y144T, Y145S, ins145N, R346K, E484K, N501Y, D614G, P681H, and D950N; (B.1.1.621) H69del, V70del, Y144del, E484K, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H; (B.1.1.7 - E484K) S13I, W152C, L452R, and D614G; (B.1.429) L452R; and D614G; (B.1.429) ·H69del;V70del;N439K;D614G;(B.1.258) ·T95I;E484K;D614G;and A701V;(B.1.526) L5F, T95I, D253G, E484K, D614G, and A701V; (B.1.526) L5F, T95I, D253G, S477N, D614G, and Q957R; (B.1.526) · F157L; V367F; Q613H; and P681R (A.23.1) ·S254F;D614G;P681R;and G769V(A.23.1) ·T478K;D614G;P681H;and T732A(B.1.1.519;Mexico) P26S, H69del, V70del, V126A, Y144del, L242del, A243del, L244del, H245Y, S477N, E484K, D614G, P681H, T1027I and D1118H; (B.1.620; Africa) ins214TDR, Q414K, N450K, D614G, and T716I; (B.1.214.2) S12F, H69del, V70del, W152R, R346S, L452R, D614G, Q677H and A899S; (C.36.3; Thailand) E484K, D614G, and V1176F; (P2) ·Q52R;A67V;H69del;V70del;F157del;R158del;E484K;D614G;Q677H and F888L;(B.1.525) ·Q52R;A67V;H69del;V70del;Y144del;E484K;D614G;Q677H and F888L;(B.1.525) ·A67V;H69del;V70del;Y144del;E484K;D614G;Q677H and F888L;(B.1.525) ·T19R;T95I;G142D;E156G;F157del;R158del;W258L;K417N;L452R;T478K;K558N;D614G;P681R;and D950N;(AY.1) ·T19R;V70F;G142D;E156G;F157del;R158del;A222V;K417N;L452R;T478K;D614G;P681R;and D950N;(AY.2) ·T19R;T95I;F157del;R158del;W258L;K417N;L452R;T478K;D614G;P681R;and D950N;or (AY.1) ·T19R;V70F;F157del;R158del;A222V;K417N;L452R;T478K;D614G;P681R;and D950N;(AY.2) H69del, V70del, and D614G; • D614G and M1229I; • A222V and D614G; • S477N and D614G; • N439K and D614G; H69del; V70del; Y453F; D614G and I692I; • Y453F and D614G; D614G and I692V; H69del; V70del; A222V; Y453F; D614G and I692I; • N501Y and D614G; ·K417N;E484K;N501Y and D614G;or • E484K and D614G Includes amino acid substitutions or deletions selected from the following.

[0115] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention has the following amino acid substitutions or deletions (relative to SEQ ID NO: 1): · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V; · E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I; · E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F; L452R, P681R, and D614G; L452R, E484Q, P681R, E154K, D614G, and Q1071H; or L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N Includes.

[0116] In a more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention has the following amino acid substitutions or deletions (relative to SEQ ID NO: 1): ·E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V Includes.

[0117] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention has the following amino acid substitutions or deletions (relative to SEQ ID NO: 1): L452R, P681R, and D614G; L452R, E484Q, P681R, E154K, D614G, and Q1071H; ·L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N; or T19R, L452R, E484Q, D614G, P681R and D950N Includes.

[0118] In a more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is sequence numbers 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959~22964, 27087~27109, 28540~28588, 2891 It comprises or consists of at least one of the following amino acid sequences, which are identical to any one of 7-28920, or are identical by at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or immunogenic fragments or immunogenic variants thereof. Therefore, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of the following sequence numbers: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. Further information regarding the amino acid sequences can also be found in Table 1 and the ST25 sequence listings for the sequence numbers of each sequence. <223> Provided under the identifier.

[0119] In some embodiments, the spike protein (S) fragment as defined herein may be encoded by the RNA of the present invention, and the fragment may be cleaved at the N-terminus to lack amino acids 1 to a maximum of 100 at the N-terminus of the full-length SARS-CoV-2 variant protein, and / or the fragment may be cleaved at the C-terminus to lack amino acids (aa)531 to a maximum of aa1273 at the C-terminus of the full-length SARS-CoV-2 variant protein. Such “spike protein (S) fragments” may further include amino acid substitutions (as described herein) and may further include at least one heterologous peptide or protein element (as described herein). In preferred embodiments, the spike protein (S) fragment may be cleaved at the C-terminus to thereby lack the C-terminal transmembrane domain (i.e., lacking aa1212 to aa1273 or aa1148 to aa1273) (amino acid positions according to reference number 1).

[0120] In other embodiments, the encoded spike protein (S) derived from SARS-CoV-2 lacks a transmembrane domain (TM) (amino acid positions aa1212-aa1273 according to reference SEQ ID NO: 1). In embodiments, the encoded spike protein (S) derived from SARS-CoV-2 lacks an extended portion of the transmembrane domain (TMflex) (amino acid positions aa1148-aa1273 according to reference SEQ ID NO: 1). Not wishing to be bound by theory, a spike protein (S) lacking a transmembrane domain (TM or TMflex) as defined herein can be suitable for vaccines, and therefore the protein is soluble and not immobilized on the cell membrane. Thus, a soluble protein can be produced (i.e., translated) at higher concentrations upon administration to a target, potentially leading to an improved immune response.

[0121] Without wishing to be bound by theory, the RBD(aa319~aa541) and CND(aa329~aa529) domains, referenced using SEQ ID NO: 1 for their amino acid positions, may be important for immunogenicity. Both regions are located in the S1 fragment of the spike protein. Therefore, in the context of the present invention, it may be preferable for the antigenic peptide or protein to contain, or consist of, the S1 fragment of the spike protein or its immunogenic fragment or immunogenic variant. Preferably, such an S1 fragment may contain at least the RBD and / or CND domains defined above. In certain embodiments, the SARS-CoV-2 spike protein CND domain encoded by the RNA of the present invention is identical to, or comprises at least one of the following amino acid sequences, or immunogenic fragments or immunogenic variants, which are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of these sequences. Therefore, in some embodiments, the SARS-CoV-2 spike protein CND domain is at least 95% identical to, any one of the following amino acid sequences. In certain embodiments, the SARS-CoV-2 spike protein CND domain is identical to, any one of the following amino acid sequences. Further information regarding the aforementioned amino acid sequences can also be found in Table 1 and the ST25 sequence listings for the respective sequence numbers. <223> Provided under the identifier.

[0122] In preferred embodiments, the encoded antigenic peptide or protein comprises or consists of a receptor-binding domain (RBD; aa319~aa541), the RBD comprising or consisting of a spike protein fragment, or an immunogenic fragment or immunogenic variant thereof. In certain embodiments, the SARS-CoV-2 spike protein RBD domain encoded by the RNA of the present invention comprises or consists of an amino acid sequence that is identical to any one of sequence numbers 27007~27046, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or at least one immunogenic fragment or immunogenic variant thereof. Therefore, in some embodiments, the SARS-CoV-2 spike protein RBD domain is at least 95% identical to any one of sequence numbers 27007-27046. In certain embodiments, the SARS-CoV-2 spike protein RBD domain is identical to any one of sequence numbers 27007-27046. Further information regarding the amino acid sequences can also be found in Table 1 and the ST25 sequence listings for the respective sequence numbers. <223> Provided under the identifier.

[0123] In a more preferred embodiment, at least one encoded antigenic peptide or protein comprises or consists of a cleaved receptor-binding domain (truncRBD; aa334~aa528), the RBD comprising or consisting of a spike protein fragment, or an immunogenic fragment or immunogenic variant thereof.

[0124] Such “spike protein (S) fragments” (RBD; aa319-541 or truncRBD, aa334-528) may further contain amino acid substitutions (as described herein) and may further contain at least one heterologous peptide or protein element (as described herein).

[0125] In a particularly preferred embodiment, the encoded antigenic peptide or protein comprises or consists of a spike protein (S), the spike protein (S) comprising or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.

[0126] In a preferred embodiment, at least one encoded antigenic peptide or protein comprises spike protein fragment S1 and lacks at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of spike protein fragment S2 (aa682-aa1273). Such embodiments may be advantageous because the S1 fragment contains a neutralizing epitope.

[0127] Rather than being constrained by theory, it may be preferable for the antigenic peptide or protein to contain or consist of spike protein fragment S1 and spike protein fragment S2 (or at least a fragment thereof), as this may promote the formation of immunogenic spike proteins.

[0128] Therefore, in a particularly preferred embodiment, the encoded antigenic peptide or protein comprises or consists of a spike protein (S), the spike protein (S) comprising or consists of a spike protein fragment S1 or its immunogenic fragment or immunogenic variant, and a spike protein fragment S2 or its immunogenic fragment or immunogenic variant.

[0129] In an alternative preferred embodiment, the encoded antigenic peptide or protein comprises or consists of a full-length spike protein or an immunogenic fragment or immunogenic variant thereof.

[0130] The term “full-length spike protein” should be understood as a spike protein derived from SARS-CoV-2 that has an amino acid sequence corresponding to the complete spike protein. Therefore, “full-length spike protein” may include aa1-aa1273 (reference protein: SEQ ID NO: 1). Thus, full-length spike protein may typically include a secreted signal peptide, spike protein fragment S1, spike protein fragment S2, receptor-binding domain (RBD), and the important neutralizing domain CND, and a transmembrane domain. In particular, variants containing certain amino acid substitutions (e.g., enabling pre-fusion stabilization of the S protein) or naturally occurring amino acid deletions are also encompassed by the term “full-length spike protein.”

[0131] In a particularly preferred embodiment, a spike protein (S) encoded by the RNA of the first embodiment is designed or adapted to stabilize the antigen in the pre-fusion structure. The pre-fusion structure is particularly advantageous in the context of efficient coronavirus vaccines because several possible epitopes of the neutralizing antibody may simply be accessible to the pre-fusion protein structure. Furthermore, the remainder of the protein in the pre-fusion structure is intended to avoid immunopathological effects, such as hypertrophic disease and / or antibody-dependent enhancement (ADE).

[0132] In preferred embodiments, administration of RNA (or a composition or vaccine) encoding a stabilized spike protein before fusion to a subject induces spike protein neutralizing antibodies but not disease-enhancing antibodies. In particular, administration of nucleic acids (or a composition or vaccine) encoding a stabilized spike protein before fusion to a subject does not induce immunopathological effects such as disease-enhancing and / or antibody-dependent potentiation (ADE).

[0133] Therefore, in preferred embodiments, the RNA of the present invention comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or induced from the SARS-CoV-2 spike protein (S), wherein the SARS-CoV-2 spike protein (S) is a pre-fusion stabilized spike protein (S_stab). Preferably, the pre-fusion stabilized spike protein comprises at least one pre-fusion stabilization mutation.

[0134] As used herein, the term “pre-fusion structure” refers to the structural conformation of the external domain of the SARS-CoV-2 S protein after processing of the mature SARS-CoV-2 S protein in the secretory system, prior to the induction of membrane fusion, which leads to the transition to the post-fusion structure of SARS-CoV-2 S.

[0135] The “pre-fusion stabilized spike protein (S_stab)” described herein includes one or more amino acid substitutions, deletions, or insertions compared to the natural SARS-CoV-2 S sequence, resulting in increased retention of the pre-fusion structure compared to the SARS-CoV-2 S external domain trimer formed from the corresponding natural SARS-CoV-2 S sequence. The “stabilization” of the pre-fusion structure by one or more amino acid substitutions, deletions, or insertions may be, for example, energy stabilization (e.g., reducing the energy of the pre-fusion structure compared to the open post-fusion structure) and / or kinetic stabilization (e.g., reducing the rate of transition from the pre-fusion structure to the post-fusion structure). In addition, the stabilization of the pre-fusion structure of the SARS-CoV-2 S external domain trimer may include increased resistance to denaturation compared to the corresponding natural SARS-CoV-2 S sequence.

[0136] Therefore, in a preferred embodiment, the SARS-CoV-2 spike protein includes one or more amino acid substitutions that stabilize the S protein in its pre-fusion structure, for example, substitutions that stabilize the membrane distal portion of the S protein (including the N-terminal region) in its pre-fusion structure.

[0137] Stabilization of the SARS-CoV-2 coronavirus spike protein can be achieved by substituting at least one amino acid at positions K986 and / or V987 with an amino acid that stabilizes the spike protein in its pre-fusion conformation (amino acid positions according to reference SEQ ID NO: 1).

[0138] In a preferred embodiment, the pre-fusion stabilization mutation includes amino acid substitutions at positions K986 and V987, where amino acids K986 and / or V987 are substituted with amino acids selected from A, I, L, M, F, V, G, or P (amino acid positions according to reference sequence number 1).

[0139] Preferably, the stabilization of the three-dimensional structure before fusion is achieved by introducing two consecutive proline substitutions at residues K986 and V987 in the spike protein (amino acid positions according to reference SEQ ID NO: 1).

[0140] Therefore, in a preferred embodiment, the stabilized spike protein (S_stab) before fusion contains at least one pre-fusion stabilization mutation, the at least one pre-fusion stabilization mutation containing the following amino acid substitutions: K986P and V987P (amino acid positions according to reference SEQ ID NO: 1).

[0141] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises at least one pre-fusion stabilization K986P and V987P mutation, and further comprises the following amino acid substitutions or deletions (amino acid positions according to reference SEQ ID NO: 1): ·E484K, N501Y, and, in some cases, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, A701V; · E484K, N501Y, and optionally L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, A701V; N501Y, P681H, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H; N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H; · E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I; · E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, V1176F; N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H; L452R, and optionally S13I, W152C, D614G; L452R, D614D, and optionally P681R; L452R, D614D, P681R, and optionally E484Q, E154K, Q1071H; L452R, D614D, P681R, and optionally T19R, L452R, D950N; L452R, D614D, P681R, and optionally T19R, F157del, T478K, D950N; E484K, and optionally Q52R, A67V, H69del, V70del, delY144, D614G, Q677H, F888L; ·E484K, and optionally A67V, H69del, V70del, Y144del, D614G, Q677H, F888L; · E484K, and optionally L5F, T95I, D253G, D614G, A701V; P681R, and optionally F157L, V367F, Q613H; P681R, and optionally S254F, D614G, G769V; L452R, P681R, and optionally, D614G; L452R, E484Q, P681R, and optionally E154K, D614G, Q1071H; L452R, P681R, and optionally T19R, F157del, R158del, T478K, D614G, D950N; · E484K, and optionally D614G, V1176F; L452Q, and optionally G75V, T76I, R246del, S247del, Y248del, L249del, T250del, P251del, G252del, F490S, D614G, T859N; ·K417N, and optionally, P681R; K417N, P681R, and optionally, D614G; K417N, L452R, P681R, and optionally, D614G; K417N, T478K, P681R, and optionally, D614G; K417N, D950N, P681R, and optionally, D614G. ·K417N, D614G, P681R, and optionally, T478K; K417N, D614G, P681R, and optionally, L452R; K417N, D614G, P681R, L452R, and optionally, T478K; ·S247del, Y248del, L249del, T250del, P251del, G252del, D253del, and optionally, D614G; S247del, Y248del, L249del, T250del, P251del, G252del, D253del, and optionally L452Q, D614G; H69del, V70del, and optionally D614G; H69del, V70del, E484K, and optionally D614G; ·H69del, V70del, N501Y and, in some cases, D614G; or H69del, V70del, N501Y, E484K and, in some cases, P681H This is a stabilized spike protein (S_stab) before fusion, which includes [the specified component].

