Viral vaccines

Abstract

The present disclosure relates to the field of viral infection prevention or treatment, and in particular, the present disclosure relates to agents for vaccinating against viral infection and inducing effective viral antigen-specific immune responses, such as antibody and / or T cell responses, as well as methods of making and using such agents. Administration of agents, such as the RNAs disclosed herein, to a subject can protect the subject from viral infection. Specifically, the present disclosure relates to amino acid sequences that include at least a portion of a viral protein having amino acid modifications found in other variants of the viral protein. Administration of RNAs encoding one or more of the amino acid sequences can provide protection against a variety of viral variants. The methods and agents described herein are particularly useful for the prevention or treatment of coronavirus infection, e.g., SARS-CoV-2 infection.

Description

[Technical Field]

[0001] The present disclosure relates to the field of preventing or treating viral infections. In particular, the present disclosure relates to agents for vaccinating against viral infections and inducing effective viral antigen-specific immune responses, such as antibody and / or T cell responses, as well as methods for making and using such agents. Administration of agents, such as the RNAs disclosed herein, to a subject can protect the subject against viral infection. Specifically, the present disclosure relates to amino acid sequences comprising at least a portion of a viral protein having amino acid modifications found in other variants of the viral protein. Administration of RNA encoding one or more of the amino acid sequences can provide protection against various viral variants. The methods and agents described herein are particularly useful for preventing or treating coronavirus infections, such as SARS-CoV-2 infections.

[0002] As a virus replicates, its genes make random "copying errors" (i.e., genetic mutations). Over time, these genetic copying errors can lead to changes in the virus's surface proteins or antigens, among other changes to the virus. Genetic mutations can cause viral antigens to "drift" - meaning that the surface of the mutant virus looks different from the original virus. If there is enough viral drift, vaccines against old strains of the virus and immunity from previous viral infections may no longer be effective against the new, drifted strain. Therefore, people may become vulnerable to newer, mutated viruses. [Background technology]

[0003] Coronaviruses are positive-sense, single-stranded RNA ((+)ssRNA) enveloped viruses that encode four structural proteins: spike protein, envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The spike protein (S protein) is responsible for receptor recognition, cell attachment, infection via the endosomal pathway, and genome release driven by fusion of the viral and endosomal membranes. Although the sequence varies among different family members, conserved regions and motifs within the S protein allow it to be divided into two subdomains, S1 and S2. The S2 domain, together with its transmembrane domain, is responsible for membrane fusion, while the S1 domain recognizes virus-specific receptors and binds to target host cells. A receptor-binding domain (RBD) has been identified in several coronavirus isolates, and the general structure of the S protein has been defined (Figure 1).

[0004] In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China, and a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was identified as the underlying cause. The genetic sequence of SARS-CoV-2 was made available to the WHO and published (MN908947.3), and the virus was classified as a member of the Betacoronavirus subfamily. Sequence analysis revealed a phylogenetic tree that showed a closer relationship to severe acute respiratory syndrome (SARS) virus isolates, namely the Middle East respiratory syndrome (MERS) virus, than to other coronaviruses that infect humans.

[0005] SARS-CoV-2 infection and the resulting disease, COVID-19, are spreading globally, affecting an increasing number of countries. On March 11, 2020, the WHO characterized the COVID-19 outbreak as a pandemic. As of December 1, 2020, there were over 63 million confirmed cases of COVID-19 worldwide, over 1.4 million deaths, and 191 countries / territories were affected. The ongoing pandemic remains a significant challenge to public health and economic stability worldwide.

[0006] There is no pre-existing immunity to SARS-CoV-2, so all individuals are at risk of infection. After infection, some, but not all, individuals develop protective immunity in terms of neutralizing antibody responses and cell-mediated immunity. However, the extent and longevity of this protection are currently unknown. According to the WHO, 80% of infected individuals recover without requiring hospitalization, while 15% develop more severe disease and 5% require intensive care. Older age and existing medical conditions are considered risk factors for developing severe disease.

[0007] Symptoms of COVID-19 generally include cough and fever, with chest radiographs showing ground-glass opacities or patchy opacities. However, many patients do not develop fever or radiographic changes, and infection may be asymptomatic, which is relevant for controlling transmission. For symptomatic subjects, disease progression can lead to acute respiratory distress syndrome requiring mechanical ventilation and subsequent multiple organ failure and death. Common symptoms in hospitalized patients (in order of most to least frequent) include fever, dry cough, shortness of breath, fatigue, myalgia, nausea / vomiting or diarrhea, headache, weakness, and rhinorrhea. Anosmia (loss of smell) or anageusia (loss of taste) may be the only presenting symptom in approximately 3% of individuals with COVID-19.

[0008] Although people of all ages can develop the disease, the case fatality rate (CFR) is particularly high in people over 60 years of age. Comorbidities, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease, are also associated with an increased CFR. Healthcare workers are overrepresented among COVID-19 patients due to occupational exposure to infected patients.

[0009] In most situations, molecular tests are used to detect SARS-CoV-2 and confirm infection. Reverse transcription-polymerase chain reaction (RT-PCR) testing, which targets SARS-CoV-2 viral RNA, is the standard in vitro method for diagnosing suspected cases of COVID-19. Samples to be tested are collected by swabbing from the nose and / or throat.

[0010] SARS-CoV-2 is an RNA virus with four structural proteins, one of which, the spike protein, is a surface protein that binds to angiotensin-converting enzyme 2 (ACE-2) present on host cells. Therefore, the spike protein is considered a relevant antigen for vaccine development.

[0011] BNT162b2 is an mRNA vaccine for the prevention of COVID-19 and has demonstrated greater than 95% efficacy in preventing COVID-19. The vaccine is made of 5'-capped mRNA encoding the full-length SARS-CoV-2 spike glycoprotein (S) encapsulated in lipid nanoparticles (LNPs). The final product is presented as an injectable dispersion concentrate containing BNT162b2 as the active substance. Other ingredients include: ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium phosphate dihydrate, sucrose, and water for injection.

[0012] The S protein sequence was selected based on the sequences of the SARS-CoV-2 isolate Wuhan-Hu-1 available at the time of program initiation: GenBank: MN908947.3 (complete genome) and GenBank: QHD43416.1 (spike surface glycoprotein). The active substance consists of a single-stranded 5' cap codon-optimized mRNA that translates into the SARS-CoV-2 spike antigen. The protein sequence contains two proline mutations (P2 S) that ensure antigenically optimal prefusion confirmation. The RNA does not contain any uridines; instead, modified N1-methylpseudouridine is used in RNA synthesis instead. The RNA contains common structural elements optimized to mediate high RNA stability and translation efficiency. LNP protects the RNA from RNAse degradation, enabling transfection of host cells after intramuscular (IM) delivery. The mRNA is translated into the SARS-CoV-2 S protein in the host cell. The S protein is then expressed on the cell surface, where it induces an adaptive immune response. The S protein has been identified as the target of neutralizing antibodies against the virus and is therefore considered a relevant vaccine component. BNT162b2 will be administered intramuscularly (IM) in two 30 μg doses of diluted vaccine solution given 21 days apart.

[0013] The recent emergence of novel circulating variants of SARS-CoV-2 has raised significant concerns about the regional and temporal effectiveness of vaccine interventions. One of the earliest variants to emerge and rapidly become dominant globally was D614G. Furthermore, recent genomic studies in the UK have demonstrated the rapid expansion of a novel lineage designated B.1.1.7 (also known as VOC-202012 / 01 or 501Y.V1). B.1.1.7 contains three amino acid deletions and seven missense mutations in the spike, including D614G and N501Y in the ACE2 receptor-binding domain (RBD). It has been shown in multiple countries to be inherently more infectious, with growth rates estimated to be 40–70% higher than other SARS-CoV-2 strains (Volz et al., 2021, Nature, https: / / doi.org / 10.1038 / s41586-021-03470-x; Washington et al., 2021, Cell https: / / doi.org / 10.1016 / j.cell.2021.03.052).

[0014] Studies have demonstrated that human sera induced by the BNT162b2 vaccine cross-neutralize the B.1.1.7 variant, suggesting that previous infection or vaccination with wild-type SARS-CoV-2 may still provide protection against the B.1.1.7 variant (Muik A. et al., 2021, Science 371(6534):1152-1153).

[0015] There have also been reports of SARS-CoV-2 transmission between humans and mink in Denmark due to a variant called mink cluster 5 or B.1.1.298, which has four missense mutations, including a two amino acid deletion in the RBD and Y453F.

[0016] Another variant that recently emerged in California, USA, designated B.1.427 / B.1.429, contains four missense mutations in the spike, one of which is a single L452R RBD mutation.

[0017] New variants arising from the B.1.1.28 lineage first described in Brazil and Japan, designated P.2 (with three spike missense mutations) and P.1 (with 12 spike missense mutations), contain the E484K mutation in the RBD, which is of particular concern; P.1 also contains, among other things, K417T and N501Y mutations in the RBD.

[0018] The emergence of multiple strains of the B.1.351 lineage (also known as 501Y.V2), which was first reported in South Africa and has since spread worldwide, is of great concern. This lineage has three RBD mutations, K417N, E484K, and N501Y, in addition to several mutations outside the RBD. Using serum induced by BNT162b2, neutralization of B.1.1.7-spike and P.1-spike viruses was reported to be roughly equivalent, while neutralization of B.1.351-spike viruses was robust but lower compared to neutralization of the USA-WA1 / 2020 strain (Liu Y. et al., 2021, N Engl J Med., doi: 10.1056 / NEJMc2102017. Epub ahead of print. PMID: 33684280). Given the emergence of new variants that appear to at least partially evade the immune response, there is a need for vaccines that are effective against SARS-CoV-2 variants.

[0019] The present disclosure provides vaccines containing epitopes of various viral protein variants and methods for providing such vaccines. Instead of reformulating existing vaccines to contain various viral protein sequences or encoding nucleic acids of different viral variants, the strategy described herein is based on combining various epitopes of different viral variants on a limited number of molecules. This allows for the administration of relatively low doses of active ingredients, particularly mRNA, while achieving a sufficiently high dose of each epitope to induce an effective immune response. It is anticipated that the vaccines described herein will be capable of eliciting broadly neutralizing antibodies and therefore suitable for resolving the ongoing SARS-CoV-2 pandemic. Summary of the Invention

[0020] overview The present invention generally relates to vaccines in which epitopes of different variants of viral proteins are combined on a single molecule. Such molecules comprise at least a portion of a viral protein containing an amino acid modification present in other variants of the viral protein. The modification generates (additional) epitopes that are specific for such other viral protein variants. Thus, the modified viral protein sequences described herein are multispecific viral protein amino acid sequences. In some embodiments, the multispecific viral protein amino acid sequence is a modified full-length viral protein. In some embodiments, the multispecific viral protein amino acid sequence is a modified portion of a viral protein.

[0021] In some embodiments, the multispecific viral protein amino acid sequences provided herein can comprise immunoreactive epitopes ((modified) amino acid residues) from a plurality (e.g., including at least two or more, e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc.) of viral protein variants, e.g., an immunoreactive epitope from a parent viral protein and an immunoreactive epitope from one or more viral protein variants that differ from the parent viral protein. In various embodiments, the multispecific viral protein amino acid sequences provided herein can comprise all of the immunoreactive epitopes ((modified) amino acid residues) or a portion thereof from one or more viral protein variants that differ from the parent viral protein. In various embodiments, the multispecific viral protein amino acid sequences provided herein can comprise a plurality (e.g., including at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc., e.g., at least two or more) immunoreactive epitopes ((modified) amino acid residues) from one or more viral protein variants that differ from the parent viral protein.

[0022] In some embodiments, the amino acid sequence comprising the polyspecific viral protein amino acid sequence is encoded by a nucleic acid such as DNA or RNA, particularly RNA. In some embodiments, a plurality of amino acid sequences each comprising a polyspecific viral protein amino acid sequence is encoded by a nucleic acid such as DNA or RNA, particularly RNA. In some embodiments, a plurality of amino acid sequences each comprising a polyspecific viral protein amino acid sequence is encoded by a plurality of nucleic acid molecules, for example, DNA or RNA molecules, particularly RNA molecules.

[0023] Given the genetic diversity of viruses, such as RNA viruses, amino acid sequences comprising the polyspecific viral protein amino acid sequences described herein and nucleic acids encoding these amino acid sequences are particularly useful for providing protection against multiple viral variants. Amino acid sequences comprising the polyspecific viral protein amino acid sequences described herein and nucleic acids encoding these amino acid sequences may provide the opportunity to obtain diverse and / or otherwise robust (e.g., persistent, e.g., detectable for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days after administration of one or more doses) neutralizing antibody and / or T cell responses, e.g., TH1-type T cell (e.g., CD4+ and / or CD8+ T cell) responses. Amino acid sequences comprising the polyspecific viral protein amino acid sequences described herein and nucleic acids encoding the amino acid sequences in a limited number of constructs are predicted to broadly and specifically neutralize multiple (e.g., at least two or more, including at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc.) viral variant strains and elicit immune responses, particularly antibody responses, that have the potential to generate protective immune responses against various viral variant strains. Generally, the limited number of constructs will contain fewer molecules (proteins and / or nucleic acid molecules) than the number of different viral protein variants, e.g., one, two, three, or four molecules, such that each molecule contains epitope sequences corresponding to multiple viral protein variants. Generally, the polyspecific viral protein amino acid sequences described herein, i.e., amino acid sequences comprising vaccine antigens and nucleic acids encoding these amino acid sequences, are useful for immunotherapeutic treatment of subjects. Vaccine antigens contain viral protein epitopes derived from multiple viral protein variants, and thus viral variant strains, that are specific for inducing an immune response thereto in a subject. In certain embodiments, the invention involves the administration of nucleic acids, such as RNA, that encode one or more of the vaccine antigens described herein, i.e., vaccine RNA.In some embodiments, the present invention involves the administration of multiple nucleic acid molecules, such as RNA molecules, encoding different vaccine antigens, for example, two, three, or four. Different vaccine antigens (likely based on the same parent viral protein sequence) may contain different modifications (which may be derived from different viral strains) and therefore different immunogenicity spectra. When administered, the RNA encoding the vaccine antigen can provide antigens for the induction, i.e., stimulation, priming, and / or expansion, of immune responses, such as antibodies and / or immune effector cells, targeted to the target antigen (viral protein, particularly different viral protein variants) or its progression products (after expression of the RNA by appropriate target cells). In some embodiments, the immune response to be induced by the present disclosure is a B cell-mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in some embodiments, the immune response to be induced by the present disclosure is a T cell-mediated immune response. In some embodiments, the immune response is an antiviral immune response. In some embodiments, the immune response is directed against multiple viral strains.

[0024] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) identifying amino acid positions in a parent viral protein that are modified compared to the corresponding amino acid positions of one or more viral protein variants; and b) providing an amino acid sequence, or a nucleotide sequence encoding the modified amino acid sequence, comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein has been modified to comprise an amino acid found at the corresponding amino acid position in one or more viral protein variants. The present invention relates to a method comprising:

[0025] In certain embodiments, the method includes repeating step b) to provide two or more of the modified amino acid sequences or two or more of the nucleotide sequences encoding two or more of the modified amino acid sequences.

[0026] In certain embodiments, the two or more modified amino acid sequences are based on the same parent viral protein.

[0027] In certain embodiments, the amino acid modifications in the two or more modified amino acid sequences are at least partially different.

[0028] In one embodiment, providing the nucleotide sequence comprises: b') substituting codons of a nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in at least one fragment of the parent viral protein is modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants. Includes.

[0029] In certain embodiments, the method comprises repeating step b') to provide two or more of the mutated nucleotide sequences encoding two or more of the amino acid sequences.

[0030] In certain embodiments, the two or more modified amino acid sequences are based on the same parent viral protein.

[0031] In certain embodiments, the amino acid modifications in the two or more modified amino acid sequences are at least partially different.

[0032] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) identifying amino acid positions in a parent viral protein that are modified compared to the corresponding amino acid positions in one or more viral protein variants; b) substituting codons of a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a first mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in the at least one fragment of the parent viral protein is modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants; and c) substituting codons of the second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a second mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in the at least one fragment of the parent viral protein is modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants. Including, wherein the amino acid modification in b) is at least partially different from the amino acid modification in c).

[0033] In certain embodiments, the method may comprise one or more further such steps of substituting codons to obtain a mutated nucleotide sequence that encodes at least a partially different amino acid modification.

[0034] In certain embodiments, at least one fragment of a parent viral protein contained in the amino acid sequence encoded by the first nucleotide sequence and at least one fragment of a parent viral protein contained in the amino acid sequence encoded by the second nucleotide sequence are identical.

[0035] In certain embodiments, the amino acid sequence encoded by the first nucleotide sequence and the amino acid sequence encoded by the second nucleotide sequence are identical.

[0036] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are identical.

[0037] In certain embodiments, one or more of the modified amino acid positions in the modified amino acid sequence encoded by the first mutated nucleotide sequence are different from the modified amino acid positions in the modified amino acid sequence encoded by the second mutated nucleotide sequence, i.e., one or more different positions are modified.

[0038] In certain embodiments, one or more amino acids at the modified amino acid positions in the modified amino acid sequence encoded by the first mutated nucleotide sequence and in the modified amino acid sequence encoded by the second mutated nucleotide sequence are different from each other, i.e., one or more of the same positions are modified but with different amino acid residues.

[0039] In certain embodiments, the amino acid sequence comprising at least a fragment of a parent viral protein comprises the amino acid sequence of a full-length viral protein.

[0040] In certain embodiments, the method further comprises providing a nucleic acid comprising a nucleotide sequence encoding the modified amino acid sequence.

[0041] In certain embodiments, the method further comprises providing a vaccine comprising a nucleic acid comprising a nucleotide sequence encoding the modified amino acid sequence.

[0042] In certain embodiments, the nucleic acid is RNA.

[0043] In some embodiments, the method is a method for producing a vaccine.

[0044] In some embodiments, the vaccine is an RNA vaccine.

[0045] In some embodiments, the vaccine has a reduced risk of immune escape.

[0046] In certain embodiments, a modified amino acid position is an amino acid position at which the amino acid sequence of one or more viral protein variants differs from the amino acid sequence of the parent viral protein.

[0047] In certain embodiments, a modified amino acid position is an amino acid position at which the amino acid sequence of one or more viral protein variants differs from the amino acid sequence of the wild-type viral protein.

[0048] In some embodiments, the modified amino acid positions are potential sites of viral escape mutations.

[0049] In some embodiments, the escape mutant of the virus is an antibody escape mutant of the virus.

[0050] In certain embodiments, viral escape mutants are resistant to neutralizing antibodies against viral proteins.

[0051] In certain embodiments, the viral proteins of the viral escape mutants exhibit reduced antibody binding.

[0052] In some embodiments, the antibodies are used to treat patients infected with a virus.

[0053] In certain embodiments, the antibodies are generated in a patient being treated with a vaccine against infection by the virus.

[0054] In certain embodiments, the parent viral protein is modified compared to the wild-type viral protein.

[0055] In some embodiments, the amino acid positions in the parent viral protein that are modified in the modified amino acid sequence relative to the wild-type viral protein are not modified.

[0056] In certain embodiments, the parental viral protein is a viral protein of a parental viral strain.

[0057] In certain embodiments, the parental viral strain is a natural isolate, or the parental viral strain is a variant of a natural isolate.

[0058] In certain embodiments, the parent virus strain is a viral variant strain that is circulating or spreading rapidly.

[0059] In certain embodiments, the parent virus strain is a virus variant that is a variant of concern.

[0060] In certain embodiments, one or more viral protein variants are modified compared to the wild-type viral protein.

[0061] In certain embodiments, one or more viral protein variants are modified compared to the parent viral protein.

[0062] In certain embodiments, the amino acid positions in one or more viral protein variants that are modified in the modified amino acid sequence compared to the wild-type viral protein and / or parent viral protein are modified.

[0063] In certain embodiments, one or more (e.g., all) of the one or more viral protein variants are viral proteins of one or more viral strains.

[0064] In certain embodiments, one or more (e.g., all) of the one or more viral strains are naturally isolated strains, or one or more (e.g., all) of the one or more viral strains are mutants of naturally isolated strains.

[0065] In certain embodiments, one or more (e.g., all) of the one or more viral strains are circulating or rapidly spreading viral variant strains.

[0066] In certain embodiments, one or more (e.g., all) of the one or more viral strains are viral variant strains that are variants of concern.

[0067] In certain embodiments, the parent virus strain and the one or more virus strains are circulating or rapidly spreading virus variant strains.

[0068] In certain embodiments, the parent virus strain and the one or more virus strains are virus variant strains that are variants of concern.

[0069] In certain embodiments, the parent viral protein and one or more viral protein variants are modified compared to the wild-type viral protein.

[0070] In certain embodiments, the one or more viral protein variants include viral protein variants of at least two viral strains.

[0071] In certain embodiments, the one or more viral protein variants in b) are different from the one or more viral protein variants in c).

[0072] In certain embodiments, in the modified amino acid sequence, the amino acid modification in the parent viral protein compared to the wild-type viral protein does not interfere with the amino acid modification at the modified amino acid position.

[0073] In some embodiments, in the modified amino acid sequence, the amino acid modification in the parent viral protein compared to the wild-type viral protein is not at the modified amino acid position or in close spatial distance to the amino acid modification in the modified amino acid position.

[0074] In some embodiments, in the modified amino acid sequence, the modification at the modified amino acid position does not result in a major structural rearrangement.

[0075] In some embodiments, the amino acid at the modified amino acid position is surface-exposed.

[0076] In some embodiments, the modified amino acid positions include one or more, e.g., at least two, amino acid positions. The modifications may correspond to one or more viral protein variants / strains and may include all modifications present in one or more viral protein variants / strains compared to a parent viral protein / strain or portion thereof.

[0077] In certain embodiments, the modified amino acid positions in b) and c) each include one or more, eg, at least two, amino acid positions.

[0078] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) providing a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parental viral protein, wherein an amino acid position in the at least one fragment of the parental viral protein is modified to include an amino acid found at a corresponding amino acid position in one or more SARS-CoV-2 S protein variants; and b) providing a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein is modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants; Including, wherein the amino acid modification in b) is at least partially different from the amino acid modification in a).

[0079] In certain embodiments, the method may comprise one or more further such steps of providing nucleic acids encoding at least partially distinct amino acid modifications.

[0080] In certain embodiments, the nucleic acid is RNA.

[0081] Further embodiments are described herein.

[0082] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein has been modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein has been modified to comprise an amino acid found at a corresponding amino acid position in one or more viral protein variants. Including, The amino acid modification in b) relates to a pharmaceutical preparation which is at least partially different from the amino acid modification in a).

[0083] In certain embodiments, the pharmaceutical formulation may include one or more additional nucleic acids encoding at least partially different amino acid modifications.

[0084] In certain embodiments, the nucleic acid is RNA.

[0085] In certain embodiments, the RNA is formulated in a lipid nanoparticle (LNP).

[0086] In certain embodiments, the pharmaceutical formulation is a pharmaceutical composition.

[0087] In certain embodiments, the pharmaceutical preparation is a vaccine.

[0088] In certain embodiments, the pharmaceutical formulation is a kit.

[0089] In certain embodiments, the pharmaceutical formulation further comprises instructions for using the pharmaceutical formulation for vaccination against infection by a virus.

[0090] In one aspect, the present invention relates to a pharmaceutical formulation for pharmaceutical use.

[0091] In some embodiments, the pharmaceutical uses include vaccination against infection by a virus.

[0092] In one aspect, the present invention relates to a method of inducing an immune response against a virus in a subject, comprising administering to the subject a pharmaceutical formulation.

[0093] In certain embodiments, the method is for prophylactic treatment against infection by a virus.

[0094] In certain embodiments, the method is a method for vaccination against infection by a virus.

[0095] Further embodiments of the above aspects are as described herein.

[0096] In some embodiments, the virus described herein is SARS-CoV-2. In some embodiments, the viral protein described herein is the SARS-CoV-2 spike protein (S protein).

[0097] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) identifying amino acid positions in the parent SARS-CoV-2 spike protein (S protein) that are modified compared to the corresponding amino acid positions in one or more SARS-CoV-2 S protein variants; and b) providing an amino acid sequence, or a nucleotide sequence encoding the modified amino acid sequence, comprising at least one fragment of the parent SARS-CoV-2 S protein, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein has been modified to include an amino acid found at the corresponding amino acid position in one or more SARS-CoV-2 S protein variants; The present invention relates to a method comprising:

[0098] In certain embodiments, the method includes repeating step b) to provide two or more of the modified amino acid sequences or two or more of the nucleotide sequences encoding two or more of the modified amino acid sequences.

[0099] In certain embodiments, the two or more modified amino acid sequences are based on the same parent SARS-CoV-2 S protein.

[0100] In certain embodiments, the amino acid modifications in the two or more modified amino acid sequences are at least partially different.

[0101] In one embodiment, providing the nucleotide sequence comprises: b') substituting codons of a nucleotide sequence encoding an amino acid sequence comprising at least one fragment of the parent SARS-CoV-2 S protein with other codons to obtain a mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in at least one fragment of the parent SARS-CoV-2 S protein is modified to include an amino acid found at the corresponding amino acid position in one or more SARS-CoV-2 S protein variants. Includes.

[0102] In certain embodiments, the method includes repeating step b') to provide two or more of the mutated nucleotide sequences encoding two or more of the modified amino acid sequences.

[0103] In certain embodiments, the two or more modified amino acid sequences are based on the same parent SARS-CoV-2 S protein.

[0104] In certain embodiments, the amino acid modifications in the two or more modified amino acid sequences are at least partially different.

[0105] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) identifying amino acid positions in the parent SARS-CoV-2 spike protein (S protein) that are modified compared to the corresponding amino acid positions in one or more SARS-CoV-2 S protein variants; b) substituting codons of a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of the parent SARS-CoV-2 S protein with other codons to obtain a first mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein is modified to include an amino acid found at a corresponding amino acid position in one or more SARS-CoV-2 S protein variants; and c) substituting codons of the second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of the parent SARS-CoV-2 S protein with other codons to obtain a second mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein is modified to include an amino acid found at the corresponding amino acid position in one or more SARS-CoV-2 S protein variants. Including, wherein the amino acid modification in b) is at least partially different from the amino acid modification in c).

[0106] In certain embodiments, the method may comprise one or more further such steps of substituting codons to obtain a mutated nucleotide sequence that encodes at least a partially different amino acid modification.

[0107] In certain embodiments, at least one fragment of the parent SARS-CoV-2 S protein contained in the amino acid sequence encoded by the first nucleotide sequence and at least one fragment of the parent SARS-CoV-2 S protein contained in the amino acid sequence encoded by the second nucleotide sequence are identical.

[0108] In certain embodiments, the amino acid sequence encoded by the first nucleotide sequence and the amino acid sequence encoded by the second nucleotide sequence are identical.

[0109] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are identical.

[0110] In certain embodiments, one or more of the modified amino acid positions in the modified amino acid sequence encoded by the first mutated nucleotide sequence are different from the modified amino acid positions in the modified amino acid sequence encoded by the second mutated nucleotide sequence.

[0111] In certain embodiments, one or more amino acids at the modified amino acid positions in the modified amino acid sequence encoded by the first mutated nucleotide sequence and in the modified amino acid sequence encoded by the second mutated nucleotide sequence are different from each other.

[0112] In certain embodiments, the amino acid sequence comprising at least a fragment of the parent SARS-CoV-2 S protein comprises the amino acid sequence of the N-terminal domain (NTD) and / or the receptor binding domain (RBD) of the SARS-CoV-2 S protein.

[0113] In certain embodiments, the amino acid sequence comprising at least a fragment of the parent SARS-CoV-2 S protein comprises the amino acid sequence of the full-length SARS-CoV-2 S protein.

[0114] In certain embodiments, the method further comprises providing a nucleic acid comprising a nucleotide sequence encoding the modified amino acid sequence.

[0115] In certain embodiments, the method further comprises providing a vaccine comprising a nucleic acid comprising a nucleotide sequence encoding the modified amino acid sequence.

[0116] In certain embodiments, the nucleic acid is RNA.

[0117] In one embodiment, the method is a method for producing a SARS-CoV-2 vaccine.

[0118] In some embodiments, the vaccine is an RNA vaccine.

[0119] In some embodiments, the vaccine has a reduced risk of immune escape.

[0120] In certain embodiments, one or more of the modified amino acid positions are located within the N-terminal domain (NTD) and / or receptor binding domain (RBD) of the SARS-CoV-2 S protein.

[0121] In certain embodiments, the modified amino acid positions are amino acid positions at which the amino acid sequence of one or more SARS-CoV-2 S protein variants differs from the amino acid sequence of the parent SARS-CoV-2 S protein.

[0122] In certain embodiments, the modified amino acid positions are amino acid positions at which the amino acid sequence of one or more SARS-CoV-2 S protein variants differs from the amino acid sequence of the wild-type SARS-CoV-2 S protein.

[0123] In some embodiments, the modified amino acid positions are potential sites of escape mutations in SARS-CoV-2.

[0124] In certain embodiments, the escape mutant of SARS-CoV-2 is an antibody escape mutant of SARS-CoV-2.

[0125] In certain embodiments, the escape mutant of SARS-CoV-2 is resistant to neutralization by antibodies against the SARS-CoV-2 S protein.

[0126] In certain embodiments, the SARS-CoV-2 S protein of the escape mutant of SARS-CoV-2 exhibits reduced antibody binding.

[0127] In certain embodiments, the antibodies are used for the treatment of patients infected with SARS-CoV-2.

[0128] In some embodiments, the antibodies are generated in patients treated with a SARS-CoV-2 vaccine.

[0129] In certain embodiments, the parent SARS-CoV-2 S protein is modified compared to the wild-type SARS-CoV-2 S protein.

[0130] In certain embodiments, the amino acid positions in the parent SARS-CoV-2 S protein that are modified in the modified amino acid sequence relative to the wild-type SARS-CoV-2 S protein are not modified.

[0131] In certain embodiments, the parent SARS-CoV-2 S protein is the S protein of a parent SARS-CoV-2 strain.

[0132] In certain embodiments, the parent SARS-CoV-2 strain is a natural isolate, or the parent SARS-CoV-2 strain is a variant of a natural isolate.

[0133] In certain embodiments, the parent SARS-CoV-2 strain is a SARS-CoV-2 variant strain that is prevalent or rapidly spreading.

[0134] In certain embodiments, the parent SARS-CoV-2 strain is a SARS-CoV-2 variant that is a variant of concern.

[0135] In some embodiments, the parent SARS-CoV-2 strain is B.1.1.7.

[0136] In certain embodiments, the one or more SARS-CoV-2 S protein variants are modified compared to the wild-type SARS-CoV-2 S protein.