[0142] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises at least one pre-fusion stabilization K986P and V987P mutation, and further comprises the following amino acid substitutions or deletions (amino acid positions according to reference SEQ ID NO: 1): · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V; · E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I; ·E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, V1176F; L452R, P681R, and D614G; L452R, E484Q, P681R, E154K, D614G, and Q1071H; or L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N It is a stabilized spike protein (S_stab) (or its fragment or variant) before fusion, which includes [the specified element].

[0143] In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is ·K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or ·K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V It is a stabilized spike protein (S_stab) (or a fragment or variant thereof) before fusion containing an amino acid substitution or deletion (amino acid position according to reference sequence number 1) selected from the above.

[0144] It should be emphasized that in embodiments within the context of the present invention, any SARS-CoV-2 coronavirus spike protein as defined herein can be mutated as described above (as illustrated with reference protein sequence number 1) to stabilize the spike protein in its pre-fusion conformation.

[0145] According to various embodiments, the RNA of the present invention encodes at least one antigenic SARS-CoV-2 spike protein as defined herein, and further, at least one heterologous peptide or protein element.

[0146] Preferably, at least one heterologous peptide or protein element may promote or enhance the secretion of the encoded antigenic SARS-CoV-2 spike protein (e.g., via a secretory signaling sequence), promote or enhance the fixation of the encoded antigenic SARS-CoV-2 spike protein in the plasma membrane (e.g., via a transmembrane element), promote or enhance the formation of an antigen complex (e.g., via a multimerizing domain or an antigen clustering element), or promote or enhance virus-like particle (VLP-forming sequence). Furthermore, the RNA of the first embodiment may further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell targeting sequence.

[0147] A suitable polymerizing domain may be selected from the amino acid sequences described in Sequence IDs 1116-1167 of WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable transmembrane element may be selected from the amino acid sequences described in Sequence IDs 1228-1343 of WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable VLP-forming sequence may be selected from the amino acid sequences described in Sequence IDs 1168-1227 of the patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable peptide linker may be selected from the amino acid sequences described in Sequence IDs 1509-1565 of the patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable self-cleaving peptide may be selected from the amino acid sequences described in SEQ ID NOs. 1434-1508 of patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable immunoadjuvant sequence may be selected from the amino acid sequences described in SEQ ID NOs. 1360-1421 of patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable dendritic cell (DC) targeting sequence may be selected from the amino acid sequences described in SEQ ID NOs. 1344-1359 of patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. A suitable secreted signal peptide may be selected from the amino acid sequences described in SEQ ID NOs. 1-1115 and SEQ ID NO. 1728 of the published PCT patent application WO2017 / 081082, or from a list of fragments or variants of these sequences.

[0148] In a preferred embodiment, the RNA encoding at least one antigenic SARS-CoV-2 spike protein further encodes at least one heterologous secreted signaling sequence and / or trimerizing element and / or antigen clustering element and / or VLP-forming sequence.

[0149] Therefore, in a preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of an amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequence numbers 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959~22964, 27087~27109, 28540~28588, or 28917~28920, or at least one immunogenic fragment or immunogenic variant thereof. Therefore, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to one of sequence numbers 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of the following sequence numbers: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. Further information regarding the amino acid sequences can also be found in Table 1 and the ST25 sequence listings for the sequence numbers of each sequence. <223> Provided under the identifier.

[0150] Therefore, in preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of an amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of these immunogenic fragments or immunogenic variants. Therefore, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of the SARS-CoV-2 spike proteins, or at least 95% identical to any In certain embodiments, the SARS-CoV-2 spike protein is identical to one of sequence numbers 27093-27095 or 28552-28558.

[0151] In a more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of at least one of the following: an amino acid sequence identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any immunogenic fragment or immunogenic variant thereof. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to, or at least 95% identical to, any one of the following: SEQ ID NOs: 27095, 28552, 28557. In a particular embodiment, the SARS-CoV-2 spike protein is identical to, or at least 95% identical to, any one of the following: SEQ ID NOs: 27095, 28552, 28557.

[0152] In a more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is identical to any one of SEQ ID NO: 27095, or comprises or consists of at least one of the following immunogenic fragments or immunogenic variants, or an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 27095. In a particular embodiment, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 27095.

[0153] In a more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of at least one amino acid sequence or amino acid coding sequence, or any immunogenic fragment or immunogenic variant thereof, that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 23090, 23091, 22960, 22961, or 28540. In certain embodiments, the SARS-CoV-2 spike protein is identical to one of sequence numbers 23090, 23091, 22960, 22961, and 28540.

[0154] In still more preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is identical to or comprises at least one of the amino acid sequences, or immunogenic fragments or immunogenic variants, of either SEQ ID NOs. 27096 or 28545, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to either SEQ ID NOs. 27096 or 28545. In certain specific embodiments, the SARS-CoV-2 spike protein is identical to either SEQ ID NOs. 27096 or 28545.

[0155] In still more preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of at least one of the following immunogenic fragments or immunogenic variants, or an amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of the sequence numbers 22959. In certain specific embodiments, the SARS-CoV-2 spike protein is identical to any one of the sequence numbers 22959.

[0156] In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises or consists of an amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of these immunogenic fragments or immunogenic variants. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of the SARS-CoV-2 spike proteins, or In certain embodiments, the SARS-CoV-2 spike protein is identical to one of sequence numbers 28541-28544 or 28917-28920.

[0157] Table 1 provides preferred antigenic peptides or proteins derived from SARS-CoV-2 as defined herein. Each column corresponds to a preferred SARS-CoV-2 spike protein construct. Column A of Table 1 provides a brief description of the preferred antigen constructs. Column B of Table 1 provides the sequence numbers for the proteins (amino acids) of each antigen construct. Column C of Table 1 provides the sequence numbers for the corresponding G / C optimized nucleic acid coding sequences (opt1, gc). Column D of Table 1 provides the sequence numbers for the corresponding G / C content modified nucleic acid coding sequences (opt10, gc mod) (see paragraph "Preferred Coded Sequences" for a detailed explanation of "Coded Sequences").

[0158] In particular, the description of the invention is in the ST25 sequence listing of this application. <223> The information provided under the identifier is explicitly included. Preferred RNA constructs containing the coding sequences in Table 1, for example, mRNA sequences containing the coding sequences in Table 1, are provided in Table 2.

[0159] [Table 1] TIFF0007894882000002.tif211168TIFF0007894882000003.tif220167TIFF0007894882000004.tif220168TIFF0007894882000005.tif20916 7TIFF0007894882000006.tif214167TIFF0007894882000007.tif215167TIFF0007894882000008.tif224167TIFF0007894882000009.tif29167

[0160] Suitable code sequence: In a preferred embodiment, the RNA of the present invention preferably comprises at least one coding sequence encoding at least one antigenic peptide or protein or fragments and variants thereof, selected from or induced from the SARS-CoV-2 spike protein as defined above. In that context, any coding sequence encoding at least one antigenic protein, the SARS-CoV-2 spike protein as defined herein, or fragments and variants thereof, may be understood as a preferred coding sequence and therefore may be included in the RNA of the present invention.

[0161] In a preferred embodiment, the RNA of the first embodiment comprises, or may comprise, at least one coding sequence encoding at least one of the SARS-CoV-2 antigenic peptides or proteins as defined herein, preferably SEQ ID NOs: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. At the nucleic acid level, is it identical to any one of the following sequence numbers: 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089~23148, 23150~23184, 27110~27247, 28589~28637, 28916, 28921~28924? It should be understood that any RNA sequence or fragment or variant thereof encoding an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the present invention may be selected as a preferred coding sequence of the present invention, and therefore understandable. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to one of sequence numbers 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916, or 28921-28924.

[0162] In a preferred embodiment, the RNA of the first embodiment comprises, or may comprise, at least one coding sequence encoding at least one antigenic peptide or protein derived from SARS-CoV-2 as defined herein, preferably SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. At the nucleic acid level, is it identical to any one of the following sequence numbers: 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089~23148, 23150~23184, 23095~23112, 27110~27247, 28589~28637, 28921~28924, or It should be understood that any RNA sequence or fragment or variant thereof encoding an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the present invention may be selected as a preferred coding sequence of the present invention, and therefore understandable. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to one of the following sequence numbers: 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, or 28921-28924.

[0163] In a preferred embodiment, the RNA of the first embodiment comprises, or may comprise, at least one coding sequence encoding at least one antigenic peptide or protein derived from SARS-CoV-2 as defined herein, preferably SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. It should be understood that any RNA sequence or fragment or variant thereof encoding an amino acid sequence that is identical at the nucleic acid level to any one of sequence numbers 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089~23148, 27110~27201, 28589~28637, or 28921~28924, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of these sequences may be selected as a preferred coding sequence for the present invention, and therefore understandable. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to one of the following sequence numbers: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, or 28921-28924.

[0164] In a preferred embodiment, the RNA of the first embodiment comprises, or may comprise, at least one coding sequence encoding at least one antigenic peptide or protein derived from SARS-CoV-2 as defined herein, preferably SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. Any RNA sequence or fragment or variant thereof encoding an amino acid sequence that is identical at the nucleic acid level to any one of sequence numbers 146, 23150-23184, or 27202-27247, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, may be selected as a preferred coding sequence of the present invention and, therefore, should be understood. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of sequence numbers 146, 23150-23184, or 27202-27247.

[0165] In a preferred embodiment, the RNA of the first embodiment includes a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the sequences described in Sequence IDs 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28921-28924, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the fragments or variants of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to one of sequence numbers 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, or 28921-28924. Further information regarding each of these preferred nucleic acid sequences can also be found in the sequence listings, in particular, the identifiers. <223> This can be derived from the details provided there.

[0166] In a preferred embodiment, the RNA of the first embodiment includes a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the sequences described in Sequence IDs 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23094, 27110-27132, 28589-28637, or 28921-28924, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the fragments or variants of these sequences. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to one of sequence numbers 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23094, 27110-27132, 28589-28637, or 28921-28924.

[0167] In a preferred embodiment, the RNA of the first embodiment includes a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any nucleic acid sequence or at least one fragment or variant of any of these sequences. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of the sequences 23150-23166 or 27202-27224.

[0168] In a preferred embodiment, the RNA of the first embodiment includes a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any nucleic acid sequence or at least one fragment or variant of any of these sequences. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of the sequences 23114-23130 or 27156-27178.

[0169] In a preferred embodiment, the RNA of the first embodiment includes a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any nucleic acid sequence or at least one fragment or variant of any of these sequences. In a particular embodiment, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of the sequences 23167-23184 or 27225-27247.

[0170] In a preferred embodiment, the RNA of the first embodiment is artificial RNA.

[0171] As used herein, the term “artificial RNA” is intended to refer to RNA that does not occur naturally. In other words, artificial RNA can be understood as a non-natural RNA molecule. Such RNA molecules may be non-natural due to their individual sequences (e.g., coding sequences with modified G / C content, UTRs) and / or other modifications of nucleotides, e.g., structural modifications. Typically, artificial RNA can be designed and / or produced by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, artificial RNA is a sequence that does not occur naturally, i.e., a sequence that differs by at least one nucleotide from the wild-type sequence / naturally occurring sequence. The term “artificial RNA” is not limited to meaning “a single RNA molecule,” but is understood to include an ensemble of essentially identical RNA molecules. Thus, it may refer to multiple essentially identical RNA molecules.

[0172] In a preferred embodiment, the RNA of the first embodiment is modified and / or stabilized RNA, preferably modified and / or stabilized artificial RNA.

[0173] Accordingly, according to preferred embodiments, the RNA of the present invention may be provided as “stabilized artificial RNA” or “stabilized coding RNA,” i.e., RNA exhibiting improved resistance to in vivo degradation and / or improved stability in vivo and / or improved translatability in vivo. Specific preferred modifications / fits in this context that are suitable for “stabilizing” RNA are described below. Preferably, the RNA of the present invention may be provided as “stabilized RNA” or “stabilized coding RNA.”

[0174] Such stabilization may be influenced by resulting in “dried RNA” and / or “purified RNA,” as further specified below. Alternatively, or in addition, such stabilization may be influenced, for example, by a modified phosphate skeleton of the RNA of the present invention. With respect to the present invention, skeletal modification is a modification in which the phosphate of the nucleotide skeleton contained in the nucleic acid is chemically modified. A nucleotide that may be used in connection therewith contains, for example, a phosphorothioate-modified phosphate skeleton, preferably containing at least one phosphate oxygen contained in a phosphate skeleton replaced by a sulfur atom. The stabilized RNA may further contain, for example, nonionic phosphate analogs, such as alkyl and aryl phosphonates, where the charged phosphonate oxygen is replaced by an alkyl or aryl group, or by phosphodiesters and alkylphosphotryesters, and the charged oxygen residue exists in an alkylated form. Such skeletal modifications typically include modifications from the group consisting of methylphosphonates, phosphoramides and phosphorothioates (e.g., cytidine-5'-O-(1-thiophosphate)), without any limitation.

[0175] The following describes preferred modifications that have the ability to "stabilize" the RNA of the present invention.

[0176] In a preferred embodiment, the RNA includes at least one codon-modified coding sequence.

[0177] In a preferred embodiment, at least one coding sequence of the RNA is a codon-modified coding sequence, where the amino acid sequence encoded by at least one codon-modified coding sequence is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence or reference coding sequence.

[0178] The term “codon-modified coding sequence” refers to a coding sequence in which at least one codon (a triplet of nucleotides encoding one amino acid) differs from the corresponding wild-type or reference coding sequence. Preferably, in the context of the present invention, a codon-modified coding sequence may exhibit improved resistance to degradation in vivo and / or improved stability in vivo and / or improved translationability in vivo. Codon modification in a broad sense uses the degeneracy of the gene code, where multiple codons may encode the same amino acid and be used interchangeably to optimize / modify the coding sequence for in vivo application.

[0179] The term "reference code sequence" refers to the code sequence that was the origin sequence to be qualified and / or optimized.

[0180] In a preferred embodiment, at least one coding sequence of the RNA is a codon-modified coding sequence, where the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon-use-compatible coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

[0181] In a preferred embodiment, at least one coding sequence of RNA has a G / C content of at least about 50%, 55%, or 60%. In a particular embodiment, at least one coding sequence of RNA of component A has a G / C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

[0182] When transfected into mammalian host cells, RNA containing codon-modified coding sequences has stability for 12–18 hours, or longer than 18 hours, for example, 24, 36, 48, 60, 72, or longer than 72 hours, and is capable of being expressed by mammalian host cells (e.g., muscle cells).

[0183] When transfected into mammalian host cells, the RNA containing the codon-modified coding sequence is translated into a protein, where the amount of protein is at least equivalent to, or preferably at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 100% more, or at least 200% more than, the amount of protein obtained by the naturally occurring, wild-type, or reference coding sequence transfected into mammalian host cells.

[0184] In some embodiments, the RNA may be modified, where the C content of at least one coding sequence may be increased, and preferably maximized, compared to the C content of the corresponding wild-type or reference coding sequence (referred to herein as the "C-maximized coding sequence"). The generation of the C-maximized nucleic acid sequence may preferably be carried out using the modification method described in WO2015 / 062738. In this context, the disclosure of WO2015 / 062738 is incorporated herein by reference.

[0185] In a preferred embodiment, the RNA may be modified, where the G / C content of at least one coding sequence may be optimized compared to the G / C content of the corresponding wild-type or reference coding sequence (referred to herein as a “G / C-optimized coding sequence”). In that context, “optimized” refers to the coding sequence, where the G / C content is preferably increased to the potentially maximum G / C content. The generation of G / C-optimized RNA sequences may be carried out using the method described in WO2002 / 098443. In this context, the disclosure of WO2002 / 098443 is included in its entirety in the present invention. <223> Throughout the entire description, including the identifier, G / C optimized code sequences are indicated by the abbreviation "opt1" or "gc".

[0186] In a preferred embodiment, the RNA may be modified so that at least one codon in the coding sequence can be adapted for human codon use (referred to herein as the "human codon-adapted coding sequence"). Codons encoding the same amino acid occur at different frequencies in humans. Therefore, the coding sequence of the nucleic acid is preferably modified so that the frequency of codons encoding the same amino acid corresponds to the naturally occurring frequency of those codons through human codon use. For example, in the case of the amino acid Ala, the wild-type or reference coding sequence is preferably adapted such that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" at a frequency of 0.28, the codon "GCA" at a frequency of 0.22, and the codon "GCG" at a frequency of 0.10. Therefore, such a method (exemplified for Ala) is adapted to each amino acid encoded by the coding sequence of the nucleic acid to obtain a human codon-adapted sequence. <223> Throughout the entire description, including the identifier, human codon-compatible code sequences are indicated by the abbreviation "opt3" or "human".

[0187] In some embodiments, the RNA may be modified, where the G / C content of at least one coding sequence may be modified compared to the G / C content of the corresponding wild-type or reference coding sequence (referred to herein as “G / C content modified coding sequence”). In this context, the terms “G / C optimization” or “G / C content modification” refer to nucleic acids containing a modified, preferably increased, number of guanosine and / or cytosine nucleotides compared to the corresponding wild-type or reference coding sequence. Such an increased number may be generated by the substitution of adenosine or thymidine nucleotide-containing codons with guanosine or cytosine nucleotide-containing codons. Advantageously, nucleic acid sequences with increased G / C content are more stable or exhibit better expression than sequences with increased A / U. Preferably, the G / C content of the nucleic acid coding sequence is increased by at least 10%, 20%, 30%, and preferably at least 40%, compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence (referred to herein as “opt 10” or “gc mod”). For example, the G / C content of the nucleic acid coding sequence is preferably increased by at least 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% compared to the G / C content of sequence number 28916.

[0188] In some embodiments, the RNA may be modified, where the codon compatibility index (CAI) may be increased or preferably maximized in at least one coding sequence (referred to herein as the “CAI-maximized coding sequence”). For example, all codons in the wild-type or reference nucleic acid sequence that are relatively rare in humans may be replaced, for example, with each of the frequent codons in humans, where the frequent codons code for the same amino acid as the relatively rare codons. Preferably, the most frequent codon is used for each amino acid of the encoded protein. Preferably, the RNA comprises at least one coding sequence, where the codon compatibility index (CAI) of at least one coding sequence is at least 0.5, at least 0.8, at least 0.9, or at least 0.95. Most preferably, the codon compatibility index (CAI) of at least one coding sequence is 1 (CAI=1). For example, in the case of the amino acid Ala, the wild-type or reference coding sequence may be fitted in a manner in which the most frequent human codon “GCC” is always used for the said amino acid. Therefore, such a method (exemplified for Ala) may be adapted to each amino acid encoded by the coding sequence of the nucleic acid to obtain the CAI-maximizing coding sequence.

[0189] In a particularly preferred embodiment, at least one coding sequence of the nucleic acid is a codon-modified coding sequence, where the codon-modified coding sequence is a G / C optimized coding sequence.

[0190] In a particularly preferred embodiment, the RNA of the first embodiment is identical to, or less than, a G / C optimized nucleic acid sequence selected from the group consisting of SEQ ID NOs: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, and 28921-28924. The invention comprises at least one coding sequence comprising a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, all of which are 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0191] In a particularly preferred embodiment, the RNA of the first embodiment includes a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, or at least one coding sequence comprising the same G / C optimized coding sequence encoding the SARS-CoV-2 antigen, which is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a G / C optimized coding sequence selected from the group consisting of SEQ ID NOs: 146, 23150-23184, and 27202-27247, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such a sequence.

[0192] In a more preferred embodiment, the RNA of the first embodiment includes a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, or at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109, 23127, 23145, 23163, 23181, 28589 (B.1.315; C.1.2), which is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or at least one coding sequence comprising such a sequence. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109, 23127, 23145, 23163, 23181, or 28589. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 23090-23091, 23162-23163, or 28589.

[0193] In a more preferred embodiment, the RNA of the first embodiment is from the group consisting of SEQ ID NOs: 27116, 27139, 27162, 27185, 27208, 27231, 27117, 27140, 27163, 27186, 27209, 27232, 27118, 27141, 27164, 27187, 27210, 27233, 28601~28607 (B.1.617;B.1.617.1;B.1.617.2;AY.1;AY.2;AY.4;AY.4.2;B.1.617.3) The selected codon is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or includes a fragment or variant of any of these sequences, or includes at least one coding sequence consisting of such a sequence. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 27116, 27139, 27162, 27185, 27208, 27231, 27117, 27140, 27163, 27186, 27209, 27232, 27118, 27141, 27164, 27187, 27210, 27233, or 28601-28607. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 27116-27118, 27208-27210, or 28601-28607.

[0194] In a more preferred embodiment, the RNA of the first embodiment includes a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, or at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27118, 27141, 27164, 27187, 27210, 27233, or 28601-28606 (B.1.617.2;AY.1;AY.2;AY.4;AY.4.2), which is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or at least one coding sequence comprising such a sequence. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 27118, 27141, 27164, 27187, 27210, 27233, or 28601-28606.

[0195] In a more preferred embodiment, the RNA of the first embodiment includes a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, or at least one coding sequence comprising the same, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27118, 27141, 27164, 27187, 27210, or 27233 (B.1.617.2). In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 27118, 27141, 27164, 27187, 27210, or 27233. In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 27118 or 27210.

[0196] In a more preferred embodiment, the RNA of the first embodiment includes a G / C optimized coding sequence encoding the SARS-CoV-2 antigen as defined herein, or a fragment or variant of any of these sequences, or at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28590-28593, 28921-28924 (B.1.1.529, Omicron), which is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28590-28593, 28921-28924 (B.1.1.529, Omicron), or at least one coding sequence comprising a codon-modified nucleic acid sequence encoding the SARS-CoV-2 antigen as defined herein, or a codon-modified coding sequence comprising at least one coding sequence comprising at least one codon-modified coding sequence comprising at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or at least 70 In some embodiments, at least one coding sequence encoding the SARS-CoV-2 antigen is at least 95% identical to sequence numbers 28590-28593 and 28921-28924.

[0197] UTR: In preferred embodiments, the RNA of the present invention comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein as defined herein, or an immunogenic fragment or immunogenic variant thereof, wherein the RNA comprises at least one heterologous untranslated region (UTR). In some embodiments, the RNA of the embodiment does not include a 3'UTR containing the sequence of SEQ ID NO: 268. In certain embodiments, the RNA of the embodiment includes a 3'UTR containing the sequence of SEQ ID NO: 268.

[0198] In preferred embodiments, the RNA of the present invention comprises a protein-coding region ("coding sequence" or "cds") and a 5'UTR and / or 3'UTR. In particular, the UTR may have regulatory sequence elements that determine nucleic acid, e.g., RNA turnover, stability, and localization. Furthermore, the UTR may have sequence elements that enhance translation. In pharmaceutical applications, the translation of RNA into at least one peptide or protein is of paramount importance to therapeutic efficacy. Certain combinations of the 3'UTR and / or 5'UTR can enhance the expression of the operably linked coding sequence encoding the peptide or protein of the present invention. RNA molecules having such UTR combinations advantageously allow for rapid and transient expression of the antigenic peptide or protein after administration to a subject, preferably after intramuscular administration. Therefore, RNAs comprising certain combinations of the 3'UTR and / or 5'UTR provided herein are particularly suitable for administration as a vaccine, and especially for administration into the muscle, dermis, or epidermis of a subject.

[0199] Preferably, the RNA of the present invention comprises at least one heterologous 5'UTR and / or at least one heterologous 3'UTR. The heterologous 5'UTR or 3'UTR may be derived from naturally occurring genes or synthesized. In a preferred embodiment, the RNA comprises at least one coding sequence as defined herein, operably ligated to at least one (heterologous) 3'UTR and / or at least one (heterologous) 5'UTR.

[0200] In preferred embodiments, the RNA comprises at least one heterologous 3'UTR and does not comprise a 3'UTR comprising the sequence of SEQ ID NO: 268. Preferably, the RNA comprises a 3'UTR that can be derived from a gene that results in an RNA having an enhanced half-life (i.e., results in a stable RNA).

[0201] In some embodiments, the 3'UTR comprises one or more of a polyadenylation signal, a binding site for a protein that affects nucleic acid stability or location in a cell, or a binding site for one or more miRNAs or miRNA.

[0202] MicroRNA (or miRNA) is a non-coding RNA 19-25 nucleotides in length that binds to the 3'UTR of a nucleic acid molecule and downregulates gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. For example, microRNAs are known to regulate protein expression in, e.g., liver (miR-122), heart (miR-1d, miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-194, miR-204), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133, miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126). The RNA may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond, for example, to any known microRNA, such as those taught in US2005 / 0261218 and US2005 / 0059005.

[0203] Thus, the miRNA or miRNA binding site defined above may be removed from the 3'UTR or introduced into the 3'UTR to match the RNA expression to a desired cell type or tissue (e.g., muscle cells).

[0204] In a preferred embodiment, the RNA comprises or consists of at least one heterologous 3'UTR that is derived from the 3'UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a homolog, fragment, or variant of any one of these genes. In some embodiments, the RNA comprises at least one heterologous 3'UTR, and the at least one heterologous 3'UTR is preferably identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence set forth in SEQ ID NOs: 253-266, 22902-22905, 22876-22895, 26996-26999, 28528-28539, or a fragment or variant of any of these, and comprises a nucleic acid sequence derived from the 3'UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a homolog, fragment, or variant of any one of these genes. Particularly preferred nucleic acid sequences in that context may be derived from published PCT application WO2019 / 077001A1, particularly from claim 9 of WO2019 / 077 OO1A1. The corresponding 3'UTR sequences of claim 9 of WO2019 / 077001A1 are incorporated herein by reference (e.g., SEQ ID NOs: 23-34 of WO2019 / 077001A1, or a fragment or variant thereof).

[0205] In further embodiments, the RNA includes a 3'UTR derived from the RPS9 gene. The 3'UTR derived from the RPS9 gene includes, or may consist of, a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence numbers 263 or 264, 22894, 22895, 22904, or 22905.

[0206] In a preferred embodiment, the RNA includes a 3'UTR derived from the PSMB3 gene. The 3'UTR derived from the PSMB3 gene includes, or may consist of, a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence numbers 253 or 254, 22892, 22893, 22902, 22903, 26996-26999, 28528-28539.

[0207] In other embodiments, the RNA comprises a nucleic acid sequence or fragment or variant thereof that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 22876-22891, 28526, and 28527, or 99% identical to them, or comprises a 3'UTR consisting of such sequences.

[0208] In other embodiments, the RNA may include 3'UTRs as described in WO2016 / 107877, the disclosures of WO2016 / 107877 relating to 3'UTR sequences are incorporated herein by reference. Preferred 3'UTRs are SEQ ID NOs. 1-24 and 49-318 of WO2016 / 107877, or fragments or variants of these sequences. In other embodiments, the nucleic acid may include 3'UTRs as described in WO2017 / 036580, the disclosures of WO2017 / 036580 relating to 3'UTR sequences are incorporated herein by reference. Preferred 3'UTRs are SEQ ID NOs. 152-204 of WO2017 / 036580, or fragments or variants of these sequences. In other embodiments, the nucleic acid comprises a 3'UTR as described in WO2016 / 022914, the disclosure of WO2016 / 022914 relating to 3'UTR sequences is incorporated herein by reference. Particularly preferred 3'UTRs are the nucleic acid sequences described in Sequence IDs 20-36 of WO2016 / 022914, or fragments or variants thereof.

[0209] In a preferred embodiment, the RNA includes at least one heterologous 5'UTR.

[0210] The terms “5' untranslated region,” “5'UTR,” or “5'UTR element” are as recognized and understood by those skilled in the art and are intended to refer, for example, to a portion of an RNA molecule located at 5' (i.e., “upstream”) of a coding sequence that is not translated into a protein. The 5'UTR may also be a portion of nucleic acid located at 5' of a coding sequence. Typically, the 5'UTR begins at the transcription start site and ends before the start codon of the coding sequence. The 5'UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5' cap structure (e.g., for mRNA).

[0211] Preferably, the RNA includes a 5'UTR which may be derived from a gene relating to RNA with an enhanced half-life (i.e., resulting in stable RNA).

[0212] In some embodiments, the 5'UTR includes a protein binding site that affects RNA stability or RNA position in a cell, or one or more miRNAs or miRNA binding sites (as defined above).