[0137] In certain embodiments, one or more SARS-CoV-2 S protein variants are modified compared to the parent SARS-CoV-2 S protein.

[0138] In certain embodiments, the modified amino acid sequence is modified at one or more amino acid positions in the SARS-CoV-2 S protein variant that are modified compared to the wild-type SARS-CoV-2 S protein and / or the parent SARS-CoV-2 S protein.

[0139] In certain embodiments, one or more (e.g., all) of the one or more SARS-CoV-2 S protein variants are S proteins of one or more SARS-CoV-2 strains.

[0140] In certain embodiments, one or more (e.g., all) of the one or more SARS-CoV-2 strains are naturally isolated strains, or one or more (e.g., all) of the one or more SARS-CoV-2 strains are mutants of naturally isolated strains.

[0141] In certain embodiments, one or more (e.g., all) of the one or more SARS-CoV-2 strains is a SARS-CoV-2 variant strain that is circulating or spreading rapidly.

[0142] In certain embodiments, one or more (e.g., all) of the one or more SARS-CoV-2 strains is a SARS-CoV-2 variant strain that is a variant of concern.

[0143] In certain embodiments, one or more of the one or more SARS-CoV-2 strains are selected from the group consisting of B.1.351, B.1.1.298, B.1.427 / B.1.429, B.1.526 and P1.

[0144] In certain embodiments, the parent SARS-CoV-2 strain and the one or more SARS-CoV-2 strains are epidemic or rapidly spreading SARS-CoV-2 variant strains.

[0145] In certain embodiments, the parent SARS-CoV-2 strain and the one or more SARS-CoV-2 strains are SARS-CoV-2 variant strains that are variants of concern.

[0146] In certain embodiments, the parent SARS-CoV-2 S protein and one or more SARS-CoV-2 S protein variants are modified compared to the wild-type SARS-CoV-2 S protein.

[0147] In certain embodiments, the parent SARS-CoV-2 strain is B.1.1.7 and one or more SARS-CoV-2 strains are selected from the group consisting of B.1.351, B.1.1.298, B.1.427 / B.1.429, B.1.526, P1.

[0148] In certain embodiments, the one or more SARS-CoV-2 S protein variants include SARS-CoV-2 S protein variants of at least two SARS-CoV-2 strains.

[0149] In certain embodiments, the one or more SARS-CoV-2 S protein variants in b) are different from the one or more SARS-CoV-2 S protein variants in c).

[0150] In certain embodiments, the one or more SARS-CoV-2 S protein variants in b) are the B.1.427 / B.1.429 and B.1.526 SARS-CoV-2 S protein variants, and the one or more SARS-CoV-2 S protein variants in c) are the B.1.351, P.1 and B.1.1.298 SARS-CoV-2 S protein variants.

[0151] In certain embodiments, in the modified amino acid sequence, the amino acid modification in the parent SARS-CoV-2 S protein compared to the wild-type SARS-CoV-2 S protein does not interfere with the amino acid modification at the modified amino acid position.

[0152] In certain embodiments, in the modified amino acid sequence, the amino acid modification in the parent SARS-CoV-2 S protein compared to the wild-type SARS-CoV-2 S protein is not at the modified amino acid position or in close spatial distance to the amino acid modification in the modified amino acid position.

[0153] In some embodiments, in the modified amino acid sequence, the modification at the modified amino acid position does not result in a major structural rearrangement.

[0154] In some embodiments, the amino acid at the modified amino acid position is surface-exposed.

[0155] In certain embodiments, the modified amino acid positions include one or more, e.g., at least two, amino acid positions. The modifications may correspond to one or more SARS-CoV-2 S protein variants / SARS-CoV-2 strains and may encompass all modifications present in one or more SARS-CoV-2 S protein variants / SARS-CoV-2 strains compared to the parent SARS-CoV-2 S protein / SARS-CoV-2 strain or portion thereof.

[0156] In certain embodiments, the modified amino acid positions in b) and c) each include one or more, eg, at least two, amino acid positions.

[0157] In some embodiments, the modified amino acid position is 18, 20, 26, 80, 138, 144, 190, 215, 246, 253, 417, 439, 452, 453, 477, 484, 501, 570, 701, 716, 140, 345, 346, 352, 378, 406, 420, 440, 441, 444, 445, 446, 450, 455, 460, 475, 478, 485, 486, 487, 489, 490, 493, 494, 499, 142, 145, 146, 147, 150, 152, 154, 156, 157, 158, 164, 247, 248, 249, 250, 251, 252, 254, 255, 258, 365, 369, 370, 374, 376, 384, 405, 408, 415, 421, 443, 447, 448, 456, 472, 473, 476, 496, 498, 500, 504 The present invention includes two or more selected from the group consisting of:

[0158] In some embodiments, the modification at the modified amino acid position is 18F, 20N, 26S, 80Y, 138Y, 144F, 190S, 215A, 246I, 253G, 417N, 439K, 452R, 453F, 477N, 484K, 501Y, 570D, 701V, 716I, 140L, 345A, 346K, 352S, 378N, 406Q, 420, 440K, 441F, 444, 445A, 446V, 450 K, 455F, 460I, 475V, 478I, 485V, 486L, 487D, 489, 490S, 493L, 494P, 499H, 142S, 145H, 146Y, 147N, 150R, 152C, 154Q, 156A, 157L, 158G, 164T, 247G, 248H, 249S, 250N, 251S, 252V, 254F, 255F, 258L, 3 65D, 369C, 370S, 374L, 376I, 384L, 405Y, 408I, 415N, 421, 443A, 447V, 448Y, 456L, 472V, 473F, 476S, 496C, 498H, 500I, 504D The present invention includes two or more selected from the group consisting of:

[0159] In some embodiments, the modification at the modified amino acid position is L18F, T20N, P26S, D80Y, D138Y, Y144F, R190S, D215A, R246I, D253G, K417N, N439K, L452R, Y453F, S477N, E484K, N501Y, A570D, A701V, T716I, F140L, T345A, R346K, A352S, K378N, E406Q, D420, N440K, L441F, K444, V445A, G446V, N45 0K, L455F, N460I, A475V, T478I, G485V, F486L, N487D, Y489, F490S, Q493L, S494P, P499H, G142S, Y145H, H146Y, K147N, K150R, W152C, E154Q, E156A, F157L, R158G, N 164T, S247G, Y248H, L249S, T250N, P251S, G252V, S254F, S255F, W258L, Y3 65D, Y369C, N370S, F374L, T376I, P384L, D405Y, R408I, T415N, Y421, S443 A, G447V, N448Y, F456L, I472V, Y473F, G476S, G496C, Q498H, T500I, G504D The present invention includes two or more selected from the group consisting of:

[0160] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) providing a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent SARS-CoV-2 S protein, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein has been modified to comprise an amino acid found at a corresponding amino acid position in one or more SARS-CoV-2 S protein variants; and b) providing a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent SARS-CoV-2 S protein, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein has been modified to comprise an amino acid found at a corresponding amino acid position in one or more SARS-CoV-2 S protein variants; Including, wherein the amino acid modification in b) is at least partially different from the amino acid modification in a).

[0161] In certain embodiments, the method may comprise one or more further such steps of providing nucleic acids encoding at least partially distinct amino acid modifications.

[0162] In certain embodiments, the nucleic acid is RNA.

[0163] Further embodiments are as described herein.

[0164] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent SARS-CoV-2 S protein, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein has been modified to comprise an amino acid found at a corresponding amino acid position in one or more SARS-CoV-2 S protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent SARS-CoV-2 S protein, wherein an amino acid position in the at least one fragment of the parent SARS-CoV-2 S protein has been modified to comprise an amino acid found at the corresponding amino acid position in one or more SARS-CoV-2 S protein variants; Including, The amino acid modification in b) relates to a pharmaceutical preparation which is at least partially different from the amino acid modification in a).

[0165] In certain embodiments, the pharmaceutical formulation may include one or more additional nucleic acids encoding at least partially different amino acid modifications.

[0166] In certain embodiments, the nucleic acid is RNA.

[0167] In certain embodiments, the RNA is formulated in a lipid nanoparticle (LNP).

[0168] In certain embodiments, the pharmaceutical formulation is a pharmaceutical composition.

[0169] In certain embodiments, the pharmaceutical preparation is a vaccine.

[0170] In certain embodiments, the pharmaceutical formulation is a kit.

[0171] In certain embodiments, the pharmaceutical formulation further comprises instructions for use of the pharmaceutical formulation for vaccination against SARS-CoV-2 infection.

[0172] In one aspect, the present invention relates to a pharmaceutical formulation for pharmaceutical use.

[0173] In certain embodiments, the pharmaceutical use includes vaccination against SARS-CoV-2 infection.

[0174] In one aspect, the present invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject, comprising administering to the subject a pharmaceutical formulation.

[0175] In certain embodiments, the method is for prophylactic treatment against SARS-CoV-2 infection.

[0176] In certain embodiments, the method is a method for vaccination against SARS-CoV-2 infection.

[0177] Further embodiments of the above aspects are as described herein.

[0178] The nucleic acids described herein may be single-stranded RNA, and the pharmaceutical preparations described herein, e.g., vaccines, contain single-stranded RNA as an active ingredient, which can be translated into a protein once it enters the recipient's cells. In addition to the wild-type or codon-optimized sequence encoding the antigen sequence, the RNA may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum RNA stability and translation efficiency. In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA contains beta-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O) ApG can be used as a specific capping structure at the 5' end of an RNA drug substance. The 5'-UTR sequence can be the 5'-UTR sequence of human alpha-globin mRNA, optionally with an optimized "Kozak sequence" (e.g., SEQ ID NO: 12) for increased translation efficiency. The 3'-UTR sequence can be a combination of two sequence elements (FI elements) from the "amino terminal enhancer of split gene" (AES) mRNA (designated F) and the mitochondrial-encoded 12S ribosomal RNA (designated I) (e.g., SEQ ID NO: 13), located between the coding sequence and the poly(A)-tail, to ensure high maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that contribute to enhanced RNA stability and total protein expression (see WO2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR can be two repeated 3'-UTRs of human beta-globin mRNA. Additionally, a 110-nucleotide-long poly(A)-tail can be used, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence (of random nucleotides) and an additional 70 adenosine residues (e.g., SEQ ID NO: 14). This poly(A)-tail sequence was designed to enhance RNA stability and translation efficiency.

[0179] Additionally, a secretory signal peptide (sec) can be fused to the antigen-encoding region, preferably in such a way that the sec is translated as an N-terminal tag. In one embodiment, the sec corresponds to the secretory signal peptide of the S protein. As commonly used in fusion proteins, a sequence encoding a short linker peptide consisting mostly of the amino acids glycine (G) and serine (S) can be used as the GS / linker.

[0180] The RNAs described herein can be combined with proteins and / or lipids, preferably lipids, to produce RNA particles for administration. If a combination of different RNAs is used, these RNAs can be combined together or separately with proteins and / or lipids to produce RNA particles for administration.

[0181] In certain embodiments, vaccine antigens comprising the multispecific SARS-CoV-2 S protein amino acid sequences described herein are capable of forming multimeric complexes, particularly trimeric complexes. In certain embodiments, vaccine antigens comprising different multispecific SARS-CoV-2 S protein amino acid sequences described herein are capable of forming multimeric complexes, particularly trimeric complexes. Thus, in embodiments of the invention involving providing a subject with vaccine antigens comprising different multispecific SARS-CoV-2 S protein amino acid sequences described herein, e.g., by administering different nucleic acids encoding vaccine antigens comprising different multispecific SARS-CoV-2 S protein amino acid sequences, the different multispecific SARS-CoV-2 S protein amino acid sequences may be capable of forming multimeric complexes, particularly trimeric complexes. To this end, the multispecific SARS-CoV-2 S protein amino acid sequences described herein may contain domains that allow the multispecific SARS-CoV-2 S protein amino acid sequences to form multimeric complexes, particularly trimeric complexes. In certain embodiments, the domain that enables formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain as described herein, e.g., a SARS-CoV-2 S protein trimerization domain. In certain embodiments, trimerization is achieved by the addition of a trimerization domain, e.g., a "fold" trimerization domain from T4 fibritin (e.g., SEQ ID NO: 10), to the multispecific SARS-CoV-2 S protein amino acid sequence, particularly when the multispecific SARS-CoV-2 S protein amino acid sequence corresponds to a portion of the SARS-CoV-2 S protein that does not include the SARS-CoV-2 S protein trimerization domain.

[0182] In certain embodiments, vaccine antigens comprising the polyspecific viral protein amino acid sequences described herein are encoded by coding sequences that are codon-optimized and / or have increased G / C content compared to the wild-type coding sequence, and the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0183] In some embodiments, the vaccine antigen comprising the polyspecific viral protein amino acid sequence is encoded by RNA. In a particularly preferred embodiment, the vaccine antigen comprising the polyspecific viral protein amino acid sequence is encoded by an isolated messenger ribonucleic acid (mRNA) polynucleotide, and the isolated mRNA polynucleotide comprises an open reading frame encoding a polypeptide comprising the vaccine antigen. In some embodiments, the isolated mRNA polynucleotide is formulated into at least one lipid nanoparticle. For example, in some embodiments, such lipid nanoparticles may comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol or steroid, and 0.5-15% polymer-conjugated lipid (e.g., PEG-modified lipid). In some embodiments, the sterol or steroid contained in the lipid nanoparticle may be or may comprise cholesterol. In some embodiments, the neutral lipid may be or may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the polymer-conjugated lipid may be or may comprise PEG2000 DMG. In some embodiments, the immunogenic composition may contain a total lipid content of about 1 mg to 10 mg, 3 mg to 8 mg, or 4 mg to 6 mg. In some embodiments, such immunogenic compositions may contain a total lipid content of about 5 mg / mL to 15 mg / mL, 7.5 mg / mL to 12.5 mg / mL, or 9 to 11 mg / mL. In certain embodiments, such isolated mRNA polynucleotides are provided in an amount effective for inducing an immune response in a subject administered the immunogenic composition at least once. In certain embodiments, such isolated mRNA polynucleotides provided in the immunogenic composition are not self-replicating RNA.

[0184] In some embodiments, the RNA described herein is a modified RNA, particularly a stabilized mRNA. In some embodiments, the RNA contains a modified nucleoside in place of at least one uridine. In some embodiments, the RNA contains a modified nucleoside in place of each uridine. In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0185] In some embodiments, the RNA contains modified nucleosides in place of uridine.

[0186] In certain embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0187] In certain embodiments, the RNA comprises a 5' cap.

[0188] In certain embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0189] In certain embodiments, the RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0190] In certain embodiments, the RNA comprises a polyA sequence.

[0191] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0192] In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14.

[0193] In certain embodiments, the RNA is or is to be formulated as a liquid, a solid, or a combination thereof.

[0194] In certain embodiments, the RNA is or is to be formulated for injection.

[0195] In certain embodiments, the RNA is or should be formulated for intramuscular administration.

[0196] In certain embodiments, the RNA is or is to be formulated as particles.

[0197] In certain embodiments, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0198] In some embodiments, the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0199] In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with liposomes. In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with lipids.

[0200] In some embodiments, the RNA is or should be formulated as a colloid. In some embodiments, the RNA is or should be formulated as particles that form the dispersed phase of the colloid. In some embodiments, 50% or more, 75% or more, or 85% or more of the RNA is present in the dispersed phase. In some embodiments, the RNA is or should be formulated as particles comprising RNA and lipids. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In some embodiments, the organic phase comprises ethanol. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In some embodiments, the lipids dispersed in the aqueous phase form liposomes.