[0213] Therefore, the miRNA or miRNA binding site defined above may be removed from or introduced into the 5'UTR to tailor nucleic acid expression to the desired cell type or tissue (e.g., muscle cells).

[0214] In a preferred embodiment, the RNA comprises at least one heterologous 5'UTR, where at least one heterologous 5'UTR comprises a nucleic acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence or fragment or variant thereof, derived from a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a 5'UTR derived from a homolog, fragment, or variant of any one of these genes. Particularly preferred nucleic acid sequences in that context can be selected from the published PCT application WO2019 / 077001A1, in particular from claim 9 of WO2019 / 077001A1. The corresponding 5'UTR sequences of claim 9 of WO2019 / 077001A1 are incorporated herein by reference (e.g., sequence numbers 1-20 of WO2019 / 077001A1, or fragments or variants thereof).

[0215] In a preferred embodiment, the RNA comprises a 5'UTR derived from the RPL31 gene, wherein the 5'UTR derived from the RPL31 gene comprises or consists of a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence numbers 243, 244, 22872, 22873.

[0216] In other embodiments, the RNA includes a 5'UTR derived from the SLC7A3 gene, wherein the 5'UTR derived from the SLC7A3 gene includes or comprises a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence numbers 245, 246, 22874, and 22875.

[0217] In a particularly preferred embodiment, the RNA comprises a 5'UTR derived from the HSD17B4 gene, wherein the 5'UTR derived from the HSD17B4 gene comprises or consists of a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence numbers 231, 232, 22870, and 22871.

[0218] In other embodiments, the RNA comprises nucleic acid sequences or fragments or variants thereof that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 22848-22867 and 28522-28525, or 99% identical to them, or comprises a 5'UTR consisting of such sequences.

[0219] In other embodiments, the RNA comprises a 5'UTR as described in WO2013 / 143700, the disclosure of WO2013 / 143700 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are nucleic acid sequences derived from SEQ ID NOs. 1-1363, SEQ ID NOs. 1395, SEQ ID NOs. 1421 and 1422 of WO2013 / 143700, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 5'UTR as described in WO2016 / 10787, the disclosure of WO2016 / 107877 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are nucleic acid sequences described in SEQ ID NOs. 25-30 and SEQ ID NOs. 319-382 of WO2016 / 107877, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 5'UTR as described in WO2017 / 036580, the disclosure of WO2017 / 036580 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 1 to 151 of WO2017 / 036580, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 5'UTR as described in 2016 / 022914, the disclosure of WO2016 / 022914 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 3 to 19 of WO2016 / 022914, or fragments or variants thereof.

[0220] Preferably, in a preferred embodiment, the RNA encodes at least one antigenic protein as defined herein, preferably one of the following 5'UTR / 3'UTR combinations (also referred to as "UTR design"): a-1 (HSD17B4 / PSMB3), a-2 (NDUFA4 / PSMB3), a-3 (SLC7A3 / PSMB3), a-4 (NOSIP / PSMB3), a-5 (MP68 / PSMB3), b-1 (UBQL N2 / RPS9), b-2(ASAH1 / RPS9), b-3(HSD17B4 / RPS9), b-4(HSD17B4 / CASP1), b-5(NOSIP / COX6B1), c-1(NDUFA4 / RPS9), c-2(NOSIP / NDUFA1), c-3(NDUFA4 / COX6B1), c-4(NDUFA4 / NDUFA1), c-5(ATP5A1 / PSMB3), d-1(Rpl31 / PSMB3), d-2(A TP5A1 / CASP1), d-3(SLC7A3 / GNAS), d-4(HSD17B4 / NDUFA1), d-5(Slc7a3 / Ndufa1), e-1(TUBB4B / RPS9), e-2(RPL31 / R PS9), e-3(MP68 / RPS9), e-4(NOSIP / RPS9), e-5(ATP5A1 / RPS9), e-6(ATP5A1 / COX6B1), f-1(ATP5A1 / GNAS), f-2(ATP 5A1 / NDUFA1), f-3(HSD17B4 / COX6B1), f-4(HSD17B4 / GNAS), f-5(MP68 / COX6B1), g-1(MP68 / NDUFA1), g-2(NDUFA4 / CA SP1), g-3(NDUFA4 / GNAS), g-4(NOSIP / CASP1), g-5(RPL31 / CASP1), h-1(RPL31 / COX6B1), h-2(RPL31 / GNAS), h-3(RP L31 / NDUFA1), h-4(Slc7a3 / CASP1), h-5(SLC7A3 / COX6B1), i-1(SLC7A3 / RPS9), i-2(RPL32 / ALB7), i-2(RPL32 / ALB7) It includes at least one coding sequence derived from SARS-CoV-2 that is operably concatenated to the 3'UTR and / or 5'UTR selected from.

[0221] In a particularly preferred embodiment, the RNA comprises at least one coding sequence as specified herein that encodes at least one antigenic protein derived from SARS-CoV-2, and the coding sequence is operably linked to the HSD17B4 5'UTR and the PSMB3 3'UTR (HSD17B4 / PSMB3 (UTR design a-1)).

[0222] This embodiment has been shown by the inventors to be particularly beneficial for inducing an immune response against SARS-CoV-2. In this context, it has been shown that a single vaccination is already sufficient to generate a viral neutralizing antibody titer.

[0223] In a more preferred embodiment, the nucleic acid preferably comprises at least one coding sequence as specified herein that encodes at least one antigenic protein as defined herein, derived from the SARS-CoV-2 (nCoV-2019) coronavirus, and the coding sequence is operably linked to the SLC7A3 5'UTR and the PSMB3 3'UTR (SLC7A3 / PSMB3 (UTR design a-3)).

[0224] In a more preferred embodiment, the nucleic acid preferably comprises at least one coding sequence as specified herein that encodes at least one antigenic protein as defined herein, derived from the SARS-CoV-2 (nCoV-2019) coronavirus, and the coding sequence is operably linked to the RPL31 5'UTR and the RPS9 3'UTR (RPL31 / RPS9 (UTR design e-2)).

[0225] In some embodiments, the RNA may be monocistronic, bicistronic, or polycistronic.

[0226] The term “monocistronic” is intended to be recognized and understood by those skilled in the art to refer to, for example, a nucleic acid containing only one coding sequence. As used herein, the terms “bisistronic” or “multicistronic” are intended to be recognized and understood by those skilled in the art to refer to a nucleic acid that may contain, for example, two (bisistronic) or more (multicistronic) coding sequences.

[0227] In a preferred embodiment, the RNA of the first embodiment is monocistronic.

[0228] In other embodiments, the RNA is monocistronic, and the coding sequence of the nucleic acid encodes at least two different antigenic peptides or proteins derived from SARS-CoV-2. Thus, the coding sequence may encode at least two, three, four, five, six, seven, or eight or more antigenic peptides or proteins derived from SARS-CoV-2, which are linked or not linked by an amino acid linker sequence, and the linker sequence may include a rigid linker, a mobile linker, a cleavable linker, or a combination thereof. Such constructs are referred to herein as “multiple antigenic constructs.”

[0229] In further embodiments, the RNA may be bicistronic or multicistronic and comprise at least two coding sequences, the at least two coding sequences encoding two or more different antigenic peptides or proteins derived from SARS-CoV-2. Thus, the coding sequences in the bicistronic or multicistronic nucleic acid preferably encode distinct antigenic proteins or peptides or their immunogenic fragments or immunogenic variants as defined herein. Preferably, the coding sequences in the bicistronic or multicistronic construct may be separated by at least one IRES (intrasequence ribosome entry site) sequence. Thus, the term “encoding two or more antigenic peptides or proteins” may mean, but is not limited to, that the bicistronic or multicistronic nucleic acid encodes at least two, three, four, five, six or more (preferably different) antigenic peptides or proteins from, for example, different SARS-CoV-2 isolates. Alternatively, a bicistronic or multicistronic nucleic acid may encode, for example, at least two, three, four, five, or six (preferably different) antigenic peptides or proteins derived from the same SARS-CoV-2. In that context, suitable IRES sequences may be selected from the nucleic acid sequences described in SEQ ID NOs. 1566-1662 of patent application WO2017 / 081082, or from a list of fragments or variants of these sequences. In this context, the disclosures of WO2017 / 081082 relating to IRES sequences are incorporated herein by reference.

[0230] In the context of the present invention, it should be understood that certain combinations of coding sequences can be generated by any combination of monocistronic, bicistronic, and multicistronic RNA constructs and / or multiple antigenic constructs to obtain a set of nucleic acids encoding multiple antigenic peptides or proteins as defined herein.

[0231] In a preferred embodiment, the A / U(A / T) content in the environment of the ribosome binding site of the RNA may be increased compared to the A / U(A / T) content in the environment of the ribosome binding site of the respective wild-type or reference RNA. This modification (increased A / U(A / T) content around the ribosome binding site) increases the efficiency of ribosome binding to the RNA. Effective binding of ribosomes to the ribosome binding site then has the effect of efficient translation RNA.

[0232] Therefore, in a particularly preferred embodiment, the RNA includes a ribosome-binding site, also referred to as a "Kozak sequence," which is identical to, or at least 80%, 85%, 90%, or 95% identical to, any one of the sequences of sequence numbers 180, 181, 22845-22847, or any fragment or variant thereof.

[0233] In a preferred embodiment, the RNA comprises at least one poly(N) sequence, for example, at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or a combination thereof.

[0234] In a preferred embodiment, the RNA of the present invention comprises at least one poly(A) sequence.

[0235] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” or “3' poly(A) tail” are intended to be recognized and understood by those skilled in the art, and are, for example, typically a sequence of adenosine nucleotides located at the 3' end of a linear (or circular) RNA (or cyclic RNA) of up to about 1000 adenosine nucleotides. Preferably, the poly(A) sequence is homopolymerized in nature, and for example, a poly(A) sequence of, for example, 100 adenosine nucleotides has a length of essentially 100 nucleotides. In other embodiments, the poly(A) sequence is interrupted by at least one nucleotide different from the adenosine nucleotides, and for example, a poly(A) sequence of, for example, 100 adenosine nucleotides may have a nucleotide length longer than 100 (including 100 adenosine nucleotides and / or the at least one nucleotide or nucleotide compartment different from the adenosine nucleotides).

[0236] The poly(A) sequence may contain about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. Preferably, the length of the poly(A) sequence may be at least about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides, or further longer than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides. In a particular embodiment, the RNA contains at least one poly(A) sequence containing 30 to 200 adenosine nucleotides, wherein the nucleotide at the 3' end of the RNA is adenosine.

[0237] In preferred embodiments, the RNA of the present invention comprises at least one poly(A) sequence containing about 30 to about 200 adenosine nucleotides. In particularly preferred embodiments, the poly(A) sequence contains about 64 adenosine nucleotides (A64). In particularly preferred embodiments, the poly(A) sequence contains about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence contains about 150 adenosine nucleotides.

[0238] In further embodiments, the RNA of the present invention comprises at least one poly(A) sequence containing about 100 adenosine nucleotides, the poly(A) sequence being interrupted by non-adenosine nucleotides, preferably 10 non-adenosine nucleotides (A30-N10-A70).

[0239] The poly(A) sequence as defined herein may be located directly at the 3' end of the RNA, preferably directly at the 3' end of the RNA.

[0240] In a preferred embodiment, the 3'-terminal nucleotide (the last 3'-terminal nucleotide in a polynucleotide chain) is the 3'-terminal A nucleotide of at least one poly(A) sequence. The term “directly located at the 3' end” should be understood as being precisely located at the 3' end, or in other words, the 3' end of the nucleic acid consists of a poly(A) sequence and terminates with an A nucleotide.

[0241] The inventors have shown that this embodiment is particularly beneficial for inducing an immune response against SARS-CoV-2. In this context, it has already been shown that a single dose of vaccination is sufficient to produce a virus-neutralizing antibody titer.

[0242] In a particularly preferred embodiment, the RNA sequence comprises a poly(A) sequence of at least 70 adenosine nucleotides, where the nucleotide at the 3' end is an adenosine nucleotide.

[0243] In this context, it has been shown that ending with an adenosine nucleotide reduces the induction of IFN-alpha by RNA vaccines. This is particularly important because the induction of IFN-alpha is considered a major factor for inducing fever in vaccinated subjects, and must, of course, be avoided.

[0244] In preferred embodiments, the poly(A) sequence of RNA is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly(A) sequence is not necessarily transcribed from a DNA template but is obtained in vitro by common chemical synthesis methods. In other embodiments, the poly(A) sequence is generated by enzymatic polyadenylation of RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively by using an immobilized poly(A) polymerase using, for example, the methods and means described in WO2016 / 174271, the entire contents of WO2016 / 174271 are incorporated herein by reference.

[0245] In some embodiments, the RNA includes a poly(A) sequence obtained by enzymatic polyadenylation, and the majority of the RNA molecule contains about 100 (+ / -20) to about 500 (+ / -50), preferably about 250 (+ / -20) adenosine nucleotides.

[0246] In other embodiments, the RNA comprises a poly(A) sequence derived from template DNA and at least one further poly(A) sequence produced by enzymatic polyadenylation, for example, as described in WO2016 / 091391, the entire contents of WO2016 / 091391 incorporated herein by reference.

[0247] In further embodiments, the RNA includes at least one poly(C) sequence.

[0248] As used herein, the term “poly(C) sequence” is intended to mean a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In preferred embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In particularly preferred embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0249] In a preferred embodiment, the RNA of the present invention comprises at least one histone stem-loop (hSL).

[0250] The term "histone stem-loop" (for example, abbreviated as "hSL" in sequence listings) is intended to refer to the nucleic acid sequence that forms the stem-loop secondary structure primarily found in histone mRNA.

[0251] The histone stem-loop sequence / structure may preferably be selected from the histone stem-loop sequences disclosed in WO2012 / 019780, the entirety of WO2012 / 01978 is incorporated herein by reference, and the disclosure relating to histone stem-loop sequences / histone stem-loop structures is incorporated herein by reference. The histone stem-loop sequences that may be used in the present invention may preferably be derived from formula (I) or (II) of WO2012 / 019780. According to a further preferred embodiment, the RNA comprises at least one histone stem-loop sequence derived from at least one of the specific formulas (Ia) or (IIa) of patent application WO2012 / 019780.

[0252] In a preferred embodiment, the RNA of the present invention comprises at least one histone stem loop, wherein the histone stem loop (hSL) comprises or consists of a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, sequence number 178 or 179.

[0253] In other embodiments, the RNA does not include histone stem loops as defined herein.

[0254] In various embodiments, the RNA includes a 3' terminal sequence element. The 3' terminal sequence element includes a poly(A) sequence and optionally a histone stem-loop sequence. Thus, the RNA of the present invention includes a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or comprises at least one 3' terminal sequence element consisting of, SEQ ID NOs: 254, 22893, 22903, 26997, 26999, 28529, 28531, 28533, 28535, 28537, and 28539, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or consisting of at least one 3' terminal sequence element consisting of, such as, SEQ ID NOs: 254, 22893, 22903, 26997, 26999, 28529, 28531, 28533, 28535, 28537, and 28539.

[0255] In a preferred embodiment, the RNA includes a 3' terminal sequence element. The 3' terminal sequence element includes a poly(A) sequence. Thus, the nucleic acid of the present invention includes a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NOs: 254, 22903, 26999, 28531, 28525, 28539, or 99% identical thereto, or includes at least one 3' terminal sequence element consisting of such sequence.

[0256] In a preferred embodiment, the RNA includes a 3' terminal sequence element. The 3' terminal sequence element includes a poly(A) sequence and a histone stem-loop sequence. Thus, the nucleic acid of the present invention includes a nucleic acid sequence or fragment or variant thereof that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NOs: 254, 22893, 26997, 28529, 28533, and 28537, or at least one 3' terminal sequence element consisting of such sequence.

[0257] In various embodiments, the RNA may include the 5' terminal sequence element described in SEQ ID NOs: 176, 177, or 22840-22844, or a fragment or variant thereof.

[0258] In further embodiments, the RNA may include the 5' terminal sequence element described in SEQ ID NOs: 176, 177, or 22840-22844, or a fragment or variant thereof. Such a 5' terminal sequence element includes, for example, a T7 RNA polymerase binding site. Furthermore, the first nucleotide of the 5' terminal start sequence may preferably include a 2'O methylation, such as 2'O methylated guanosine or 2'O methylated adenosine.

[0259] In preferred embodiments, at least one heterologous 5'UTR contains or consists of a nucleic acid sequence derived from a 5'UTR of HSD17B4, and at least one heterologous 3'UTR contains or consists of a nucleic acid sequence derived from a 3'UTR of PSMB3. In certain embodiments, the 5'UTR derived from HSD17B4 is at least about 95%, 96%, 97%, 98%–99% identical to SEQ ID NO: 232. In some embodiments, the 3'UTR of PSMB3 is at least about 95%, 96%, 97%, 98%–99% identical to SEQ ID NO: 254. In a particularly preferred embodiment, the RNA comprises, from 5' to 3', i) a 5' cap 1 structure; ii) a 5' UTR derived from the 5' UTR of the HSD17B4 gene, preferably the 5' UTR described in SEQ ID NO: 232; iii) at least one coding sequence (encoding the SARS-CoV spike antigen of the embodiment); iv) a 3' UTR derived from the 3' UTR of the PSMB3 gene, preferably the 3' UTR described in SEQ ID NO: 254; v) optionally a histone stem-loop sequence; and vi) a poly(A) sequence containing about 100 A nucleotides, wherein the nucleotide at the 3' end of the RNA is adenosine.

[0260] Preferably, the RNA contains about 50 to about 20,000 nucleotides, or about 500 to about 10,000 nucleotides, or about 1,000 to about 10,000 nucleotides, or preferably about 1,000 to about 5,000 nucleotides, or more preferably about 2,000 to about 5,000 nucleotides.

[0261] In a particularly preferred embodiment, the RNA is coding RNA. In a preferred embodiment, the coding RNA may be selected from mRNA, (coding) self-replicating RNA, (coding) circular RNA, (coding) viral RNA, or (coding) replicon RNA.

[0262] In other embodiments, the coding RNA is circular RNA. As used herein, “circular RNA” or “circRNA” should be understood as a circular polynucleotide construct encoding at least one antigenic peptide or protein as defined herein. Preferably, such circRNA is a single-stranded RNA molecule. In preferred embodiments, the circRNA comprises at least one coding sequence encoding at least one antigenic protein derived from SARS-CoV-2 coronavirus, or an immunogenic fragment or immunogenic variant thereof.

[0263] In further embodiments, the coding RNA is replicon RNA. The term “replicon RNA” is intended to be, for example, an optimized self-replicating RNA, as recognized and understood by those skilled in the art. Such a construct may include, for example, substitution of a replicase element and structural viral protein with the nucleic acid of interest (i.e., a coding sequence encoding an antigenic peptide or protein of the SARS-CoV-2 coronavirus) derived from an alphavirus (e.g., SFV, SIN, VEE, or RRV). Alternatively, the replicase may be provided in an independent coding RNA construct or a coding DNA construct. Downstream of the replicase may be a subgenome promoter that controls the replication of the replicon RNA.

[0264] In a particularly preferred embodiment, at least one nucleic acid is neither a replicon RNA nor a self-replicating RNA.

[0265] In a particularly preferred embodiment, the RNA of the present invention is mRNA.

[0266] Preferably, the mRNA does not contain replicase elements (e.g., nucleic acids that encode replicases).

[0267] The terms "RNA" and "mRNA" are intended to be recognized and understood by those skilled in the art, for example, as ribonucleic acid molecules, i.e., polymers composed of nucleotides. These nucleotides are typically adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate, and cytidine-monophosphate monomers, linked to one another along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar of an adjacent first monomer, i.e., ribose, and the phosphate portion of a second monomer. A particular sequence of monomers is called an RNA sequence. mRNA (messenger RNA) results in a nucleotide coding sequence that can be translated into the amino acid sequence of a particular peptide or protein.

[0268] In the context of the present invention, the RNA, preferably mRNA, gives rise to at least one coding sequence that encodes an antigenic protein derived from the SARS-CoV-2 spike protein as defined herein, which is translated into a (functional) antigen after administration (e.g., after administration to a subject, e.g., a human subject).

[0269] In preferred embodiments, the RNA, preferably mRNA, is a SARS-CoV-2 vaccine, preferably the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B Suitable for SARS-CoV-2 vaccines against at least one of the following strains: B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0270] In a particularly preferred embodiment, RNA, preferably mRNA, is suitable for a SARS-CoV-2 vaccine, preferably against B.1.351 (South Africa) or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and BA.1_v5).

[0271] Preferably, the RNA may be modified by the addition of a 5' cap structure, which preferably stabilizes the RNA and / or enhances the expression of the encoded antigen and / or reduces stimulation of the innate immune system (after administration to the subject). The 5' cap structure is particularly important in embodiments where the RNA is linear coding RNA, such as linear mRNA or linear coding replicon RNA.

[0272] Therefore, in preferred embodiments, the RNA, particularly mRNA, includes a 5' cap structure, preferably a cap 0, cap 1, cap 2, modified cap 0, or modified cap 1 structure.

[0273] As used herein, the term “5' cap structure” is intended to be recognized and understood by those skilled in the art and to refer to, for example, a 5' modified nucleotide located at the 5' end of RNA, such as mRNA, particularly a guanine nucleotide. Preferably, the 5' cap structure is attached to the RNA via a 5'-5'-triphosphate bond.

[0274] Suitable 5' cap structures in the context of the present invention include cap 0 (methylation of the first nucleic acid base, e.g., m7GpppN), cap 1 (further methylation of the ribose of the nucleotide adjacent to m7GpppN), cap 2 (further methylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (further methylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (further methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0275] The 5' cap (cap 0 or cap 1) structure can be formed by chemical RNA synthesis or by RNA in vitro transcription (simultaneous transcription capping) using a cap analog.

[0276] As used herein, the term “capped analog” is intended to refer to a nonpolymerizable dinucleotide or trinucleotide that has capping functionality in that it promotes translation or localization and / or prevents the degradation of nucleic acid molecules, particularly RNA molecules, when incorporated at the 5' end of a nucleic acid molecule, as recognized and understood by those skilled in the art. Nonpolymerizable means that the capped analog does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by template-dependent polymerases, particularly by template-dependent RNA polymerases, and is therefore incorporated only at the 5' end. Examples of capped analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; non-methylated capped analogs (e.g., GpppG); dimethylated capped analogs (e.g., m2,7GpppG); trimethylated capped analogs (e.g., m2,2,7GpppG); dimethylated symmetric capped analogs (e.g., m7Gpppm7G); or anti-reverse capped analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). Further capping analogs are described previously (WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475). Further preferred capping analogs in that context are described in WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 053297, WO2017 / 066782, WO2018 / 075827, and WO2017 / 066797, and disclosures relating to capping analogs are incorporated herein by reference.

[0277] In some embodiments, the modified cap 1 structure is generated using the tri-nucleotide cap analogs disclosed in WO2017 / 053297, WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 066782, WO2018 / 075827 and WO2017 / 066797, and the entirety of the aforementioned PCT applications is incorporated herein by reference. In particular, the modified cap structure 1 may be generated co-transferred by appropriately using any cap structure derivable from the structures disclosed in claims 1 to 5 of WO2017 / 053297. Furthermore, the modified cap structure 1 may be generated co-transferred by appropriately using any cap structure derivable from the structures defined in claim 1 or claim 21 of WO2018 / 075827.

[0278] In preferred embodiments, RNA, particularly mRNA, includes a cap-1 structure.

[0279] In preferred embodiments, the 5' cap structure may preferably be added co-transcribed using a tri-nucleotide cap analog as defined herein in an RNA in vitro transcription reaction as defined herein.

[0280] In a preferred embodiment, the cap 1 structure of the coding RNA of the present invention is formed by co-transcriptional capping using the tri-nucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. The preferred cap 1 analog in that context is m7G(5')ppp(5')(2'OMeA)pG.

[0281] In another preferred embodiment, the RNA cap 1 structure of the present invention is formed using co-transcriptional capping with the tri-nucleotide capping analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0282] In other embodiments, the RNA cap 0 structure of the present invention is formed using simultaneous transcription capping with the cap analog 3'OMe-m7G(5')ppp(5')G.

[0283] In other embodiments, the 5' cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase) to generate the cap 0, cap 1, or cap 2 structure. The 5' cap structure (cap 0 or cap 1) may also be added using an immobilized capping enzyme and / or cap-dependent 2'-O methyltransferase using the methods and means disclosed in WO2016 / 193226, the entire contents of WO2016 / 193226 being incorporated herein by reference.

[0284] In preferred embodiments, approximately 70%, 75%, 80%, 85%, 90%, and 95% of RNA(species) contain a cap 1 structure determined using a capping assay. In preferred embodiments, less than approximately 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of RNA(species) do not contain a cap 1 structure determined using a capping assay. In other preferred embodiments, approximately 70%, 75%, 80%, 85%, 90%, and 95% of RNA(species) contain a cap 0 structure determined using a capping assay. In preferred embodiments, less than approximately 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of RNA(species) do not contain a cap 0 structure determined using a capping assay.

[0285] The term "RNA species" is not limited to meaning "a single molecule," but is understood to include an ensemble of essentially identical RNA molecules. Therefore, it can refer to multiple essentially identical (coding) RNA molecules.

[0286] To determine the presence or absence of a cap 0 or cap 1 structure, a capping assay described in published PCT application WO 2015 / 101416, in particular claims 27-46 of published PCT application WO 2015 / 101416, whose entire contents are incorporated herein by reference, can be used. Other capping assays that may be used to determine the presence or absence of a cap 0 or cap 1 structure in RNA are described in PCT / EP2018 / 08667, or published PCT applications WO2014 / 152673 and WO2014 / 152659, whose entire contents are incorporated herein by reference.

[0287] In preferred embodiments, the RNA contains an m7G(5')ppp(5')(2'OMeA) cap structure. In such embodiments, the coding RNA contains an m7G cap at the 5' end and further methylation of the ribose of the nucleotides adjacent to m7GpppN, in which case 2'O methylated adenosine. Preferably, about 70%, 75%, 80%, 85%, 90%, and 95% of the RNA(species) contain such cap structures as determined by a capping assay.

[0288] In other preferred embodiments, the RNA contains an m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the coding RNA contains an m7G cap at the 5' end and further methylation of the ribose of the adjacent nucleotide, in this case, 2'O methylated guanosine. Preferably, about 70%, 75%, 80%, 85%, 90%, and 95% of the coding RNA(s) contain such cap structures as determined by a capping assay.

[0289] Therefore, the first nucleotide of the RNA or mRNA sequence, i.e., the nucleotide downstream of the m7G(5')ppp structure, may be 2'O-methylated guanosine or 2'O-methylated adenosine.

[0290] According to some embodiments, RNA is modified RNA, and the modifications refer to chemical modifications including skeletal modifications and sugar or base modifications.

[0291] Modified RNA may include nucleotide analogs / modifications, such as skeletal modifications, sugar modifications, or base modifications. In the context of the present invention, skeletal modification is a modification in which the phosphate of the nucleotide backbone of RNA is chemically modified. Sugar modification in the context of the present invention is a chemical modification of the sugar of the nucleotide of RNA. Furthermore, base modification in the context of the present invention is a chemical modification of the base portion of the nucleotide of RNA. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0292] In particularly preferred embodiments, the nucleotide analogs / modifications that can be incorporated into the modified RNA described herein are preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, and 2'-O-methylinosine-5'-triphosphate. 4-Thiouridine-5'-triphosphate, 5-Aminoallylcytidine-5'-triphosphate, 5-Aminoallyluridine-5'-triphosphate, 5-Bromocytidine-5'-triphosphate, 5-Bromouridine-5'-triphosphate, 5-Bromo-2'-deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5'-triphosphate, 5-Iodocytidine-5'-triphosphate, 5-Iodo-2'-deoxycytidine-5'-triphosphate, 5-Iodouridine-5'-triphosphate, 5-Iodo-2'-deoxyuridine-5'-triphosphate, 5-Methylcytidine-5'-triphosphate, 5-Methyluridine-5'-triphosphate, 5-Propynyl-2'-deoxycytidine-5'-triphosphate, 5-Propynyl-2' - Selected from deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridine-4-onribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseuduridine, 2-thio-pseuduridine,5-Hydroxyuridine, 3-Methyluridine, 5-Carboxymethyluridine, 1-Carboxymethyl-Pseudouridine, 5-Propynyluridine, 1-Propynyl-Pseudouridine, 5-Taurinomethyluridine, 1-Taurinomethyl-Pseudouridine, 5-Taurinomethyl-2-Thio-uridine, 1-Taurinomethyl-4-Thio-uridine, 5-Methyluridine, 1-Methyl-Pseudouridine, 4-Thio-1-Methyl-Pseudouridine, 2-Thio-1-Methyl-Pseudouridine, 1-Methyl-1-Deaza-Pseudouridine N, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, and 4-methoxy-2-thio-pseuduridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine , pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, and 4-Methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-adenine, and 2-Methoxyadenine, Inosine, 1-Methylinosine, Waiosine, Waibutosine, 7-Deaza-Guanosine, 7-Deaza-8-Aza-Guanosine, 6-Thio-Guanosine, 6-Thio-7-Deaza-Guanosine, 6-Thio-7-Deaza-8-Aza-Guanosine, 7-Methyl -Guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate) (Axate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudolidine, 6-azacytidine, 2-thiocytidine, alpha-thiocytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudolidine, 5,6-dihydrouridine, alpha-thiouridine, 4-thiouridine, 6-aza-uridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine Nucleotides for base modification are particularly preferred, selected from the group of base-modified nucleotides consisting of n, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0293] In some embodiments, at least one modified nucleotide is selected from pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0294] In some embodiments, 100% of the uracil in the coding sequence as defined herein has a chemical modification, preferably the chemical modification is located at the 5' position of the uracil.