[0201] In certain embodiments, the RNA is mRNA or saRNA.

[0202] In certain embodiments, the compositions or pharmaceutical formulations described herein are pharmaceutical compositions.

[0203] In certain embodiments, the compositions or pharmaceutical preparations described herein are vaccines.

[0204] In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0205] In some embodiments, the compositions or pharmaceutical formulations described herein are kits.

[0206] In some embodiments, the RNA and optionally the particle-forming components are contained in separate vials.

[0207] In certain embodiments, the kit further comprises instructions for using the composition or pharmaceutical preparation to induce an immune response to a virus, e.g., a coronavirus, in a subject.

[0208] In certain aspects, the present invention relates to a composition or pharmaceutical formulation described herein for pharmaceutical use.

[0209] In some embodiments, the pharmaceutical use includes inducing an immune response in a subject against a virus, e.g., a coronavirus.

[0210] In some embodiments, medical uses include therapeutic or prophylactic treatment of viral infections, e.g., coronavirus infections.

[0211] In certain embodiments, the compositions or pharmaceutical formulations described herein are for administration to humans.

[0212] In some embodiments, the coronavirus is a betacoronavirus.

[0213] In some embodiments, the coronavirus is a sarbecovirus.

[0214] In some embodiments, the coronavirus is SARS-CoV-2.

[0215] In certain embodiments, the invention relates to methods of inducing an immune response to coronavirus in a subject, comprising administering to the subject a composition described herein, e.g., a vaccine antigen comprising a polyspecific SARS-CoV-2 S protein amino acid sequence, or a nucleic acid, e.g., RNA, encoding the vaccine antigen. In certain embodiments, the method comprises administering a plurality of such vaccine antigens or nucleic acids.

[0216] In some embodiments, the method is a method for vaccination against a coronavirus.

[0217] In certain embodiments, the method is for the therapeutic or prophylactic treatment of a coronavirus infection.

[0218] In certain embodiments, the subject is a human.

[0219] In some embodiments, the coronavirus is a betacoronavirus.

[0220] In some embodiments, the coronavirus is a sarbecovirus.

[0221] In some embodiments, the coronavirus is SARS-CoV-2.

[0222] In some embodiments of the methods described herein, the composition is a composition described herein.

[0223] In certain aspects, the invention relates to a composition or pharmaceutical formulation described herein for use in a method described herein.

[0224] In certain aspects, the invention relates to compositions, e.g., protein or nucleic acid compositions, obtainable by carrying out the methods described herein. [Brief explanation of the drawings]

[0225] [Figure 1] Schematic representation of the SARS-CoV-2 S protein. The S1 subunit sequence consists of a signal sequence (SS) and a receptor-binding domain (RBD), which is an important subunit within the S protein involved in binding to the human cell receptor ACE2. The S2 subunit contains the S2 protease cleavage site (S2'), a fusion peptide (FP) for subsequent membrane fusion, heptad repeat regions (HR1 and HR2) with a central helix (CH) domain, a transmembrane domain (TM), and a cytoplasmic tail (CT). [Figure 2]Constructs for SARS-CoV-2 vaccine development. The compositions described here were designed based on the complete and wild-type S protein and could be based on different constructs containing (1) the complete protein with mutations in the first seventh repeat (HRP1) region, including stabilizing mutations that preserve the neutralization-sensitive site, (2) the S1 domain, or (3) only the RB domain (RBD). Additionally, a fibritin domain (F) was fused to the C-terminus to stabilize the protein fragment. All constructs began with a signal peptide (SP) to ensure Golgi transport to the plasma membrane. [Figure 3] General structure of RNA that can be designed based on the constructs described here. Schematic representation of the general structure of an RNA vaccine, with a 5' cap, 5' and 3' untranslated regions, coding sequence with endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. UTR = untranslated region; sec = secretory signal peptide; RBD = receptor binding domain; GS = glycine-serine linker. [Figure 4] General structure of RNA that can be designed based on the constructs described herein. Schematic representation of the general structure of a drug substance RNA, with a 5' cap, 5' and 3' untranslated regions, coding sequence with endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. GS = glycine-serine linker; UTR = untranslated region; Sec = secretory signal peptide; RBD = receptor binding domain. [Figure 5]General structure of RNA that can be designed based on the constructs described here. Schematic representation of the general structure of an RNA vaccine: 5' cap, 5' and 3' untranslated regions, coding sequence for SARS-CoV-2 antigen with Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase, and endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. UTR = untranslated region; Sec = secretory signal peptide; RBD = receptor-binding domain; GS = glycine-serine linker. [Figure 6A] An exemplary novel bivalent vaccine design based on the B.1.1.7 strain spike protein. Cryo-EM structure of the SARS-CoV-2 spike protein with one upright RBD illustrating the surface-exposed mutation sites in vaccine sequences (A) S-Seq1 and (B) S-Seq2. The crystal structure of the SARS-CoV-2 furin-cleaved spike protein (PDB ID: 6ZGG) was obtained from the RCSB PDB database and visualized using PyMol v.2.4.1. Yellow sites indicate amino acid residues targeted by strain B.1.1.7 mutations. Red sites indicate amino acid residues targeted by additional non-B.1.1.7 strain mutations. [Figure 6B] An exemplary novel bivalent vaccine design based on the B.1.1.7 strain spike protein. Cryo-EM structure of the SARS-CoV-2 spike protein with one upright RBD illustrating the surface-exposed mutation sites in vaccine sequences (A) S-Seq1 and (B) S-Seq2. The crystal structure of the SARS-CoV-2 furin-cleaved spike protein (PDB ID: 6ZGG) was obtained from the RCSB PDB database and visualized using PyMol v.2.4.1. Yellow sites indicate amino acid residues targeted by strain B.1.1.7 mutations. Red sites indicate amino acid residues targeted by additional non-B.1.1.7 strain mutations. [Figure 7]Vaccine candidate expression in HEK293T cells. Expression of RNA-encoded variant SARS-CoV-2 spike (S) proteins in HEK293T cells after transfection with 0.15 μg / mL of modRNA (A and B) encoding BNT162b2 and BNT162b2(alpha+SA) using commercially available transfection reagents, respectively, or 0.15 μg / mL of LNP-formulated modRNA (C and D) encoding BNT162b2, BNT162b2(alpha), and BNT162b2(alpha; L452R+E484Q). Surface expression of the variant S proteins was detected by flow cytometry using human recombinant ACE-2 protein fused to a mouse Fc-tag and a secondary fluorescently tagged anti-mouse antibody. The percentage of S protein-expressing cells in the HEK293T population per vaccine candidate (in A and C) and median fluorescence intensity (MFI) (in B and D) are shown. Data shown are the mean + SD of HEK293T transfections performed in triplicate. [Figure 8] Kinetics of anti-S1 (B.1.1.7) IgG antibody responses in the serum of Balb / C mice after a single immunization with mRNA vaccine candidates encoding P2 S constructs derived from different SARS-CoV-2 alpha variants. Anti-S1 (B.1.1.7) IgG antibody titers after a single immunization with 1 μg LNP-formulated modRNA encoding BNT162b2(alpha), BNT162b2(alpha+SA), and 0.9% sodium chloride as a buffer control are shown at days 7 (A), 14 (B), 21 (C), and 28 (D) post-immunization. The longitudinal trajectory of serum antibody responses is shown in E. Data are presented as the mean ± SEM of all animals measured in duplicate (n=5). Statistical analysis of the different time points is presented in Table 8. [Figure 9]Kinetics of anti-RBD (B.1.351) IgG antibody responses in the serum of Balb / C mice after a single immunization with mRNA vaccine candidates encoding P2 S constructs derived from different SARS-CoV-2 alpha variants. Anti-RBD (B.1.351) IgG antibody titers after a single immunization with 1 μg LNP-formulated modRNA encoding BNT162b2(alpha), BNT162b2(alpha+SA), and 0.9% sodium chloride as a buffer control are shown at days 7 (A), 14 (B), 21 (C), and 28 (D) after immunization. The longitudinal trajectory of antibody responses in the serum is shown in E. Data are presented as the mean ± SEM of all animals measured in duplicate (n=5). Statistical analysis of the different time points is shown in Table 9. [Figure 10] Pseudovirus 50% neutralization (pVN50) titers in the serum of Balb / C mice 28 days after a single immunization with mRNA vaccine candidates encoding P2 S constructs derived from different SARS-CoV-2 alpha variants. Pseudovirus 50% neutralization antibody (pVN50) titers after a single immunization with 1 μg LNP-formulated modRNA encoding BNT162b2(alpha), BNT162b2(alpha+SA), and 0.9% sodium chloride as a buffer control, 28 days after immunization. The VSV-SARS-CoV-2 pseudoviruses used in the analysis carried SARS-CoV-2 S proteins specific for either the ancestral Wuhan strain (Wuhan), the B.1.1.7 (alpha) variant, or the B.1.351 (beta) variant. Each individual's serum (n=5 per group) was tested in duplicate, and the geometric mean pVN50 titers were plotted. For values ​​below the limit of detection (LOD; 12), the LOD / 2 value is plotted. Group geometric mean titers (values ​​above the horizontal line and bar) with 95% confidence intervals are shown. [Figure 11]Pseudovirus 50% neutralizing (pVN50) titers in the serum of Balb / C mice 28 days after a single immunization with BNT16b2(alpha) or BNT162b2(alpha; L452R+E484Q). Pseudovirus 50% neutralizing antibody (pVN50) titers after a single immunization with 1 μg LNP-formulated modRNA encoding BNT162b2(alpha), BNT162b2(alpha; L452R+E484Q), and 0.9% sodium chloride as a buffer control, 28 days after immunization. The VSV-SARS-CoV-2 pseudoviruses used in the analysis have SARS-CoV-2 S proteins specific for either the B.1.1.7 (alpha), B.1.617.1 (kappa), or B.1.617.2 (delta) variants. Each individual serum (n = 5 per group) was tested in duplicate, and the geometric mean pVN50 titer was plotted. For values ​​below the limit of detection (LOD; 12), the LOD / 2 value was plotted. Group geometric mean titers (values ​​above the horizontal line and bar) with 95% confidence intervals are shown. [Figure 12] IgG responses to different recombinant S proteins in the serum of Balb / c mice 28 days after a single immunization with BNT16b2(alpha) or BNT162b2(alpha; L452R+E484Q) measured by multiplex analysis. ECL signals 28 days after immunization following a single immunization with 1 μg LNP-formulated modRNA encoding BNT162b2(alpha), BNT162b2(alpha; L452R+E484Q), and 0.9% sodium chloride as a buffer control. The ECL signal was analyzed for the linear range (indicated by the dotted lines in the different graphs), and responses to SARS-CoV-2 S (B.1.1.7; 1:5400 serum dilution) (A), SARS-CoV-2 S (B.1.1.529; BA.1; 1:1800 serum dilution) (B), and SARS-CoV-2 S (BA.1 + L452R; 1:1800 serum dilution) (C) are shown. Data shown represent sera from individual animals measured in duplicate, and horizontal lines indicate group mean ± SEM (n = 5 per group; n = 3 for buffer group only). Statistical analysis of the different time points is shown in Table 12. DETAILED DESCRIPTION OF THE INVENTION

[0226] Detailed Description Although the present invention is described in detail below, it is understood that this invention is not limited to particular methodologies, protocols, and reagents, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0227] Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0228] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0229] Below, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the invention to only the specifically described embodiments. The description should be understood to disclose and encompass embodiments combining any number of the disclosed elements with the specifically described embodiments. Furthermore, all permutations and combinations of all described elements should be considered disclosed by this description unless the context dictates otherwise.

[0230] Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0231] definition The following definitions are provided which apply to all aspects of the present invention. The following terms have the following meanings unless otherwise specified: Any undefined terms have their art-recognized meanings.

[0232] The term "about" means approximately or approximation, and in the context of a numerical value or range herein, means, in certain embodiments, ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0233] In the context of describing the present invention (particularly in the context of the claims), the singular "a," "an," "the ...

[0234] Unless otherwise indicated, the term "comprising" is used herein to indicate that additional members may optionally be present in addition to the listed members introduced by "comprising." However, it is contemplated that the term "comprising" may be a specific embodiment of the present invention that does not exclude the possibility of additional members being present, i.e., for the purposes of this embodiment, "comprising" is understood to have the meaning of "consisting of" or "consisting essentially of."

[0235] As used herein, the terms "one or more" or "at least one" refer to one or more, including at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more.

[0236] The terms "two or more," "at least two," "multiple," or "poly" refer to two or more, including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more. As used herein, terms such as "reduce," "reduce," "inhibit," or "impair" refer to an overall decrease or the ability to cause an overall decrease in a level, preferably at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or more. These terms include complete or essentially complete inhibition, i.e., a reduction to or essentially to zero.

[0237] The term "multispecific" when referring to a viral protein amino acid sequence, e.g., a SARS-CoV-2 S protein amino acid sequence, refers to an amino acid sequence based on different variants of a viral protein, e.g., an amino acid sequence in which multiple epitopes normally present in different naturally occurring strains of said viral protein are combined in a single amino acid sequence.

[0238] Terms such as "increase," "enhancement," or "exceed" preferably relate to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500% or more.

[0239] According to this disclosure, the term "peptide" includes oligo- and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids joined together by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 150 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used interchangeably herein.

[0240] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic protein has therapeutic or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of such a disease or reduce the severity of such a disease or condition. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, vaccination antigens and immunostimulants such as cytokines.

[0241] A "fragment" or "portion" in reference to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains a start codon that serves for translation initiation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of the amino acid sequence.

[0242] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence or may be a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0243] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic versions. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0244] For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes total mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0245] Amino acid insertion variants include the insertion of one, two, or more amino acids into a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications that substitute amino acid positions and / or amino acids that are not conserved in homologous proteins or peptides with others having similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of members of a family of amino acids related in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0246] Preferably, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably displayed over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0247] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0248] The terms "% identical", "% identity" or similar terms refer specifically to the percentage of nucleotides or amino acids that are identical in optimal alignment of the compared sequences. The percentage is purely statistical, and the differences between the two sequences are not necessarily randomly distributed throughout the compared sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In one embodiment, the algorithm parameters used in the BLASTN algorithm on the NCBI website include (i) a prediction threshold setting of 10; (ii) a word size setting of 28; (iii) a maximum match within the query range setting of 0; (iv) a match / mismatch score setting of 1 or -2; (v) a gap cost setting of linear; and (vi) use of a filter for low complexity regions. In one embodiment, the algorithm parameters used in the BLASTP algorithm on the NCBI website include (i) a prediction threshold setting of 10; (ii) a word size setting of 3; (iii) a maximum match within the query range setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap costs set to presence: 11 and extension: 1; and (vi) a conditional compositional score matrix adjustment.

[0249] Percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying the result by 100.

[0250] In some embodiments, the degree of similarity or identity is expressed over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is expressed over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference sequence.

[0251] Homologous amino acid sequences according to the present disclosure exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0252] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, such as, for example, by solid-phase synthesis and similar methods.

[0253] In some embodiments, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigenic sequence, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been changed compared to the amino acid sequence of the parent molecule or sequence, yet still fulfills one or more functions of the parent molecule or sequence, such as inducing an immune response. In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0254] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be altered in sequence to differ from the naturally occurring or native sequence from which they are derived while maintaining the desired activity of the native sequence.