[0295] In the context of the present invention, pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine are particularly preferred.

[0296] However, in some embodiments, the RNA of the present invention does not include the N1-methylpseudridine (m1Ψ) substituted position. In further embodiments, the RNA of the embodiment does not include the pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine substituted positions. Still in further embodiments, the RNA of the present invention includes a coding sequence consisting only of G, C, A, and U nucleotides.

[0297] The incorporation of modified nucleotides, such as pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine, into the RNA coding sequence may be advantageous as an unwanted innate immune response (in the event of administration of coding RNA or vaccine) that can be modulated or reduced (if necessary).

[0298] In some embodiments, the RNA comprises at least one coding sequence encoding a SARS-CoV-2 antigenic protein as defined herein, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudridine (m1ψ), preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0299] In preferred embodiments, the RNA does not contain the N1-methylpseudridine (m1Ψ) substituted position. In further embodiments, the RNA does not contain the pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine substituted positions.

[0300] In a preferred embodiment, the RNA comprises a coding sequence consisting only of G, C, A, and U nucleotides, and therefore does not contain modified nucleotides (except for the 5' terminal cap structure, e.g., cap 1).

[0301] Nucleic acids suitable for coronavirus vaccines, preferably mRNA constructs: In various embodiments, RNA, preferably mRNA, preferably has the following elements in the 5' to 3' direction: A) 5' cap structure, preferably a 5' cap structure as specified herein; B) A 5'-terminated start element, preferably a 5'-terminated start element as specified herein; C) Where applicable, 5'UTR, preferably the 5'UTR as specified herein; D) Ribosome binding sites, preferably ribosome binding sites as specified herein; E) At least one code sequence, preferably at least one code sequence as specified herein; F) 3'UTR, preferably the 3'UTR as specified herein; G) Depending on the case, a poly(A) sequence, preferably a poly(A) sequence as specified herein; H) Depending on the case, a poly(C) sequence, preferably a poly(C) sequence as specified herein; I) In some cases, histone stem loops, preferably histone stem loops as specified herein; J) In some cases, 3' terminal array elements, preferably 3' terminal array elements as specified herein. Includes.

[0302] In a particularly preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap1 structure as defined in this specification; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089~23148, 23150~23184, 27110~27247, 28589~28637, 28916, 28921~28924 or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; E) Histone stem loop selected from sequence number 178 or 179; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0303] In a further preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089~23148, 23150~23184, 27110~27247, 28589~28637, 28916, 28921~28924 or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0304] In a particularly preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 27118, 27141, 27164, 27187, 27210, 27233, 28601-28606 or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; E) Histone stem loop selected from sequence number 178 or 179; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0305] In a further preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 27118, 27141, 27164, 27187, 27210, 27233, 28601-28606 or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0306] In a particularly preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 27118, 27141, 27164, 27187, 27210, 27233, or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; E) Histone stem loop selected from sequence number 178 or 179; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0307] In a further preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 27118, 27141, 27164, 27187, 27210, 27233, or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0308] In a particularly preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 28590-28593, 28921-28924, or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; E) Histone stem loop selected from sequence number 178 or 179; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0309] In a further preferred embodiment, the nucleic acid, preferably mRNA, has the following elements in the 5' to 3' direction: A) Cap 1 structure as defined herein; B) A 5'UTR derived from the HSD17B4 gene as defined herein, preferably as described in SEQ ID NO: 231 or 232; C) Code sequences selected from sequence numbers 28590-28593, 28921-28924, or their fragments or variants; D) A 3'UTR derived from the PSMB3 gene as defined herein, preferably the 3'UTR described in SEQ ID NO: 253 or 254; F) A poly(A) sequence containing approximately 100 A nucleotides, preferably representing the 3' end. Includes.

[0310] Preferred RNA sequences, preferably mRNA sequences, of the present invention are provided in Table 2. Thereafter, each column represents a specific preferred SARS-CoV-2 construct of the present invention, where a description of the SARS-CoV-2 skeletal construct is shown in column A of Table 2, and the corresponding RNA sequence, in particular the mRNA sequence, containing a preferred coding sequence is provided in columns C-G. Table 2a provides RNA sequences containing HSD17B4 / PSMB3-hSL-A100(a-1). Table 2b provides RNA sequences containing HSD17B4 / PSMB3-A100(a-1).

[0311] [Table 2a] TIFF0007894882000011.tif221167TIFF0007894882000012.tif220167TIFF0007894882000013.tif215167TIFF0007894882 000014.tif220167TIFF0007894882000015.tif215167TIFF0007894882000016.tif224167TIFF0007894882000017.tif90167

[0312] [Table 2b] TIFF0007894882000019.tif210168TIFF0007894882000020.tif220167TIFF0007894882000021.tif219168TIFF00078948820 00022.tif220168TIFF0007894882000023.tif215167TIFF0007894882000024.tif220167TIFF0007894882000025.tif138168

[0313] In certain embodiments, RNA, preferably mRNA, is sequence numbers 148, 149, 151, 162, 163, 165, 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835, 22837, 22839, 23309~23368, 23370~23404, 23529~ The RNA sequence is identical to, or comprises, a nucleic acid sequence selected from the group consisting of 23588, 23590-23624, 24837-24944, 27248-27907, 28638-28915, and 28925-28940, or is identical to, or comprises, a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0314] In certain embodiments, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of sequence numbers provided in columns B to G of Table 2a or Table 2b, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0315] In certain embodiments, RNA, preferably mRNA, is sequence numbers 27256, 27279, 27302, 27325, 27348, 27371, 27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601, 27624, 27647, 27688, 27729, 27770, 27811, 27852, 27893, 28650~28655, 28699~ The RNA sequence is identical to, or comprises, a nucleic acid sequence selected from the group consisting of 28704, 28762, 28789-28794, 28852, and 28879-28884, or is identical by at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0316] In certain embodiments, RNA, preferably mRNA, is sequence numbers 27256, 27279, 27302, 27325, 27348, 27371, 27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601, 27624, 27647, 27688, 27729, 27770, 27811, 27852, The RNA sequence is identical to, or comprises, a nucleic acid sequence selected from the group consisting of 27893, 28762, and 28852, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0317] In certain embodiments, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28639-28642, 28778-28781, 28688-28691, 28868-28871, and 28925-28940, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0318] In a preferred embodiment, RNA, preferably mRNA, is sequence numbers 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-2868. 6. A nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, or consists of such a sequence. Further information regarding each nucleic acid sequence can be found in the sequence listing for each sequence number. <223> This information is provided below the identifier and in Table 2 (see columns B to G in particular).

[0319] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27394, 27417, 27486, 28762, 28650-28655, and 28789-28794, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0320] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27394, 27417, 27486, and 28762, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0321] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28639-28642, 28778-28781, 28925-28928, and 28933-28936, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0322] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, and 28937-28940, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0323] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27532, 27555, 27624, 28852, 28699-28704, and 28879-28884, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0324] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27532, 27555, 27624, and 28852, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0325] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28688-28691, 28868-28871, 28929-28932, and 28937-28940, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences.

[0326] In a preferred embodiment, RNA, preferably mRNA, is selected from the group consisting of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, and 28933-28936. A nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or comprising a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0327] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27394, 27417, 27486, 28762, 28650-28655, and 28789-28794, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0328] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27394, 27417, 27486, and 28762, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0329] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28639-28642, 28778-28781, 28925-28928, and 28933-28936, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0330] In a preferred embodiment, RNA, preferably mRNA, is identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940, or at least 70%, 80%, 85%, 86%, 8%. The RNA sequence comprises or consists of nucleic acid sequences that are 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or fragments or variants of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0331] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27532, 27555, 27624, 28852, 28699-28704, and 28879-28884, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0332] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27532, 27555, 27624, and 28852, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0333] In a preferred embodiment, the RNA, preferably mRNA, is identical to, or comprises, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28688-28691, 28868-28871, 28929-28932, and 28937-28940, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0334] In certain embodiments, the RNA of the present invention may be prepared using any method known in the art, including chemical synthesis, such as solid-phase RNA synthesis, and in vitro methods, such as RNA in vitro transcription reactions. Therefore, in preferred embodiments, the RNA is obtained by RNA in vitro transcription.

[0335] Therefore, in preferred embodiments, the RNA of the present invention is preferably RNA transcribed in vitro.

[0336] The term “RNA in vitro transcription” or “in vitro transcription” refers to the process by which RNA is synthesized in a cell-free system (in vitro). RNA may be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which is a linear plasmid DNA template or a PCR-amplified DNA template, according to the present invention. A promoter for regulating RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerase. In a preferred embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme, and then subjected to RNA in vitro transcription.

[0337] The reagents used in RNA in vitro transcription typically include: a DNA template (linear plasmid DNA or PCR product) having a promoter sequence with high binding affinity to its respective RNA polymerase, e.g., bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for four bases (adenine, cytosine, guanine, and uracil); optionally, cap analogs as defined herein; optionally, further modified nucleotides as defined herein; DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) capable of binding to the promoter sequence in the DNA template; optionally, ribonuclease (RNase) inhibitors to inactivate any potentially contaminating RNases; optionally, pyrophosphatases to degrade pyrophosphates that may inhibit RNA in vitro transcription; and Mg as a cofactor for polymerases. 2+The solution includes a buffer (TRIS or HEPES) for maintaining a suitable pH value, which may also contain a buffer system containing MgCl2 to supply ions; an antioxidant (e.g., DTT); and / or a polyamine, such as spermidine at an optimal concentration, such as TRIS citrate as disclosed in WO2017 / 109161.

[0338] In a preferred embodiment, the cap 1 structure of the RNA of the present invention is formed by co-transcriptional capping using the tri-nucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred cap 1 analog that can be suitably used in the production of the coding RNA of the present invention is m7G(5')ppp(5')(2'OMeA)pG.

[0339] In a particularly preferred embodiment, the RNA cap 1 structure of the present invention is formed by co-transcriptional capping using the tri-nucleotide capping analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0340] In other embodiments, the RNA cap O structure of the present invention is formed using simultaneous transcription capping with the cap analog 3'OMe-m7G(5')ppp(5')G.

[0341] In additional embodiments, the nucleotide mixture used for RNA in vitro transcription may further include modified nucleotides as defined herein. In that context, preferred modified nucleotides may be selected from pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In certain embodiments, uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) with pseudouridine (ψ) and / or N1-methylpseudridine (m1ψ) to obtain modified RNA.

[0342] In preferred embodiments, the nucleotide mixture used for RNA in vitro transcription does not contain modified nucleotides as defined herein. In preferred embodiments, the nucleotide mixture used for RNA in vitro transcription contains only G, C, A, and U nucleotides, and optionally cap analogs as defined herein.

[0343] In a preferred embodiment, the nucleotide mixture used for the RNA in vitro transcription reaction (i.e., fractions of each nucleotide in the mixture) may be optimized to a predetermined RNA sequence, preferably a predetermined RNA sequence described in WO2015 / 188933, the entire contents of which are incorporated herein by reference.

[0344] In this context, in vitro transcription is performed in the presence of a sequence-optimized nucleotide mixture and optionally a capping analog, preferably where the sequence-optimized nucleotide mixture does not contain chemically modified nucleotides.

[0345] In this context, a sequence-optimized nucleoside triphosphate (NTP) mix is ​​a mixture of nucleoside triphosphates (NTPs) for use in in vitro transcription reactions of RNA molecules of a given sequence, including four nucleoside triphosphates (NTPs) GTP, ATP, CTP, and UTP, where each fraction of the four nucleoside triphosphates (NTPs) in the sequence-optimized nucleoside triphosphate (NTP) mix corresponds to the fraction of each nucleotide in the RNA molecule. If a ribonucleotide is not present in the RNA molecule, the corresponding nucleoside triphosphate is also not present in the sequence-optimized nucleoside triphosphate (NTP) mix.

[0346] In embodiments where more than one distinct RNA as defined herein must be produced, for example, two, three, four, five, six, seven, eight, nine, ten or even more distinct RNAs must be produced (see Second Embodiment), the method described in WO2017 / 109134 may be preferably used.

[0347] In the context of RNA-based vaccine production, it may be required to provide GMP-grade nucleic acids, such as GMP-grade RNA. GMP-grade RNA can be produced using a manufacturing process approved by the regulatory authority. Therefore, in a particularly preferred embodiment, RNA production is carried out under current Good Manufacturing Practices (GMP) that perform various quality control steps at the DNA (template) and RNA levels, preferably the quality control steps described in WO2016 / 180430. In a preferred embodiment, the RNA of the present invention is GMP-grade RNA, in particular GMP-grade mRNA. Therefore, RNA for vaccines is preferably GMP-grade RNA.

[0348] The obtained RNA product is preferably purified using PureMessenger® (RP-HPLC as described in CureVac, Tübingen, Germany; WO2008 / 077592) and / or tangent flow filtration (as described in WO2016 / 193206) and / or oligo-d(T) purification (see WO2016 / 180430).

[0349] In a further preferred embodiment, RNA is freeze-dried (e.g., as described in WO2016 / 165831 or WO2011 / 069586, the entirety of both PCT applications is incorporated herein by reference) to obtain temperature-stable dried RNA (powder). RNA may also be dried using spray drying or spray freeze-drying (e.g., as described in WO2016 / 184575 or WO2016 / 184576) to obtain temperature-stable RNA (powder) as defined herein. Accordingly, in the context of the production and purification of nucleic acids, particularly RNA, the disclosures of WO2017 / 109161, WO2015 / 188933, WO2016 / 180430, WO2008 / 077592, WO2016 / 193206, WO2016 / 165831, WO2011 / 069586, WO2016 / 184575, and WO2016 / 184576 are incorporated herein by reference.

[0350] Therefore, in a preferred embodiment, the RNA is dried RNA.

[0351] As used herein, the term “dried RNA” should be understood as RNA that has been freeze-dried, spray-dried, or spray-freeze-dried, as defined above, in order to obtain temperature-stable dried RNA (powder).

[0352] In preferred embodiments, the nucleic acid of the present invention is purified nucleic acid, particularly purified RNA.

[0353] As used herein, the term “purified nucleic acid” should be understood as nucleic acid having a higher purity than the starting material after a certain purification process. Essentially, typical impurities not present in purified nucleic acid include peptides or proteins, spermidine, BSA, incomplete nucleic acid sequences, nucleic acid fragments, free nucleotides, bacterial impurities, or impurities derived from the purification method. Therefore, a “degree of nucleic acid purity” as close to 100% as possible is desirable. It is also desirable that the amount of full-length nucleic acid be as close to 100% as possible. Thus, as used herein, “purified nucleic acid” has a purity higher than 75%, 80%, 85%, and more specifically, higher than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably 99% or higher. The degree of purity may also be determined, for example, by analytical HPLC, where the percentages obtained above correspond to the ratio between the area of ​​the peak of the target nucleic acid and the sum of the ranges of all peaks representing by-products. Alternatively, the degree of purity may be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0354] In a preferred embodiment, the nucleic acid of the present invention is purified RNA.

[0355] As used herein, the terms “purified RNA” or “purified mRNA” should be understood as RNA that has a higher purity than the starting material (e.g., RNA transcribed in vitro) after a particular purification step (e.g., HPLC, TFF, oligo-d(T) purification, precipitation step). Typical impurities not inherently present in purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, e.g., RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, incomplete RNA sequences, RNA fragments (short double-stranded RNA fragments, incomplete sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, capping analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other possible impurities that may be induced from fermentation methods, for example, include bacterial impurities (bioburden, bacterial DNA) or impurities induced from purification methods (e.g., organic solvents). Therefore, it is desirable that the “degree of RNA purity” be as close to 100% as possible. It is also desirable that the amount of full-length RNA transcript be as close to 100% as possible in terms of RNA purity. Therefore, as used herein, “purified RNA” has a purity of 99% or higher, more specifically, higher than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. The degree of purity may be determined, for example, by analytical HPLC, where the percentages obtained above correspond to the ratio between the peak area of ​​the target RNA and the total range of all peaks representing byproducts. Alternatively, the degree of purity may be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0356] In a particularly preferred embodiment, the RNA is purified by RP-HPLC and / or TFF to remove double-stranded RNA, uncapped RNA, and / or RNA fragments.

[0357] For example, the formation of double-stranded RNA as a byproduct during RNA in vitro transcription can lead to the induction of innate immune responses, particularly IFN-alpha, a major factor inducing fever in vaccinated subjects, which is, of course, an undesirable side effect. Current techniques for immunoblotting of dsRNA (via dot blotting, serologically specific electron microscopy (SSEM), or ELISA, etc.) are used to detect and size-classify dsRNA species from a mixture of nucleic acids.

[0358] Preferably, the RNA of the present invention is purified by RP-HPLC and / or TFF as described herein to reduce the amount of dsRNA.

[0359] Preferably, the RNA according to the present invention is purified using RP-HPLC, preferably using reverse-phase high-pressure liquid chromatography (RP-HPLC) with a macroporous styrene / divinylbenzene column (e.g., particle size 30 μm, pore size 4000 Å), and further using a filter cassette equipped with a cellulose-based membrane having a molecular weight cutoff of about 100 kDa.

[0360] In this context, it is particularly preferable that the purified RNA is purified by RP-HPLC and / or TFF, yielding approximately 5%, 10%, or 20% less double-stranded RNA byproducts than those in RNA that has not been purified by RP-HPLC and / or TFF. Therefore, the RNA of the present invention contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than those in RNA that has not been purified by RP-HPLC and / or TFF.

[0361] Alternatively, the purified RNA obtained by RP-HPLC and / or TFF contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than the byproducts in RNA purified using oligo-dT purification, precipitation, filtration, and / or anion exchange chromatography. Therefore, the RP-HPLC and / or TFF-purified RNA of the present invention contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than the byproducts in RNA purified using oligo-dT purification, precipitation, filtration, and / or AEX.

[0362] In embodiments, the nucleic acids of the present invention may be produced and purified using an automated apparatus for RNA in vitro transcription. Such apparatus may also be used to produce compositions or vaccines (see embodiments 2 and 3). Preferably, the apparatus described in WO2020 / 002598 (the entire contents of which are incorporated herein by reference), in particular the apparatus described in claims 1 to 59 and / or 68 to 76 (and Figures 1 to 18) of WO2020 / 002598, can be suitably used.

[0363] The methods described herein may preferably be applied to methods for producing RNA compositions or vaccines, which are described in more detail below.

[0364] Compositions, pharmaceutical compositions: The second aspect relates to a composition comprising at least one RNA of the first aspect.

[0365] In particular, embodiments relating to the composition of the second embodiment may similarly be read and understood as preferred embodiments of the vaccine of the third embodiment. Similarly, embodiments relating to the vaccine of the third embodiment may similarly be read and understood as preferred embodiments of the composition of the second embodiment (containing at least one RNA of the first embodiment). Furthermore, features and embodiments described in the context of the first embodiment (RNA of the present invention) should be read and understood as preferred embodiments of the composition of the second embodiment.

[0366] In preferred embodiments, the composition comprises at least one RNA in a first embodiment that encodes at least one antigenic peptide or protein, which is either the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, or derived therefrom.

[0367] In a preferred embodiment, the composition comprises at least one RNA encoding at least one antigenic peptide or protein selected from or induced from the SARS-CoV-2 spike protein according to the first embodiment, or its immunogenic fragment or immunogenic variant, wherein the composition is preferably administered intramuscularly or intradermally.

[0368] Preferably, intramuscular or intradermal administration of the composition results in the expression of the encoded SARS-CoV-2 spike protein construct in the subject. In a preferred embodiment, administration of the composition results in RNA translation, leading to the production of the encoded SARS-CoV-2 spike protein in the subject.

[0369] Preferably, the composition of the second embodiment is suitable for a vaccine, and in particular for a SARS-CoV-2 vaccine, preferably the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India) Suitable for SARS-CoV-2 vaccines against at least one of the following strains: B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, United Kingdom), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0370] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against B.1.351 (beta, South Africa).

[0371] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against B.1.617.2, AY.1, AY.2, AY.4, or AY.4.2.

[0372] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against B.1.617.2.

[0373] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against B.1.1.529, B.1.1.529.1 / BA.1 (Omicron) and / or B.1.1.529.2 / BA.2.

[0374] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and / or BA.1_v5.

[0375] In a particularly preferred embodiment, the composition of the second embodiment is suitable for a SARS-CoV-2 vaccine against B.1.1.529 and B.1.617.2.

[0376] In the context of the present invention, “composition” means any type of composition in which a specific component (e.g., RNA encoding at least one antigenic peptide or protein selected or derived from SARS-CoV-2, for example, in association with a lipid-based carrier) can be incorporated, optionally together with any further components, typically together with at least one pharmaceutically acceptable carrier or excipient. The composition may be a dry composition, e.g., in powder or granule form, or in solid units, e.g., in lyophilized form. Alternatively, the composition may be in liquid form, and each component may be incorporated independently in dissolved or dispersed (e.g., suspended or emulsified) form.

[0377] In a preferred embodiment of the second aspect, the composition comprises at least one RNA of the first aspect and optionally at least one pharmaceutically acceptable carrier or excipient.

[0378] As used herein, the terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” preferably include the liquid or non-liquid main component of the composition for administration. If the composition is provided in liquid form, the carrier may be water, e.g., pyrogen-free water; isotonic saline or buffer (water) solution, e.g., phosphate, citrate, etc. Water or preferably a buffer, more preferably an aqueous buffer, containing a sodium salt, preferably at least 50 mM sodium salt, a calcium salt, preferably at least 0.01 mM calcium salt, and optionally a potassium salt, preferably at least 3 mM potassium salt. According to preferred embodiments, sodium, calcium, and optionally potassium salts may be in the form of their halides, e.g., chlorides, iodides, or bromides; or their hydroxides, carbonates, bicarbonates, or sulfates. Examples of sodium salts include NaCl, NaI, NaBr, Na2CO3, NaHCO3, and Na2SO4. Examples of potassium salts, depending on the case, include KCl, KI, KBr, K2CO3, KHCO3, and K2SO4. Examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2.

[0379] Furthermore, the aforementioned cationic organic anions may be in a buffer. Therefore, in embodiments, the nucleic acid composition may include pharmaceutically acceptable carriers or excipients, for example, one or more pharmaceutically acceptable carriers or excipients for increasing in vivo stability, increasing cell transfection, enabling persistence or delay, increasing translation of the encoded coronavirus protein, and / or altering the in vivo release profile of the encoded coronavirus protein. In addition to traditional excipients, e.g., any and all solvents, dispersions, diluents, or other liquid vehicles, the dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, and excipients of the present invention may, without limitation, include lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, polynucleotide-transfected cells, hyaluronidases, nanoparticle mimics, and combinations thereof. In embodiments, one or more suitable solid or liquid fillers or diluents or encapsulation compounds may also be used, and they are suitable for administration to a subject. As used herein, the term “suitability” means that the components of a composition are capable of being mixed with at least one, and possibly more, nucleic acids of the composition in such a manner that no interactions occur that substantially reduce the biological activity or pharmaceutically effectiveness of the composition under typical conditions of use (e.g., intramuscular or intradermal administration). A pharmaceutically acceptable carrier or excipient must be sufficiently pure and sufficiently low in toxicity to be suitable for administration to the target to be treated.Compounds that can be used as pharmaceutically acceptable carriers or excipients include sugars, e.g., lactose, glucose, trehalose, mannose, and sucrose; starches, e.g., corn starch or potato starch; dextrose; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; tragacanth powder; malt; gelatin; animal fat; solid lubricants, e.g., stearic acid, magnesium stearate; calcium sulfate; vegetable oils, e.g., peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa-derived oils; polyols, e.g., polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0380] At least one pharmaceutically acceptable carrier or excipient of the composition may be preferably selected to be suitable for intramuscular or intradermal delivery / administration of the composition. Therefore, the composition is preferably a pharmaceutical composition, preferably a composition for intramuscular administration.

[0381] The compositions, preferably the subjects to whom the pharmaceutical compositions are intended to be administered, include, but are not limited to, humans and / or other primates; commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or commercially relevant birds, such as poultry, chickens, ducks, geese, and / or turkeys.

[0382] The pharmaceutical composition of the present invention may preferably be sterile and / or pyrogen-free.

[0383] The polyvalent composition of the present invention: In embodiments, the compositions defined herein (e.g., polyvalent compositions) may contain more than one RNA species as defined in the context of the first embodiment of the present invention. Preferably, the compositions defined herein may each contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 different RNA species as defined in the context of the first embodiment.

[0384] In the embodiment, the composition (e.g., a polyvalent composition) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different RNA species as defined in the context of the first embodiment, each encoding at least one different SARS-CoV-2 spike protein (as defined in the context of the first embodiment).

[0385] In this context, different SARS-CoV-2 spike proteins or stabilized spike proteins before fusion, ·K986, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, A67V, T95I, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, T19I, L24del, P25del, P26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, D796Y, Q954H, and N969K; ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, N440K, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, and L981F; ·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V; · E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I; · E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F; L452R, P681R, and D614G; L452R, E484Q, P681R, E154K, D614G, and Q1071H; or L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N It is even more preferable that the spike protein contains amino acid changes.

[0386] In this context, different SARS-CoV-2 spike proteins or stabilized spike proteins before fusion, ·E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or · E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V It is even more preferable that the spike protein contains amino acid changes.

[0387] In this context, different SARS-CoV-2 spike proteins or stabilized spike proteins before fusion, The following amino acid changes in spike proteins: K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F At least one SARS-CoV-2 spike protein or a stabilized spike protein before fusion having; and The following amino acid changes in spike proteins: L452R, E484Q, P681R, E154K, D614G, and Q1071H; or L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N At least one SARS-CoV-2 spike protein or a stabilized spike protein before fusion having It is even more preferable that the spike protein contains amino acid changes.

[0388] In a preferred embodiment, the composition (e.g., a polyvalent composition) comprises two, three, four, or five RNA species and optionally at least one pharmaceutically acceptable carrier or excipient, wherein the RNA species are SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 287 The nucleic acid sequence is identical to, or comprises, a nucleic acid sequence selected from the group consisting of 36-28776, 28638-28686, 28777-28825, 28925-28928, and 28933-28936, or is identical to, or comprises, a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, where each of the 2, 3, 4, or 5 nucleic acid species encodes a different SARS-CoV-2 spike protein.

[0389] In a preferred embodiment, the composition (e.g., a polyvalent composition) comprises two, three, four, or five RNA species and optionally at least one pharmaceutically acceptable carrier or excipient, wherein the RNA species are sequence numbers 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867- A nucleic acid sequence selected from the group consisting of 28915, 28937-28940, which is identical to, or comprises, a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, where each of the 2, 3, 4, or 5 nucleic acid species encodes a different SARS-CoV-2 spike protein.

[0390] Particularly preferred embodiments of the polyvalent composition are provided below.

[0391] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that is identical to any one of SEQ ID NOs: 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 22960-22961 or 28540; and / or iii) A single RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 27093-27095 or 28552-28558, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 27095, 28552-28558; and / or vii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27095, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or viii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 28541-28544 or 28917-28920, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence. It includes at least two, three, or four additional RNA species selected from the following.

[0392] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that encodes an amino acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27093-27095 or 28552-28558, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 22960-22961 or 28540; and / or iii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) One RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 28541-28544 or 28917-28920, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. It includes at least two, three, or four additional RNA species selected from the following.

[0393] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of SEQ ID NOs. 27095, 28552-28558, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 22960-22961 or 28540; and / or iii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) One RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 28541-28544 or 28917-28920, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. It includes at least two, three, or four additional RNA species selected from the following.

[0394] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that is identical to any one of Sequence ID No. 27095, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 22960-22961 or 28540; and / or iii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) One RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 28541-28544 or 28917-28920, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. It includes at least two, three, or four additional RNA species selected from the following.

[0395] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of SEQ ID NOs. 22960-22961, 28540, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or iii) A single RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 27093-27095 or 28552-28558, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 27095, 28552-28558; and / or vii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27095, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or viii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 28541-28544 or 28917-28920, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence. It includes at least two, three, or four additional RNA species selected from the following.