[0255] As used herein, "instruction material" or "instructions" includes publications, records, diagrams, or any other medium of representation that can be used to communicate the utility of the compositions and methods of the invention. The instruction material of the kits of the invention can be, for example, attached to or shipped with a container containing a composition of the invention. Alternatively, the instruction material can be shipped separately from the container, with the intention that the instruction material and the composition be used cooperatively by the recipient.

[0256] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0257] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant entity," such as a recombinant nucleic acid, in the context of the present invention is not naturally occurring.

[0258] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus), can be isolated from a natural source, and has not been artificially modified in a laboratory is naturally occurring.

[0259] As used herein, "physiological pH" refers to a pH of about 7.5.

[0260] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids described herein can be present in vitro or in vivo; for example, the cells may form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during transfection are usually not integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells capable of episomal amplification of nucleic acids have a greatly reduced dilution rate. If transfected nucleic acid is desired to remain in the genome of the cell and its daughter cells, stable transfection should be performed.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding antigen is transiently transfected into cells.RNA can be transfected so that cells transiently express the protein it encodes.

[0261] coronavirus Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They possess the largest genomes (26–32 kb) among known RNA viruses and are phylogenetically classified into four genera (α, β, γ, and Δ), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory illness, although the severity of the illness can be greater in infants, the elderly, and immunocompromised individuals. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), which belong to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses have emerged from animal reservoirs into human populations within the last 15 years and caused highly lethal pandemics. A severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak, which causes atypical pneumonia (coronavirus disease 2019; COVID-19), emerged in China in mid-December 2019 and has developed into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the betacoronavirus lineage B and shares at least 70% sequence similarity with SARS-CoV.

[0262] Generally, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in virus assembly, while the N protein is required for viral RNA synthesis. A key glycoprotein is responsible for virus binding and entry into target cells. The S protein is synthesized as a single-chain inactive precursor and is cleaved into two noncovalently linked subunits, S1 and S2, by a furin-like host protease in the producing cell. The S1 subunit contains a receptor-binding domain (RBD) that recognizes host cell receptors. The S2 subunit contains a fusion peptide, two heptad repeat regions, and a transmembrane domain, all of which undergo large conformational rearrangements to mediate fusion between the virus and host cell membranes. The S1 and S2 subunits trimerize to form the large prefusion spike.

[0263] The SARS-CoV-2 S precursor protein is proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit contains the receptor-binding domain (RBD) that mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0264] antigen Described herein are amino acid sequences containing sequences derived from viral proteins, e.g., viral surface proteins, e.g., SARS-CoV-2 S protein. The viral protein-derived sequences correspond to at least a portion of a viral protein containing amino acid modifications present in other variants of the viral protein, e.g., viral protein variants of other strains. The modifications generate (additional) epitopes that are specific for such other viral protein variants. Thus, the modified viral protein sequences described herein are multispecific viral protein amino acid sequences because they contain (modified) amino acid residues characteristic of multiple viral protein variants and / or viral strains. Also described herein are nucleic acids, e.g., RNAs, encoding amino acid sequences containing viral protein-derived sequences. The amino acid sequences and nucleic acids described herein are useful for inducing immune responses in subjects against viral proteins, particularly viral proteins of different viral strains. Amino acid sequences containing the multispecific viral protein amino acid sequences (i.e., antigenic peptides or proteins) described herein are also referred to herein as "vaccine antigens," "peptide and protein antigens," "antigenic molecules," or simply "antigens." The polyspecific viral protein amino acid sequences are also referred to herein as "antigenic peptides or proteins" or "antigenic sequences."

[0265] The SARS-CoV-2 coronavirus full-length spike (S) protein consists of 1273 amino acids and has the amino acid sequence of SEQ ID NO: 1:

[0266] For purposes of this disclosure, the above sequence is considered the wild-type SARS-CoV-2 S protein amino acid sequence. Position numbering in the SARS-CoV-2 S protein shown herein is relative to the amino acid sequence of SEQ ID NO: 1 and corresponds to positions in SARS-CoV-2 S protein variants.

[0267] The full-length spike (S) protein of SEQ ID NO: 1 can be modified in such a way that the prototype pre-fusion conformation is stabilized. Stabilization of the pre-fusion conformation can be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 of the full-length spike protein. In particular, a spike (S) protein stabilized protein variant is obtained in such a way that amino acid residue 986 is replaced with proline and amino acid residue 987 is also replaced with proline. In one embodiment, a SARS-CoV-2 S protein variant in which the prototype pre-fusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 7: (SEQ ID NO: 7)

[0268] In some embodiments, the compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity across a panel of SARS-CoV-2 spike variants (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or more). In some embodiments, such SARS-CoV-2 spike variants comprise mutations in the N-terminal domain (NTD) and / or receptor-binding domain (RBD).

[0269] In some embodiments, the compositions described herein are characterized in that the amino acid sequence, including the polyspecific SARS-CoV-2 S protein amino acid sequence, comprises one or more amino acid modifications (and thus epitopes) that are characteristic of a panel of SARS-CoV-2 spike variants (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or more). In some embodiments, such modifications that are characteristic of SARS-CoV-2 spike variants comprise mutations in the N-terminal domain (NTD) and / or receptor binding domain (RBD).

[0270] Modifications in the N-terminal domain (NTD) include: 18, 20, 26, 80, 138, 144, 190, 215, 246, 253, 140, 142, 145, 146, 147, 150, 152, 154, 156, 157, 158, 164, 247, 248, 249, 250, 251, 252, 254, 255, 258 Modifications at positions selected from the group consisting of:

[0271] Specific modifications in the N-terminal domain (NTD) include: L18F, T20N, P26S, D80Y, D138Y, Y144F, R190S, D215A, R246I, D253G, F140L, G142S, Y145H, H146Y, K147N, K150R, W152C, E154Q, E156A, F157L, R158G , N164T, S247G, Y248H, L249S, T250N, P251S, G252V, S254F, S255F, W258L The modifications include those selected from the group consisting of:

[0272] Modifications in the receptor binding domain (RBD) include: 417, 439, 452, 453, 477, 484, 501, 345, 346, 352, 378, 406, 420, 440, 441, 444, 445, 446, 450, 455, 460, 475, 478, 485, 486, 487, 489, 490, 493, 494, 499, 365, 369, 370, 374, 376, 384, 405, 408, 415, 421, 443, 447, 448, 456, 472, 473, 476, 496, 498, 500, 504 Modifications at positions selected from the group consisting of:

[0273] Specific modifications in the receptor binding domain (RBD) include: K417N, N439K, L452R, Y453F, S477N, E484K, N501Y, T345A, R346K, A352S, K378N, E406Q, D420, N440K, L441F, K444, V445A, G446V, N450K, L455F, N460I, A475V, T478I, G485V, F486L, N487D, Y489, F490S, Q493L, S494P, P499H, Y365D, Y369C, N370S, F374L, T376I, P384L, D405Y, R408I, T415N, Y421, S44 3A, G447V, N448Y, F456L, I472V, Y473F, G476S, G496C, Q498H, T500I, G504D The modifications include those selected from the group consisting of:

[0274] In some embodiments, the compositions described herein are characterized in that the amino acid sequence comprising the polyspecific SARS-CoV-2 S protein amino acid sequence is based on a parent SARS-CoV-2 S protein amino acid sequence (e.g., of the parent SARS-CoV-2 strain) that contains one or more amino acid modifications compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, and contains one or more additional amino acid modifications compared to the wild-type SARS-CoV-2 S protein amino acid sequence that are present in one or more other SARS-CoV-2 S protein amino acid sequences (e.g., of one or more other SARS-CoV-2 strains), e.g., as compared to SEQ ID NO: 1. Thus, in some embodiments, the compositions described herein are characterized in that the amino acid sequence comprising the polyspecific SARS-CoV-2 S protein amino acid sequence can include one or more amino acid modifications present in a parent SARS-CoV-2 S protein amino acid sequence compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, and one or more additional amino acid modifications present in one or more other SARS-CoV-2 S protein amino acid sequences compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1.

[0275] For example, in some embodiments, the compositions described herein may be characterized in that the amino acid sequence comprising the polyspecific SARS-CoV-2 S protein amino acid sequence may be based on the SARS-CoV-2 S protein amino acid sequence of strain B.1.1.7 as the parental SARS-CoV-2 S protein amino acid sequence and, therefore, may contain, as compared to the wild-type SARS-CoV-2 S protein amino acid sequence, amino acid modifications present in the parental SARS-CoV-2 S protein amino acid sequence as compared to, for example, SEQ ID NO: 1, namely, the H69 / V70 deletion, the Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. Additionally, an amino acid sequence comprising a polyspecific SARS-CoV-2 S protein amino acid sequence can include, compared to the wild-type SARS-CoV-2 S protein amino acid sequence, one or more amino acid modifications of, e.g., one or more strains other than the parental strain, that are present in one or more other SARS-CoV-2 S protein amino acid sequences, e.g., as compared to SEQ ID NO: 1. For example, such one or more strains other than the parental strain can be selected from the group consisting of B.1.351, B.1.1.298, B.1.427 / B.1.429, B.1.526, and P.1.

[0276] In certain embodiments, an amino acid sequence comprising a polyspecific SARS-CoV-2 S protein amino acid sequence can include one or more of the amino acid modifications present in B.1.526(NY) and one or more of the amino acid modifications present in B.1.427 / B.1.429(CAL) (e.g., in addition to the amino acid modifications present in the parent SARS-CoV-2 S protein amino acid sequence of strain B.1.1.7). One or more of the amino acid modifications present in B.1.526(NY) can be D253G and A701V. One or more of the amino acid modifications present in B.1.427 / B.1.429(CAL) can be L452R. Additional modifications can include L18F (e.g., as present in B.1.351 and P.1), N439K, and S477N.

[0277] In certain embodiments, an amino acid sequence comprising a polyspecific SARS-CoV-2 S protein amino acid sequence can include one or more of the amino acid modifications present in B.1.351(SA) (e.g., in addition to the amino acid modifications present in the parent SARS-CoV-2 S protein amino acid sequence of strain B.1.1.7). One or more of the amino acid modifications present in B.1.351(SA) can be D80A, D215G, R246I, K417N, and E484K.

[0278] Both of the above different amino acid sequences (or encoding nucleic acids), including the polyspecific SARS-CoV-2 S protein amino acid sequence, can be used in combination to induce neutralizing activity across at least the SARS-CoV-2 spike variants of strains B.1.351, B.1.1.298, B.1.427 / B.1.429, B.1.526 and P.1.

[0279] Those skilled in the art will be aware of various spike variants and / or resources describing same. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences, and in particular their modifications compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein:

[0280] B.1.1.7 ("Variant of Concern 202012 / 01" (VOC-202012 / 01) B.1.1.7 is a SARS-CoV-2 variant first detected in October 2020 in samples collected in the previous month during the COVID-19 pandemic in the UK and began to spread rapidly by mid-December. It has been associated with a significant increase in COVID-19 infection rates in the UK, an increase thought to be due, at least in part, to a mutation, N501Y, within the receptor-binding domain of the spike glycoprotein, which is required for binding to ACE2 in human cells. The B.1.1.7 variant is defined by 23 mutations: 13 nonsynonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., 17 mutations that alter the protein and 6 that do not). Spike protein alterations in B.1.1.7 include deletions 69-70, 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0281] B.1.351(501.V2) The B.1.351 lineage, colloquially known as the South African COVID-19 variant, is a variant of SARS-CoV-2. Preliminary results indicate that this variant may have increased infectivity. The B.1.351 variant is defined by multiple spike protein alterations, including L18F, D80A, D215G, deletions 242-244, R246I, K417N, E484K, N501Y, D614G, and A701V. There are three specifically targeted mutations in the spike region of the B.1.351 genome: K417N, E484K, and N501Y.

[0282] B.1.1.298 (Cluster 5) B.1.1.298 was discovered in North Denmark and is believed to have spread from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been identified. Specific mutations include deletions 69-70, Y453F, D614G, I692V, M1229I, and possibly S1147L.

[0283] P.1(B.1.1.248) Lineage B.1.1.248, also known as the Brazil variant, is one of the SARS-CoV-2 variants designated the P.1 lineage. P.1 has several S-protein modifications [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F] and is similar to the South African variant B.1.351 at certain critical RBD positions (K417, E484, and N501).

[0284] B.1.427 / B.1.429(CAL.20C) Lineage B.1.427 / B.1.429, also known as CAL.20C, is defined by the following modifications in the S-protein: S13I, W152C, L452R, and D614G, of which the L452R modification is of particular concern. The CDC has listed B.1.427 / B.1.429 as a "variant of concern."

[0285] B.1.525 B.1.525 carries the same E484K modification as found in the P.1 and B.1.351 variants, and also carries the same ΔH69 / ΔV70 deletion as found in B.1.1.7 and B.1.1.298. It also carries the modifications D614G, Q677H, and F888L.

[0286] B.1.526 B.1.526 was detected as an emerging lineage of virus isolates in the New York area that shares mutations with previously reported variants. The most common set of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

[0287] The table below provides an overview of circulating SARS-CoV-2 strains that are VOI / VOC.

[0288] [Table 1]

[0289] In certain embodiments, the vaccine antigens described herein comprise, consist essentially of, or consist of an amino acid sequence that is a modified variant of the spike protein (S) of SARS-CoV-2, or a fragment thereof.

[0290] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0291] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0292] In certain embodiments, the vaccine antigen comprises, consists essentially of, or consists of an amino acid sequence or fragment thereof that is a modified variant of the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein (S) of SARS-CoV-2).

[0293] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:1.

[0294] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9. and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:1.

[0295] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0296] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9. and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0297] In some embodiments, the vaccine antigen comprises, consists essentially of, or consists of an amino acid sequence, or a fragment thereof, that is a modified variant of the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of SARS-CoV-2. The amino acid sequence of amino acids 327-528 of SEQ ID NO: 1, or a fragment thereof, is also referred to herein as "RBD" or "RBD domain."

[0298] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1.

[0299] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1.

[0300] According to certain embodiments, the signal peptide is fused directly or via a linker to an amino acid sequence of a modified variant of the SARS-CoV-2 S protein or a fragment thereof, i.e., an antigenic peptide or protein. Thus, in certain embodiments, the signal peptide is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the vaccine antigen.

[0301] Such signal peptides generally have a length of about 15-30 amino acids and are preferably, but not limited to, sequences located at the N-terminus of an antigenic peptide or protein. A signal peptide as defined herein preferably enables transport of an antigenic peptide or protein encoded by an RNA to a predetermined intracellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment. In certain embodiments, a signal peptide sequence as defined herein includes, but is not limited to, the signal peptide sequence of the SARS-CoV-2 S protein, particularly a sequence comprising amino acids 1-16 or 1-19 of SEQ ID NO: 1, or a functional variant thereof.

[0302] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1.

[0303] In certain embodiments, the RNA encoding the signal sequence comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1.

[0304] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-19 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-19 of SEQ ID NO:1.

[0305] In certain embodiments, the RNA encoding the signal sequence comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-57 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1.

[0306] The signal peptide sequence defined herein further includes, but is not limited to, an immunoglobulin signal peptide sequence, for example, the signal peptide sequence of an immunoglobulin heavy chain variable region, where the immunoglobulin may be a human immunoglobulin. In particular, the signal peptide sequence defined herein includes a sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functional variant thereof.

[0307] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31.

[0308] In certain embodiments, the RNA encoding the signal sequence is (i) nucleotides 54 to 119 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32 or the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 54-119 of SEQ ID NO: 32; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31.