[0396] In a preferred embodiment, the polyvalent composition comprises one RNA species containing a coding sequence that encodes an amino acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920, wherein the polyvalent composition further comprises i) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27108-27109, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence; and / or ii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 10, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or iii) A single RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 27093-27095 or 28552-28558, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NOs: 27096 or 28545; and / or v) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 22959, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or vi) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of sequence numbers 27095, 28552-28558; and / or vii) A single RNA species containing a coding sequence that is identical to any one of sequence numbers 27095, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences; and / or viii) One RNA species containing a coding sequence that encodes an amino acid sequence identical to any one of sequence numbers 22960-22961 or 28540, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. It includes at least two, three, or four additional RNA species selected from the following.

[0397] In preferred embodiments, the composition, preferably a polyvalent composition, includes C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, USA), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Bay Suitable for vaccines against B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427 / B.1.429 (Epsilon, California, USA), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Iota, New York, USA), A.23.1 (Uganda), B.1.617.1 (Copper, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and / or B.1.621 (Mu, Colombia).

[0398] In the embodiment, the RNA contained in the composition is provided in amounts of about 100 ng to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 5 μg to about 100 μg, preferably about 10 μg to about 50 μg, specifically about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0399] If the composition contains more than one or more RNA species as defined herein (a polyvalent composition), the amount of RNA for each RNA species is approximately 100 ng to approximately 500 μg, approximately 1 μg to approximately 200 μg, approximately 1 μg to approximately 100 μg, approximately 5 μg to approximately 100 μg, preferably approximately 10 μg to approximately 50 μg, specifically, It is provided in quantities of approximately 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0400] In some embodiments, the amount of RNA for each RNA species is essentially equal in mass. In other embodiments, the amount of RNA for each RNA species is selected to be equimolar.

[0401] Complexation: In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, is complexed or associated with a further compound to obtain a complexed formulation. The complexed formulation may have the function of a transfection agent. The complexed formulation may also have the function of protecting RNA and / or mRNA from degradation.

[0402] In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, and optionally at least one further RNA, is complexed or associated, or at least partially complexed or partially associated, with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof.

[0403] As used herein, the term “cationic or polycationic compound” is intended to refer to a charged molecule that is recognized and understood by those skilled in the art and is positively charged at a pH range of about 1 to 9, about 3 to 8, about 4 to 8, about 5 to 8, more preferably about 6 to 8, even more preferably about 7 to 8, and most preferably, at a physiological pH range of about 7.2 to 7.5. Accordingly, cationic components, such as cationic peptides, cationic proteins, cationic polymers, cationic polysaccharides, and cationic lipids, may be any positively charged compound or polymer that is positively charged under physiological conditions. “Cationic or polycationic peptide or protein” may contain, for example, at least one positively charged amino acid selected from Arg, His, Lys, or Orn, or one or more positively charged amino acids. Therefore, the "polycationic" component is also within the range of exhibiting one or more positive charges under given conditions.

[0404] In this context, particularly preferred cationic or polycationic compounds are cationic or polycationic peptides or proteins or fragments thereof from the following list: protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, HIV-binding peptides, HIV-1 Tat (HIV), Tat-inducing peptides, penetratin, cell-permeable peptides (CPPs) including VP22-inducing or analog peptides, and HSV. The following can be selected: VP22 (herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG peptide, Pep-1, L-oligomer, calcitonin peptide, Antennapedia-inducing peptide, pAntp, pIsl, FGF, lactoferrin, transportan, buphorin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-inducing peptide, SAP, or histone. More preferably, nucleic acids (e.g., DNA or RNA), for example, coding RNA, preferably mRNA, are complexed with one or more polycations, preferably protamine or oligofectamine, most preferably protamine.

[0405] More preferred cationic or polycationic compounds that can be used as transfection or complexing agents include cationic polysaccharides, e.g., chitosan, polybrene; cationic lipids, e.g., DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or polycationic polymers, e.g., modified polyamino acids, e.g., beta-amino acid polymers or inverse polyamides; modified polyethylene, e.g., PVP; modified acrylates, e.g., pDMAEMA; and modified The materials may include modified amide amines, such as pAMAM; modified polybeta-aminoesters (PBAEs), such as 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers with modified diamine terminals; dendrimers, such as polypropylamine dendrimers or pAMAM-based dendrimers; polyimines, such as PEI, poly(propyleneimine), polyallylamines; sugar skeleton-based polymers, such as cyclodextrin-based polymers or dextran-based polymers; silane skeleton-based polymers, such as PMOXA-PDMS copolymers; and block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0406] According to various embodiments, the composition of the present invention comprises at least one RNA, preferably at least one mRNA as defined in the context of the first embodiment, and a polymeric carrier.

[0407] As used herein, the term “polymeric carrier” is intended to be recognized and understood by those skilled in the art and to refer to a compound that facilitates, for example, the transport and / or complexation of another compound (e.g., cargo nucleic acid). A polymeric carrier is typically a carrier formed from a polymer. A polymeric carrier may associate with its cargo (e.g., DNA or RNA) by covalent or noncovalent interactions. The polymer may be based on different subunits, for example, copolymers.

[0408] Suitable polymeric carriers in that context include, for example, polyacrylates, polyhydroxyanoacrylates, polylactic acid, polylactic acid-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrin, protamine, pegylated protamine, pegylated PLL, and polyethyleneimine (PEI), dithiobis(succinimidylpropionate) (DSP), dimethyl-3,3'-dithiobispropionimidate (DTBP), and poly(ethyleneimine). Biscarbamate (PEIC), poly(L-lysine) (PLL), histidine-modified PLL, poly(N-vinylpyrrolidone) (PVP), poly(propyleneimine) (PPI), poly(amideamine) (PAMAM), poly(amideethyleneimine) (SS-PAEI), triethylenetetramine (TETA), poly(β-aminoester), poly(4-hydroxy-L-proine ester) (PHP), poly(allylamine), poly(α-[4-aminobutyl]-L-glycolic acid) (PAGA), poly(D,L-lactic acid) Co-glycolic acid (PLGA), poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene) (PPZ), poly(phosphoester) (PPE), poly(phosphoramide) (PPA), poly(N-2-hydroxypropyl methacrylamide) (pHPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) (PPE_EA), galactosylated chitosan, N-dodecylated chitosan, histones, collagen, and dextran -May contain spermine. In one embodiment, the polymer may be an inert polymer, for example, PEG, but not limited to PEG. In one embodiment, the polymer may be a cationic polymer, for example, PLL, TETA, poly(allylamine), poly(N-ethyl-4-vinylpyridinium bromide), pHPMA, and pDMAEMA, but not limited to PEG. In one embodiment, the polymer may be a biodegradable PEI, for example, DSP, DTBP, and PEIC, but not limited to PEG.In one embodiment, the polymer may be biodegradable, for example, histidine-modified PLL, SS-PAEI, poly(β-aminoester), PHP, PAGA, PLGA, PPZ, PPE, PPA, and PPE-EA, but are not limited to these.

[0409] Inclusion / complexification within LNPs: In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, and optionally at least one further RNA, are complexed with one or more lipids (e.g., cationic lipids and / or neutral lipids), encapsulated, partially encapsulated, or associated with each other, thereby forming a lipid-based carrier, such as a liposome, lipid nanoparticles (LNPs), lipoplex, and / or nanoliposome.

[0410] RNA incorporated into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be located entirely or partially within the internal space of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes within the lipid layer / membrane, or may associate with the external surface of the lipid layer / membrane.

[0411] The incorporation of RNA into liposomes / LNPs is also referred to herein as “encapsulation,” where the RNA is completely contained within the internal space of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. The purpose of incorporating RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes is to protect the RNA from environments that may contain enzymes, chemicals, or conditions that degrade nucleic acids and / or systems or receptors, causing rapid efflux of nucleic acids. Furthermore, incorporating RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may enhance RNA incorporation and thus enhance the therapeutic effect of RNA encoding the antigenic SARS-CoV-2 spike protein. Therefore, incorporating at least one RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be particularly suitable for SARS-CoV-2 vaccines, e.g., intramuscular and / or intradermal administration.

[0412] In this context, the terms “complexed” or “associated” refer to an intrinsically stable combination of RNA with one or more lipids to form a larger complex or aggregate without covalent bonding.

[0413] The term "lipid nanoparticles," also known as "LNPs," is not limited to any specific form and includes any form that arises when cationic lipids and, optionally, one or more further lipids are combined, for example, in an aqueous environment and / or in the presence of RNA. For example, liposomes, lipid complexes, lipoplexes, etc., fall within the scope of lipid nanoparticles (LNPs).

[0414] Liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes can be of different sizes, for example, multilayer vesicles (MLVs) which may have a diameter of several hundred nanometers and contain a series of concentric bilayers separated by narrow aqueous compartments, small cell vesicles (SUVs) which may have a diameter less than 50 nm, and large monolayer vesicles (LUVs) which may have a diameter of 50 nm to 500 nm.

[0415] The LNPs of the present invention are preferably characterized as microscopic vesicles having an internal aqueous space isolated from an outer medium by one or more bilayer membranes. The bilayer membrane of the LNP is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, LNPs typically function to transport at least one RNA to a target tissue.

[0416] Therefore, in a preferred embodiment of the second aspect, at least one RNA is complexed with one or more lipids, thereby forming lipid nanoparticles (LNPs). Preferably, the LNPs are particularly suitable for intramuscular and / or intradermal administration. The LNPs typically comprise cationic lipids and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., pegylated lipids). At least one RNA may be encapsulated in an aqueous space enclosed by the lipid portion of the LNP or by part or all of the lipid portion of the LNP. The RNA or its portion may also associate with and complex with the LNP. The LNPs may comprise any lipids capable of binding RNA or forming particles in which one or more RNA species are encapsulated. Preferably, the RNA-containing LNPs comprise one or more cationic lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and pegylated lipids.

[0417] Preferably, the LNP of the present invention is (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) comprising at least one polymer-conjugated lipid, preferably a PEG lipid, Here, (i) to (iv) represent the molar ratios of approximately 20-60% cationic lipids, 5-25% neutral lipids, 25-55% sterols, and 0.5-15% polymer-conjugated lipids.

[0418] The cationic lipids of LNPs may be cationizable, meaning they are protonated when the pH drops below the pK of the lipid's ionizable group, and progressively become more neutral at higher pH values. Then, at pH values ​​below pK, the lipids can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipids include zwitterionic lipids that become positively charged with decreasing pH.

[0419] Such lipids are not limited to, but include DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleo Xy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleylcarbamoyloxy-3-dimethylamine Dilinaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethic 1,2-Dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), ICE (imidazole-based), HGT5000, HGT5001, DMDMA, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC(2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane)HGT4003, 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A) 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analog, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethyl (Tylamino)butanoate (MC3), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanegiyl)zide Decane-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16 -diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropane-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,The following are examples of cationic liposomes: 31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamideglycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.); or any combination of any of the above. Further preferred cationic lipids for use in the compositio...

Claims

1. (a) Below: (i) At least one coding sequence encoding the SARS-CoV-2 spike protein (S), which is a pre-fusion stabilized spike protein (S_stab) containing at least 10 of the following mutations: S373P, S375F, S477N, T478K, Q498R, N501Y, Y505H, D614G, H655Y, N764K, D796Y, Q954H, and N969K, and is at least 90% identical to SEQ ID NO: 10, and containing K986P and V987P stabilizing mutations to SEQ ID NO: 10, and N969K, and (ii) at least one heterogeneous untranslated region (UTR) mRNA containing, and (b) at least one pharmaceutically acceptable carrier A composition comprising the mRNA being complexed with or associated with lipid nanoparticles (LNPs), and the LNPs (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one PEG-lipid A composition comprising, where (i) to (iv) are in a molar ratio of approximately 20 to 60% at least one cationic lipid, 5 to 25% at least one neutral lipid, 25 to 55% at least one steroid or steroid analog, and 0.5 to 5% at least one PEG-lipid.

2. The composition according to claim 1, wherein the mRNA comprises at least one poly(A) sequence containing 30 to 200 adenosine nucleotides and a 5' cap structure.

3. The composition according to claim 1, wherein at least one coding sequence of mRNA has a G / C content of at least about 50%.

4. The composition according to claim 3, wherein at least one coding sequence of mRNA has a G / C content of at least about 55%.

5. The composition according to claim 1, wherein the mRNA contains a sequence that is at least 90% identical to sequence number 28590.

6. The composition according to claim 1, wherein at least one heterogeneous UTR is selected from at least one heterogeneous 5'UTR and / or at least one heterogeneous 3'UTR.

7. The composition according to claim 6, wherein at least one heterologous 3'UTR comprises or consists of a nucleic acid sequence derived from the 3'UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or at least one heterologous 5'UTR comprises or consists of a nucleic acid sequence derived from the 5'UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2.

8. The composition according to claim 1, wherein the mRNA comprises at least one histone stem loop.

9. The composition according to claim 1, wherein the mRNA comprises a nucleotide analog substitution.

10. The composition according to claim 9, wherein the mRNA comprises a pseudouridine or 1-methylpseudridine substitution at the uridine position.

11. The composition according to claim 1, wherein the mRNA contains a 1-methylpseudridine substitution at the uridine position.

12. The composition according to claim 1, wherein the mRNA has at least about 50% RNA integrity.

13. The composition according to claim 1, wherein the mRNA is purified mRNA obtained by reverse-phase high-pressure liquid chromatography and / or tangent flow filtration.

14. The composition according to claim 13, wherein the mRNA is purified mRNA purified by reverse-phase high-pressure liquid chromatography and / or tangent flow filtration, and comprises about 5%, 10%, or 20% less double-stranded RNA byproducts compared to RNA not purified by reverse-phase high-pressure liquid chromatography and / or tangent flow filtration.

15. LNP (i) at least one neutral lipid containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and (ii) at least one sterol containing cholesterol The composition according to claim 1, comprising:

16. The composition according to claim 15, wherein the mRNA contains at least 90% identical sequences to sequence number 163.

17. The composition according to claim 1, further comprising a lioprotectant.

18. The composition according to claim 17, wherein the lioprotectant comprises sucrose.

19. The composition according to claim 1, comprising less than approximately 20% free mRNA.

20. The composition according to claim 1, wherein the LNP has an average diameter of about 60 nm to 200 nm.

21. The composition according to claim 1, having a molar ratio (N / P ratio) of lipids to RNA of about 2 to about 12.

22. The composition according to claim 13, having a molar ratio (N / P ratio) of lipids to RNA of about 2 to about 12.

23. A kit comprising the composition described in claim 1, optionally comprising a liquid vehicle for dissolution, and optionally comprising a technical instruction manual providing information on dosage and administration for use.

24. The composition according to claim 1 for the treatment or prevention of a disease.

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