[0309] Such signal peptides are preferably used to facilitate secretion of the encoded antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to the encoded antigenic peptide or protein as defined herein.

[0310] Thus, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, the signal peptide preferably being fused to the N-terminus of the antigenic peptide or protein, more preferably an antigenic peptide or protein described herein.

[0311] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7.

[0312] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence that (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8, or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8, or 9; and / or (ii) comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7.

[0313] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO:7 or a fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO:7.

[0314] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence that (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and / or (ii) comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or a fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.

[0315] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1.

[0316] In some embodiments, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

[0317] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1.

[0318] In some embodiments, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.

[0319] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3 or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3.

[0320] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence comprising: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:4, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:4; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3.

[0321] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO:29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO:29.

[0322] In some embodiments, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-716 of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-716 of SEQ ID NO: 30; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO: 29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO: 29.

[0323] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.

[0324] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-725 of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-725 of SEQ ID NO: 32, and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO: 31.

[0325] According to certain embodiments, the trimerization domain is fused directly or via a linker to an amino acid sequence that is a modified variant of the SARS-CoV-2 S protein or a fragment thereof, i.e., an antigenic peptide or protein. Thus, in certain embodiments, the trimerization domain is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the vaccine antigen (which may optionally be fused to a signal peptide, as described above).

[0326] Such trimerization domains are preferably located at the C-terminus of the antigenic peptide or protein, but are not limited thereto. The trimerization domains defined herein preferably enable trimerization of antigenic peptides or proteins encoded by RNA. Examples of trimerization domains defined herein include, but are not limited to, Foldon, the natural trimerization domain of T4 fibritin. The C-terminal domain of T4 fibritin (Foldon) is essential for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. In certain embodiments, trimerization domains defined herein include, but are not limited to, a sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10, or a functional variant thereof. In certain embodiments, trimerization domains defined herein include, but are not limited to, a sequence comprising the amino acid sequence of SEQ ID NO: 10, or a functional variant thereof.

[0327] In certain embodiments, the trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10. In certain embodiments, the trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.

[0328] In certain embodiments, the RNA encoding the trimerization domain is selected from the group consisting of: i) the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11 or the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10. In certain embodiments, the RNA encoding the trimerization domain i) comprises the nucleotide sequence of nucleotides 7-87 of SEQ ID NO: 11; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10.

[0329] In certain embodiments, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 10.

[0330] In certain embodiments, the RNA encoding the trimerization domain i) comprises the nucleotide sequence of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11, or a fragment of the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the RNA encoding the trimerization domain (i) comprises the nucleotide sequence of SEQ ID NO:11; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:10.

[0331] Such trimerization domains are preferably used to promote trimerization of the encoded antigenic peptide or protein, and more preferably, a trimerization domain as defined herein is fused to an antigenic peptide or protein as defined herein.

[0332] Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerization domain as defined herein, wherein the trimerization domain is fused to the antigenic peptide or protein, more preferably to the C-terminus of the antigenic peptide or protein.

[0333] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5 or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

[0334] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence comprising: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.

[0335] In one embodiment, the RNA encoding the vaccine antigen (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and / or (ii) encodes an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.

[0336] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:18 or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:18.

[0337] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29.

[0338] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, and / or (ii) an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.

[0339] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.

[0340] In some embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.

[0341] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO:29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO:29.

[0342] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, and / or (ii) an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.

[0343] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

[0344] In some embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

[0345] According to certain embodiments, the transmembrane domain is fused to an amino acid sequence of a modified variant of the SARS-CoV-2 S protein or a fragment thereof, i.e., an antigenic peptide or protein, either directly or via a linker, e.g., a glycine / serine linker. Thus, in certain embodiments, the transmembrane domain is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the vaccine antigen (which may optionally be fused to a signal peptide and / or a trimerization domain, as described above).

[0346] Such a transmembrane domain is preferably located at the C-terminus of the antigenic peptide or protein, but is not limited thereto. Preferably, such a transmembrane domain, if present, is located at the C-terminus of the trimerization domain, but is not limited thereto. In some embodiments, the trimerization domain is located between the amino acid sequence of the modified variant of SARS-CoV-2 S protein or its fragment, i.e., the antigenic peptide or protein, and the transmembrane domain.

[0347] A transmembrane domain as defined herein preferably allows the anchoring of an antigenic peptide or protein to a cell membrane.

[0348] In certain embodiments, the transmembrane domain sequence defined herein includes, but is not limited to, the transmembrane domain sequence of the SARS-CoV-2 S protein, particularly a sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional variant thereof.

[0349] In certain embodiments, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In certain embodiments, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

[0350] In certain embodiments, the RNA encoding the transmembrane domain sequence is (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8, or 9 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

[0351] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

[0352] In some embodiments, the RNA encoding the vaccine antigen (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO: 30, and / or (ii) encodes an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-311 of SEQ ID NO: 29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-311 of SEQ ID NO: 29.

[0353] In certain embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.

[0354] In certain embodiments, the RNA encoding the vaccine antigen (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-995 of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-995 of SEQ ID NO: 32, and / or (ii) encodes an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-314 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-314 of SEQ ID NO: 31.

[0355] In some embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29.

[0356] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO: 30, and / or (ii) an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO: 29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO: 29.

[0357] In some embodiments, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.

[0358] In some embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-995 of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-995 of SEQ ID NO: 32, and / or (ii) an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO: 31.

[0359] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence comprising: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29.

[0360] In one embodiment, the RNA encoding the vaccine antigen encodes an amino acid sequence comprising: (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 31, or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 31.

[0361] In one embodiment, the vaccine antigen comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28 or an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28.

[0362] In certain embodiments, the RNA encoding the vaccine antigen (i) comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27, and / or (ii) encodes an immunogenic fragment of an amino acid sequence comprising an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28.

[0363] In certain embodiments, the vaccine antigen comprises an amino acid sequence that is a modified variant of a contiguous sequence of the SARS-CoV-2 coronavirus spike (S) protein, consisting of, or consisting essentially of, an amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) comprised by the vaccine antigen. In certain embodiments, the vaccine antigen comprises an amino acid sequence that is a modified variant of a contiguous sequence of the SARS-CoV-2 coronavirus spike (S) protein that does not exceed 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.

[0364] In some embodiments, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) that is a modified variant of the RNA described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2). In some embodiments, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) that is a modified variant of the RNA described herein as RBP020.2.

[0365] In one embodiment, the RNA encoding the vaccine antigen is nucleoside-modified messenger RNA (modRNA) and encodes an amino acid sequence comprising (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.

[0366] In one embodiment, the RNA encoding the vaccine antigen is nucleoside-modified messenger RNA (modRNA) and encodes an amino acid sequence comprising (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.

[0367] In one embodiment, the RNA encoding the vaccine antigen is nucleoside-modified messenger RNA (modRNA) and encodes an amino acid sequence comprising (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20, and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.

[0368] As used herein, the term "vaccine" refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

[0369] In some embodiments, the RNA encoding the antigen molecule is expressed in the subject's cells to provide the antigen molecule. In some embodiments, the expression of the antigen molecule occurs on the cell surface or in the extracellular space. In some embodiments, the antigen molecule is presented in the context of MHC. In some embodiments, the RNA encoding the antigen molecule is transiently expressed in the subject's cells. In some embodiments, after administration of the RNA encoding the antigen molecule, particularly after intramuscular administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule occurs in the muscle. In some embodiments, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule occurs in the spleen. In some embodiments, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule occurs in antigen-presenting cells, preferably specialized antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, after administration of the RNA encoding the antigen molecule, no or essentially no expression of the RNA encoding the antigen molecule occurs in the lung and / or liver. In some embodiments, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in the spleen is at least 5 times higher than in the lung.

[0370] In some embodiments, the methods and agents described herein, e.g., mRNA compositions, result in delivery of RNA encoding a vaccine antigen to lymph nodes and / or the spleen after administration to a subject, particularly after intramuscular administration. In some embodiments, the RNA encoding a vaccine antigen is detectable in the lymph nodes and / or the spleen from 6 hours after administration onward, and preferably for up to 6 days or more.

[0371] In certain embodiments, the methods and agents, e.g., mRNA compositions, described herein, upon administration to a subject, particularly upon intramuscular administration, result in delivery of RNA encoding a vaccine antigen to B cell follicles, subcapsular sinuses and / or T cell zones, particularly B cell follicles and / or subcapsular sinuses of lymph nodes.

[0372] In certain embodiments, the methods and agents described herein, e.g., mRNA compositions, after administration to a subject, particularly after intramuscular administration, can enhance the expression of B cells (e.g., CD19 + ), subcapsular sinus macrophages (CD169 + ) and / or dendritic cells in the T cell zone (CD11c + ) and the intermediate sinus of lymph nodes, especially B cells (CD19 + ) and / or subcapsular sinus macrophages of lymph nodes (CD169 + ) resulting in delivery of RNA encoding a vaccine antigen.

[0373] In certain embodiments, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to the splenic white pulp following administration to a subject, particularly following intramuscular administration.

[0374] In certain embodiments, the methods and agents described herein, e.g., mRNA compositions, after administration to a subject, particularly after intramuscular administration, can increase the number of B cells, DCs (CD11c + ), especially B cells surrounding the body and / or splenic macrophages, especially B cells and / or DCs (CD11c + ) resulting in delivery of RNA encoding a vaccine antigen.

[0375] In one embodiment, the vaccine antigen is expressed in the lymph nodes and / or spleen, particularly in cells of said lymph nodes and / or spleen.

[0376] An antigenic molecule or its advanced products, such as fragments thereof, can bind to an antigen receptor, such as a BCR or TCR, or an antibody carried by an immune effector cell.

[0377] For example, peptide and protein antigens, i.e., peptide and protein antigens provided to a subject according to the present invention by administration of RNA encoding the vaccine antigen, preferably result in the induction of an immune response, e.g., a humoral and / or cellular immune response, in the subject to whom the peptide or protein antigen is provided, which immune response is preferably directed against the target antigen, particularly the coronavirus S protein, and in particular the SARS-CoV-2 S protein.

[0378] A vaccine antigen may comprise a target antigen, a variant thereof, or a fragment thereof. In certain embodiments, such a fragment or variant is immunologically equivalent to the target antigen. The term "antigen fragment" or "antigen variant" refers to an agent that results in the induction of an immune response, which immune response targets an antigen, i.e., a target antigen. Thus, a vaccine antigen may correspond to or comprise a target antigen, a fragment of a target antigen, or an antigen that is homologous to the target antigen or its fragment. A vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence that is homologous to an immunogenic fragment of a target antigen. An "immunogenic fragment of an antigen" preferably relates to a fragment of an antigen that can induce an immune response against the target antigen. A vaccine antigen may be a recombinant antigen.

[0379] The term "immunologically equivalent" refers to an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, that exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, the amino acid sequence induces an immune response with specificity that reacts with the reference amino acid sequence.

[0380] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers to immune effector cells that undergo cell division, among other things.

[0381] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0382] The term "clonal expansion" or "expansion" refers to the process by which a specific entity reproduces. In the present invention, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize the antigen are amplified. Preferably, clonal expansion induces differentiation of immune effector cells.

[0383] The term "antigen" refers to an agent containing an epitope against which an immune response can be generated. The term "antigen" particularly includes proteins and peptides. In some embodiments, the antigen is presented by immune system cells, for example, antigen-presenting cells such as dendritic cells or macrophages. In some embodiments, the antigen or its processed product, such as a T cell epitope, is bound by an immunoglobulin molecule, such as a T cell or B cell receptor or antibody. Thus, the antigen or its processed product can specifically react with an antibody or T lymphocyte (T cell). In some embodiments, the antigen is a viral antigen, such as a coronavirus S protein, for example, a SARS-CoV-2 S protein, and the epitope is derived from such an antigen.

[0384] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. A viral antigen can be a coronavirus S protein, for example, a SARS-CoV-2 S protein.

[0385] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from the extracellular surface, for example, by an antibody located on the outside of the cell. In this context, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. Association can be direct or indirect. For example, association can be via one or more transmembrane domains, one or more lipid anchors, or by interaction with some other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule that associates with the cell surface can be a transmembrane protein having an extracellular portion, or a protein that associates with the cell surface by interacting with another protein that is a transmembrane protein.

[0386] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible for binding by proteins and other molecules. If an antigen is located on the cell surface, it is expressed on the surface of the cell and is accessible for binding by, for example, an antigen-specific antibody added to the cell.

[0387] The term "extracellular portion" or "exodomain" according to the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, for example by a binding molecule, such as an antibody, that is located on the outside of the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0388] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0389] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is generally about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, the binding peptides are generally about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may be effective.

[0390] Peptide and protein antigens can be, for example, 2 to 100 amino acids, including 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0391] The peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to develop antibody and T cell responses against the peptide or protein.

[0392] In one embodiment, the vaccine antigen is recognizable by immune effector cells. Preferably, once recognized by immune effector cells, the vaccine antigen, in the presence of an appropriate costimulatory signal, can induce stimulation, priming, and / or proliferation of immune effector cells bearing an antigen receptor that recognizes the vaccine antigen. In the context of this embodiment of the present invention, the vaccine antigen is preferably presented or presented on a cell surface, preferably an antigen-presenting cell. In one embodiment, the antigen is presented by a diseased cell, such as a virus-infected cell. In one embodiment, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of an MHC. In one embodiment, TCR binding, when expressed by a T cell and / or presented on a T cell by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or proliferation of the T cell. In one embodiment, TCR binding, when presented by a T cell to an antigen expressed by a T cell and / or presented on a diseased cell, results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0393] In certain embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope on the antigen. In certain embodiments, the antibody or B cell receptor binds to a natural epitope on the antigen.

[0394] nucleic acid As used herein, the term "polynucleotide" or "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. In the present invention, polynucleotides are preferably isolated.

[0395] The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.

[0396] In certain embodiments of all aspects of the invention, RNA encoding the vaccine antigen is expressed in cells, such as cells of an antigen-presenting subject, that have been engineered to produce the vaccine antigen.

[0397] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0398] In the present invention, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or the ends of the RNA. It is also contemplated herein that the nucleotides of the RNA may be chemically synthesized nucleotides or non-standard nucleotides such as deoxynucleotides. In the context of the present invention, these modified RNAs are considered analogs of naturally occurring RNA.

[0399] In some embodiments of the present invention, RNA is messenger RNA (mRNA), which is related to RNA transcripts that code for peptides or proteins.As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region and a 3' untranslated region (3'-UTR).In some embodiments, RNA is produced by in vitro transcription or chemical synthesis.In some embodiments, mRNA is produced by in vitro transcription using DNA template, where DNA refers to nucleic acid that comprises deoxyribonucleotides.

[0400] In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for transcription control can be any promoter of any RNA polymerase. The DNA template for in vitro transcription involves cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0401] In certain embodiments of the present invention, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In certain particularly preferred embodiments, the replicon or self-replicating RNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses generally ranges from 11,000 to 12,000 nucleotides, and the genomic RNA generally has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (forming viral particles). The genome generally contains two open reading frames (ORFs). The four nonstructural proteins (nsP1-nsP4) are generally co-encoded by a first ORF that begins near the 5' end of the genome, while the alphavirus structural proteins are co-encoded by a second ORF that is found downstream of the first ORF and extends toward the 3' end of the genome. Generally, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins can be translated from subgenomic transcripts, which are RNA molecules that mimic eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., early in the viral life cycle, ( +The alphavirus-based trans-replication system relies on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule is capable of replicating the replicase in trans (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules capable of being replicated in trans by the replicase must contain certain alphavirus sequence elements to allow recognition and RNA synthesis by the alphavirus replicase.

[0402] In some embodiments, the RNA described herein can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridines.

[0403] As used herein, the term "uracil" refers to one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is:

[0404] [ka]

[0405] As used herein, the term "uridine" refers to one of the nucleosides that can occur in RNA. The structure of uridine is:

[0406] [ka]

[0407] UTP (uridine 5'-triphosphate) has the following structure:

[0408] [ka]

[0409] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0410] [ka]

[0411] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0412] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the following structure:

[0413] [ka]

[0414] N1-methyl-pseudo-UTP has the following structure:

[0415] [ka]

[0416] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure:

[0417] [ka]

[0418] In some embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0419] In certain embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In certain embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0420] In certain embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In certain embodiments, the modified nucleoside comprises pseudouridine (ψ). In certain embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In certain embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In certain embodiments, the RNA may comprise more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In certain embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0421] In certain embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0422] In some embodiments, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine is partially or completely, preferably completely, substituted for cytidine in the RNA. In some embodiments, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the RNA contains 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0423] In some embodiments, the RNA of the present invention includes a 5' cap. In some embodiments, the RNA of the present invention does not have an uncapped 5' triphosphate. In some embodiments, the RNA can be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via a 5'-to-5' triphosphate linkage. In some embodiments, this guanosine is methylated at position 7. Adding a 5' cap or 5' cap analog to RNA can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be added to the RNA post-transcriptionally using a capping enzyme.

[0424] In one embodiment, the component cap for RNA is m2 having the following structure: 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’OG(5')ppp(5')m 2’-O (sometimes called ApG).

[0425] [ka]

[0426] Below are RNA and m2 7,3’O G(5')ppp(5')m 2’-O An exemplary cap 1 RNA containing ApG.

[0427] [ka]

[0428] Below is another exemplary Cap1 RNA (no cap analog):

[0429] [ka]

[0430] In some embodiments, the RNA comprises a cap analog anti-inversion cap (ARCA cap (m2)) having the structure: 7,3’O G(5')ppp(5')G)) is modified with a "cap 0" structure.

[0431] [ka]

[0432] Below are RNA and m2 7,3’O An exemplary cap 0 RNA containing G(5')ppp(5')G.

[0433] [ka]

[0434] In one embodiment, the "cap 0" structure is the cap analog beta-S-ARCA (m2 7,2’O G(5')ppSp(5')G).

[0435] [ka]

[0436] Below is the Beta-S-ARCA(m2 7,2’O G(5')ppSp(5')G) and RNA.

[0437] [ka]

[0438] The "D1" diastereomer of beta-S-ARCA or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column, and therefore has a shorter retention time, compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) (see WO 2011 / 015347, incorporated herein by reference).

[0439] A particularly preferred cap is beta-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O )ApG.

[0440] In some embodiments, the RNA of the present invention comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0441] In certain embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0442] In certain embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0443] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 12. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 13.

[0444] In certain embodiments, the RNA of the invention comprises a 3'-poly(A) sequence.

[0445] As used herein, the term "poly(A) sequence" or "poly(A tail)" generally refers to an uninterrupted or interrupted sequence of adenylic acid residues located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylic acid residues. In nature, uninterrupted poly(A) sequences are typical. The RNAs disclosed herein may have a poly(A) sequence attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0446] Poly(A) sequences of approximately 120 A nucleotides have been shown to beneficially affect the levels of RNA and protein translated from open reading frames located upstream (5') of the poly(A) sequence in transfected eukaryotic cells (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0447] The poly(A) sequence can be of any length. In certain embodiments, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, 400, 300, 200, or 150 A nucleotides, and particularly up to about 120 A nucleotides. In this context, "essentially consisting" means that the majority of the nucleotides in the poly(A) sequence, generally at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly(A) sequence, are A nucleotides, while the remaining nucleotides can be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0448] In some embodiments, poly(A) sequences are attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

[0449] In one embodiment, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 can be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences in which the four nucleotides (dA, dC, dG, and dT) are evenly distributed, e.g., 5 to 50 nucleotides in length, exhibit consistent growth of plasmid DNA in E. coli at the DNA level and further include beneficial properties related to supporting RNA stability and translation efficiency at the RNA level. As a result, in certain embodiments, the poly(A) sequences contained in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0450] In certain embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at the 3' end, i.e., the poly(A) sequence is not masked or followed by a nucleotide other than A at the 3' end.

[0451] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0452] In some embodiments, the RNA comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO:14 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:14.

[0453] A particularly preferred poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0454] According to the present disclosure, vaccine antigens are preferably administered as single-stranded, 5'-capped mRNA, which is translated into the respective protein upon entry into the cells of the subject receiving the RNA. Preferably, the RNA contains structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence) that optimize the maximum effectiveness of the RNA in terms of stability and translation efficiency.

[0455] In some embodiments, beta-S-ARCA (D1) is utilized as a specific capping structure at the 5' end of RNA. 7,3’-O Gppp(m1 2’-O) ApG is used as a specific capping structure at the 5' end of the RNA. In one embodiment, the 5'-UTR sequence is derived from human alpha-globin mRNA, optionally with an optimized 'Kozak sequence' to enhance translation efficiency. In one embodiment, a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of cleavage" (AES) mRNA (designated F) and the mitochondrial-encoded 12S ribosomal RNA (designated I) is placed between the coding sequence and the poly(A) sequence to ensure high maximum protein levels and long-term mRNA persistence. In one embodiment, two repeated 3'-UTRs derived from human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure high maximum protein levels and long-term mRNA persistence. In one embodiment, a 110-nucleotide-long poly(A) sequence is used, consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to enhance RNA stability and translation efficiency.

[0456] In some embodiments of all aspects of the invention, RNA encoding the vaccine antigen is expressed in the cells of the subject to be treated to provide the vaccine antigen. In some embodiments of all aspects of the invention, the RNA is transiently expressed in the cells of the subject. In some embodiments of all aspects of the invention, the RNA is in vitro transcribed RNA. In some embodiments of all aspects of the invention, the vaccine antigen is expressed on the cell surface. In some embodiments of all aspects of the invention, the vaccine antigen is expressed and presented in the context of MHC. In some embodiments of all aspects of the invention, the vaccine antigen is expressed in the extracellular space, i.e., the vaccine antigen is secreted.

[0457] In the present invention, the term "transcription" relates to the process by which a gene encoded by a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0458] According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied to produce the transcript. These cloning vectors are commonly referred to as transcription vectors and are encompassed by the term "vector" according to the present invention. According to the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by the T7 or SP6 promoter. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and then introduced into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0459] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes whereby an mRNA chain directs the assembly of an amino acid sequence to produce a peptide or protein.

[0460] In some embodiments, after administration of the RNA described herein, for example, formulated as an RNA-lipid particle, at least a portion of the RNA is delivered to the target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein it encodes. In some embodiments, the target cell is a splenocyte. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or macrophage. RNA particles, such as the RNA-lipid particles described herein, can be used to deliver RNA to such target cells. Thus, the present invention also relates to a method for delivering RNA to a target cell in a subject, comprising administering to the subject the RNA particles described herein. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.

[0461] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template for the synthesis of other polymers and macromolecules in biological processes that have a predetermined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0462] In some embodiments, the nucleic acid compositions described herein, e.g., compositions comprising lipid nanoparticle-encapsulated mRNA, are characterized by sustained expression of the encoded polypeptide (e.g., when administered to a subject). For example, in some embodiments, such compositions, when administered to a human, achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some embodiments, are characterized by such expression persisting for a period of at least 36 hours or more, including, for example, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours or more.

[0463] In some embodiments, the RNA encoding the vaccine antigen administered according to the present invention is non-immunogenic. RNA encoding an immunostimulatory agent can be administered according to the present invention to provide an adjuvant effect. The RNA encoding the immunostimulatory agent can be standard RNA or non-immunogenic RNA.

[0464] As used herein, the term "non-immunogenic RNA" refers to, for example, RNA that, upon administration to a mammal, either does not induce a response by the immune system or induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to the modifications and processing that render the non-immunogenic RNA non-immunogenic, i.e., standard RNA (stdRNA). In a preferred embodiment, the non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is rendered non-immunogenic by the incorporation of modified nucleosides into the RNA and the elimination of double-stranded RNA (dsRNA) to inhibit RNA-mediated activation of innate immune receptors.

[0465] Regarding rendering non-immunogenic RNA non-immunogenic by incorporating a modified nucleoside, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises the substitution of one or more uridines with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside containing a modified nucleobase is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 In certain particularly preferred embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ) or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0466] In certain embodiments, substitution of one or more uridines with nucleosides comprising modified nucleobases includes substitution of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of uridines.

[0467] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, a significant amount of aberrant products, including double-stranded RNA (dsRNA), is produced due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to protein synthesis inhibition. dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC, for example, using a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, using Escherichia coli RNase III to specifically hydrolyze dsRNA but not ssRNA, thereby removing dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA using cellulosic materials. In one embodiment, an RNA preparation is contacted with cellulosic materials, and ssRNA is separated from the cellulosic materials under conditions that allow dsRNA to bind to the cellulosic materials but not ssRNA.

[0468] As used herein, the terms "remove" or "removal" refer to the characterization of a population of a first substance, such as non-immunogenic RNA, that is separated from a nearby population of a second substance, such as dsRNA, where the population of the first substance is not necessarily free of the second substance, and the population of the second substance is not necessarily free of the first substance. However, a population of a first substance that is characterized by the removal of the population of the second substance will have a measurably lower content of the second substance compared to a mixture in which the first and second substances have not been separated.

[0469] In some embodiments, removing dsRNA from non-immunogenic RNA comprises removing dsRNA, so that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3% or less than 0.1% of the RNA in non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA is free or essentially free of dsRNA.In some embodiments, non-immunogenic RNA composition comprises purified preparation of single-stranded nucleoside modified RNA.For example, in some embodiments, purified preparation of single-stranded nucleoside modified RNA is substantially free of double-stranded RNA (dsRNA). In certain embodiments, a purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0470] In some embodiments, non-immunogenic RNA is translated in cells more efficiently than standard RNA having the same sequence. In some embodiments, translation is enhanced by up to a factor of 2-fold relative to the unmodified counterpart. In some embodiments, translation is enhanced by up to a factor of 3-fold. In some embodiments, translation is enhanced by up to a factor of 4-fold. In some embodiments, translation is enhanced by up to a factor of 5-fold. In some embodiments, translation is enhanced by up to a factor of 6-fold. In some embodiments, translation is enhanced by up to a factor of 7-fold. In some embodiments, translation is enhanced by up to a factor of 8-fold. In some embodiments, translation is enhanced by up to a factor of 9-fold. In some embodiments, translation is enhanced by up to a factor of 10-fold. In some embodiments, translation is enhanced by up to a factor of 15-fold. In some embodiments, translation is enhanced by up to a factor of 20-fold. In some embodiments, translation is enhanced by up to a factor of 50-fold. In some embodiments, translation is enhanced by up to a factor of 100-fold. In some embodiments, translation is enhanced by up to a factor of 200-fold. In some embodiments, translation is enhanced by a factor of 500-fold. In some embodiments, translation is enhanced by a factor of 1000-fold. In some embodiments, translation is enhanced by a factor of 2000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, the factor is any other significant amount or range of amounts.

[0471] In some embodiments, non-immunogenic RNA exhibits significantly lower natural immunogenicity than standard RNA with the same sequence. In some embodiments, non-immunogenic RNA exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, natural immunogenicity is reduced by a factor of 3-fold. In some embodiments, natural immunogenicity is reduced by a factor of 4-fold. In some embodiments, natural immunogenicity is reduced by a factor of 5-fold. In some embodiments, natural immunogenicity is reduced by a factor of 6-fold. In some embodiments, natural immunogenicity is reduced by a factor of 7-fold. In some embodiments, natural immunogenicity is reduced by a factor of 8-fold. In some embodiments, natural immunogenicity is reduced by a factor of 9-fold. In some embodiments, natural immunogenicity is reduced by a factor of 10-fold. In some embodiments, natural immunogenicity is reduced by a factor of 15-fold. In some embodiments, natural immunogenicity is reduced by a factor of 20-fold. In some embodiments, natural immunogenicity is reduced by a factor of 50-fold. In some embodiments, natural immunogenicity is reduced by a factor of 100-fold. In some embodiments, natural immunogenicity is reduced by a factor of 200-fold. In some embodiments, natural immunogenicity is reduced by a factor of 500-fold. In some embodiments, natural immunogenicity is reduced by a factor of 1000-fold. In some embodiments, natural immunogenicity is reduced by a factor of 2000-fold.

[0472] The term "exhibiting significantly lower natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of the non-immunogenic RNA can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA can be repeatedly administered without eliciting a natural immune response sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA.

[0473] "Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively nonspecific and immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0474] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0475] As used herein, the term "exogenous" refers to any substance that comes from or is produced outside an organism, cell, tissue or system.

[0476] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0477] As used herein, the terms "link," "fuse," or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements, components, or domains.

[0478] Codon optimization / increased G / C content In some embodiments, amino acid sequences, including the polyspecific viral protein amino acid sequences described herein, are encoded by coding sequences that are codon-optimized and / or have an increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In certain embodiments, the codon optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0479] The term "codon optimization" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the present invention, the coding region is preferably codon-optimized for optimal expression in a subject treated with the RNA molecule described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNAs in a cell. Thus, the sequence of an RNA can have codons modified so that frequently occurring tRNAs are available in place of "rare codons."

[0480] In one embodiment of the present invention, the guanosine / cytosine (G / C) content of the coding region of the RNA described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that mRNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode the same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to the wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons encoding the same amino acid but without A and / or U or with a reduced content of A and / or U nucleotides.

[0481] In various embodiments, the G / C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% or more compared to the G / C content of the coding region of the wild-type RNA.

[0482] RNA Administration Embodiments In some embodiments, the present disclosure provides an RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide comprising at least a portion of a SARS-CoV-2 S protein, wherein at least a portion of the SARS-CoV-2 S protein is modified to incorporate an amino acid modification found in other SARS-CoV-2 S protein variants. The RNA may comprise multiple different RNA molecules encoding polypeptides with different ones of the modifications. The RNA is suitable for intracellular expression of the polypeptide. In some embodiments, such encoded polypeptides do not comprise a sequence corresponding to the entire S protein. In some embodiments, the encoded polypeptides comprise a sequence corresponding to a receptor binding domain (RBD). In certain embodiments, such RNA (e.g., mRNA) can be complexed with a polycationic polymer, polyplex, protein, or peptide. In certain embodiments, such RNA can be formulated into lipid nanoparticles (e.g., those described herein). In certain embodiments, such RNA (e.g., mRNA) is used as or for use in an immunogenic composition (e.g., a vaccine) and / or to induce an immunological effect (e.g., SARS-CoV-2 neutralizing antibody and / or T cell response (e.g., CD4 + and / or CD8 + In certain embodiments, such RNA (e.g., mRNA) may be useful for vaccinating humans (e.g., including humans known to have been exposed to and / or infected with SARS-CoV-2 and / or humans with no known exposure to SARS-CoV-2).

[0483] In certain embodiments, the mRNA constructs described herein comprise a nucleic acid sequence encoding an amino acid sequence corresponding to a full-length SARS-CoV-2 spike protein (including, e.g., embodiments in which such encoded amino acid sequence may include at least one or more amino acid substitutions, e.g., proline substitutions, as described herein and / or embodiments in which the mRNA sequence has been codon-optimized for a mammalian, e.g., human, subject). In certain embodiments, an mRNA construct encoding an amino acid sequence corresponding to a full-length SARS-CoV-2 S protein may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine) in a particular subject population (e.g., a particular age population, e.g., an age population as described herein).

[0484] In some embodiments, the compositions or pharmaceutical formulations described herein comprise RNA encoding an amino acid sequence that is a modified variant of the SARS-CoV-2 S protein or an amino acid sequence comprising an immunogenic fragment of the SARS-CoV-2 S protein. Similarly, the methods described herein include administering such RNA.

[0485] The active platform used herein may be based on an antigen-encoding RNA vaccine that induces robust neutralizing antibodies and concomitant / associated T cell responses, preferably to achieve protective immunization with a minimal vaccine dose. The administered RNA is preferably in vitro transcribed RNA.

[0486] Three different RNA platforms are particularly preferred: unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying RNA (saRNA). In one particularly preferred embodiment, the RNA is in vitro transcribed RNA.

[0487] Below, we describe embodiments of these three different RNA platforms, where certain terms used when describing the elements thereof have the following meanings. S1S2 protein / S1S2 RBD: Sequences encoding each (modified) antigen of SARS-CoV-2.

[0488] nsP1, nsP2, nsP3, and nsP4: wild-type sequences encoding the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase and the subgenomic promoter and conserved sequence elements supporting replication and translation.

[0489] virUTR: viral untranslated region encoding part of the subgenomic promoter and replication and translation support sequence elements.

[0490] hAg-Kozak: 5'-UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

[0491] Sec:Sec corresponds to the endogenous S1S2 protein secretory signal peptide (sec), which directs translocation of the nascent polypeptide chain into the endoplasmic reticulum.

[0492] Glycine-serine linker (GS): A sequence encoding a short linker peptide consisting mostly of the amino acids glycine (G) and serine (S), as commonly used in fusion proteins.

[0493] Fibritin: A partial sequence of T4 fibritin (Foldon) used as an artificial trimerization domain.

[0494] The TM:TM sequence corresponds to the transmembrane portion of the S1S2 protein.

[0495] The FI element:3'-UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of cleavage" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified during an ex vivo selection process for sequences that contribute to RNA stability and enhance total protein expression.

[0496] A30L70: a 110-nucleotide-long poly(A) tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues, designed to enhance RNA stability and translation efficiency in dendritic cells.

[0497] Generally, the vaccine RNAs described herein may contain, from 5' to 3', one of the following structures: Cap-5'-UTR-vaccine antigen coding sequence-3'-UTR-poly(A) or Cap-hAg-Kozak-vaccine antigen coding sequence-FI-A30L70.

[0498] Generally, the vaccine antigens described herein may comprise, from N-terminus to C-terminus, one of the following structures: Signal sequence-RBD-trimerization domain or Signal sequence-RBD-trimerization domain-transmembrane domain.

[0499] The RBD and trimerization domain may be separated by a linker, particularly a GS linker, such as a linker having the amino acid sequence GSPGSGSGS. The trimerization domain and transmembrane domain may be separated by a linker, particularly a GS linker, such as a linker having the amino acid sequence GSGSGS.

[0500] The signal sequence can be a signal sequence described herein. The RBD can be a (modified) RBD domain described herein. The trimerization domain can be a trimerization domain described herein. The transmembrane domain can be a transmembrane domain described herein.

[0501] In one embodiment, The signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence. The RBD comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1. The trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto; and The transmembrane domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to this amino acid sequence.

[0502] In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31; The trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10; and The transmembrane domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO:1.

[0503] The RNA described herein or the RNA encoding the vaccine antigen described herein can be unmodified uridine-containing RNA (uRNA), nucleoside-modified mRNA (modRNA), or self-amplifying RNA (saRNA). In some embodiments, the RNA described herein or the RNA encoding the vaccine antigen described herein is nucleoside-modified mRNA (modRNA).

[0504] Unmodified uridine messenger RNA (uRNA) The active entity of unmodified messenger RNA (uRNA) drug substances is a single-stranded mRNA that is translated upon insertion into cells. In addition to the sequence encoding the vaccine antigen (i.e., open reading frame), each uRNA preferably contains consensus structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) optimized for maximum RNA efficacy with respect to stability and translation efficiency. A preferred 5' cap structure is beta-S-ARCA(D1)(m2 7,2’-O The preferred 5'-UTR and 3'-UTR comprise the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The preferred poly(A) tail comprises the sequence of SEQ ID NO: 14.

[0505] Different embodiments of this platform are sequences derived from and modified variants of: [Table 2]

[0506] FIG. 3 illustrates the general structure of an antigen-encoding RNA.

[0507] Nucleoside-modified messenger RNA (modRNA) The active component of nucleoside-modified messenger RNA (modRNA) drug substances is similarly a single-stranded mRNA that is translated upon insertion into cells. In addition to the sequence encoding the vaccine antigen (i.e., open reading frame), each modRNA contains common structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) to optimize the RNA's maximum efficacy as a uRNA. Compared to uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred 5' cap structure is m2 7,3’-O Gppp(m1 2’-O) ApG. Preferred 5'-UTR and 3'-UTR comprise the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively. A preferred poly(A) tail comprises the sequence of SEQ ID NO: 14. Further purification steps are applied to the modRNA to reduce dsRNA contaminants produced during the in vitro transcription reaction.

[0508] Different embodiments of this platform are sequences derived from and modified variants of: [Table 3]

[0509] FIG. 4 illustrates the general structure of an antigen-encoding RNA. [Table 4]

[0510] Self-amplifying RNA (saRNA) The active component of self-amplifying mRNA (saRNA) drug substances is a single-stranded RNA that self-amplifies upon entry into cells, leading to subsequent translation of vaccine antigens. In contrast to uRNA and modRNA, which preferably encode a single protein, the coding region of saRNA contains two open reading frames (ORFs). The 5'-ORF encodes an RNA-dependent RNA polymerase, such as the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). The replicase ORF is followed 3' by a subgenomic promoter and a second ORF encoding the antigen. Furthermore, the saRNA UTR contains 5' and 3' conserved sequence elements (CSEs) required for self-amplification. saRNAs contain consensus structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) to optimize the RNA's maximal efficacy as a uRNA. Preferably, saRNAs contain a uridine. A preferred 5'-cap structure is beta-S-ARCA(D1)(m2 7,2’-O GppSpG).

[0511] Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle. However, saRNA does not encode alphavirus structural proteins required for genome packaging or cell entry, and therefore, production of replication-competent viral particles is highly unlikely. Replication does not involve any intermediate steps that produce DNA. saRNA use / uptake, therefore, does not pose the risk of genomic integration or other permanent genetic modification within target cells. Furthermore, saRNA itself effectively activates the innate immune response through recognition of dsRNA intermediates, preventing their continued replication.

[0512] Different embodiments of this platform are sequences derived from and modified variants of: [Table 5]

[0513] FIG. 5 illustrates the general structure of an antigen-encoding RNA.

[0514] In some embodiments, a vaccine RNA described herein comprises a nucleotide sequence that is a modified variant of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 19, 20, 21, 24, 25, 26, 27, 30, and 32. Particularly preferred vaccine RNAs described herein comprise a nucleotide sequence that is a modified variant of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 25, 26, 30, and 32, e.g., a modified variant of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 26, 30, and 32.

[0515] The RNA described herein is preferably formulated into lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and RNA. In some embodiments, the cationic lipid is ALC-0315, the neutral lipid is DSPC, the steroid is cholesterol, and the polymer-conjugated lipid is ALC-0159. A preferred method of administration is intramuscular administration, more preferably in an aqueous cryoprotectant buffer for intramuscular administration. The formulation is preferably a preservative-free, sterile dispersion of RNA-lipid nanoparticles (LNPs) in an aqueous cryoprotectant buffer for intramuscular administration.

[0516] In various embodiments, the formulation contains the following ingredients, preferably in the proportions or concentrations set forth below:

[0517] [Table 6] ALC-0315:

[0518] [ka] ALC-0159:

[0519] [ka] DSPC:

[0520] [ka] cholesterol:

[0521] [ka]

[0522] In certain embodiments, the ratio of mRNA to total lipids (N / P) is 6.0 to 6.5, for example, about 6.0 or about 6.3.

[0523] Nucleic acid containing particles The nucleic acids described herein, such as RNA encoding vaccine antigens, can be formulated and administered as particles.

[0524] In the context of the present disclosure, the term "particle" refers to a structured entity formed by molecules or molecular complexes. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized small structure dispersed in a medium. In some embodiments, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

[0525] The electrostatic interaction between positively charged molecules such as polymers and lipids and negatively charged nucleic acids is involved in particle formation. This leads to the complexation and spontaneous formation of nucleic acid particles. In some embodiments, the nucleic acid particles are nanoparticles.

[0526] As used herein, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

[0527] "Nucleic acid particles" can be used to deliver nucleic acids to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionizable lipid or lipid-like substance, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0528] Without intending to be bound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like substances and / or cationic polymers form aggregates with nucleic acids, which aggregates result in colloidally stable particles.

[0529] In some embodiments, the particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

[0530] In some embodiments, a nucleic acid particle comprises more than one type of nucleic acid molecule, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, such as with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions or other properties.

[0531] The nucleic acid particles described herein can, in certain embodiments, have an average diameter ranging from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

[0532] The nucleic acid particles described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index ranging from about 0.1 to about 0.3, or from about 0.2 to about 0.3.

[0533] For RNA-lipid particles, the N / P ratio indicates the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the RNA. This correlates with the charge ratio, since nitrogen atoms (depending on the pH) are usually positively charged and phosphate groups are negatively charged. The N / P ratio is pH-dependent when charge balance exists. Lipid formulations are often formed with an N / P ratio greater than 4, up to 12, because positively charged nanoparticles are thought to be favorable for transfection. In this case, the RNA is thought to be fully bound to the nanoparticles.

[0534] The nucleic acid particles described herein can be produced using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0535] As used herein, the term "colloid" refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have particle sizes between 1 and 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the entire suspension.

[0536] For the preparation of colloids containing at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, methods conventionally used for the preparation of liposome vesicles are applicable and adapted as appropriate. The most commonly used methods for liposome vesicle preparation share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

[0537] In the film hydration method, lipids are first dissolved in a suitable organic solvent and allowed to dry to form a thin film on the bottom of a flask. The resulting lipid film is hydrated using a suitable aqueous medium to produce a liposome dispersion. Further miniaturization steps may be included.

[0538] Reverse phase evaporation, which involves the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase, is another method of film hydration for liposomal vesicle preparation. Brief sonication of the mixture is required to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel, which then transforms into a liposomal suspension.

[0539] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids in the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in some embodiments as follows: an ethanol solution containing lipids, such as a cationic lipid and an additional lipid, is injected into an aqueous solution under stirring. In some embodiments, the RNA lipoplex particles described herein can be obtained without using an extrusion step.

[0540] The terms "extrude" or "extrusion" refer to the production of particles having a fixed cross-sectional profile, particularly the reduction of particle size by forcing the particles through a filter of defined pore size.

[0541] Other methods with organic solvent-free characteristics may also be used according to the present invention for the preparation of colloids.

[0542] LNPs generally contain four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a polyethylene glycol (PEG)-lipid. Each component is responsible for protecting the payload and enabling effective intracellular delivery. LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0543] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in the so-called Z dimension together with the linear dimension and the dimensionless polydispersity index (PI). 平均 (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is defined as Z 平均Used synonymously with this value of

[0544] The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis as described in the definition of "average diameter". Under certain conditions, it can be interpreted as an indicator of the size distribution of an ensemble of nanoparticles.

[0545] Various types of nucleic acid-containing particles have previously been described as suitable for delivery of nucleic acids in particulate form (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids can physically protect the nucleic acid from degradation and, through specific chemistries, aid in cellular uptake and endosomal escape.

[0546] The present invention describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer bound to the nucleic acid to form a nucleic acid particle, and compositions comprising such particles. The nucleic acid particle may contain nucleic acid complexed to the particle in different forms by non-covalent interactions. The particles described herein are not virus particles, particularly infectious virus particles, i.e., they cannot infect cells virally.

[0547] Suitable cationic or cationically ionizable lipids or lipid-like substances and cationic polymers form nucleic acid particles and are encompassed by the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that binds with nucleic acid to form nucleic acid particles. Such components include any component that can be part of a nucleic acid particle.

[0548] Some embodiments described herein relate to compositions, methods and uses comprising one or more, e.g., two, three, four, five, six or more, nucleic acid species, e.g., RNA species, such as: a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein has been modified to comprise an amino acid found in a corresponding amino acid position in one or more viral protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein has been modified to comprise an amino acid found in a corresponding amino acid position in one or more viral protein variants.

[0549] In some embodiments, a set of nucleic acids is provided, each comprising a nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parental viral protein, wherein each encoded fragment of the set is substantially identical to the other encoded fragments of the set and to the parental encoded fragment, except that modifications are present in the encoded fragment of the set compared to the parental encoded fragment that occurred in the circulating viral variant.

[0550] In microparticle preparations, each nucleic acid species can be individually formulated as an individual microparticle preparation.In this case, each individual microparticle preparation contains one nucleic acid species.Each individual microparticle preparation can be present as a separate entity, for example, in a separate container.This type of preparation can be obtained by providing each nucleic acid species separately (usually each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing particles to form.Each particle exclusively contains the specific nucleic acid species that is provided when the particle is formed (individual microparticle preparation).

[0551] In some embodiments, compositions, for example, pharmaceutical compositions, comprise more than one individual particle formulation.Each pharmaceutical composition is called a mixed microparticle formulation.The mixed microparticle formulation according to the present invention can be obtained by forming each individual microparticle formulation separately as described above, and then mixing the individual microparticle formulations.By mixing, a formulation comprising a mixed population of nucleic acid-containing particles can be obtained.The individual particle populations can be combined into one container that contains a mixed population of individual microparticle formulations.

[0552] Alternatively, different nucleic acid species can be formulated together as complex microparticle preparations.This kind of preparation can be obtained by providing the complex preparation (usually complex solution) of different RNA species together with particle forming agent, thereby allowing particle formation.In contrast to mixed microparticle preparations, complex microparticle preparations usually comprise particles that contain more than one RNA species.In complex particle compositions, different RNA species usually exist together in a single particle.

[0553] cationic polymer Given their high chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Cationic polymers are typically used to electrostatically concentrate negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines, which are thought to change protonation state in the pH range of 5.5 to 7.5, leading to an ionic imbalance that results in endosomal rupture. Polymers such as poly-l-lysine, polyamidoamine, protamine, polyethyleneimine, and naturally occurring polymers such as chitosan are all applicable to nucleic acid delivery and are suitable as cationic polymers. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have been widely used for nucleic acid delivery due to their ease of synthesis and biodegradability. These synthetic polymers are also suitable as cationic polymers.

[0554] As used herein, "polymer" has its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) covalently linked. The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties as described herein, may also be present in the polymer.

[0555] If more than one type of repeat unit is present in a polymer, the polymer may be referred to as a "copolymer." It is understood that a polymer, as used herein, may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeat units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block) and one or more regions each comprising a second repeat unit (e.g., a second block). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more different blocks.

[0556] In some embodiments, the polymer is biocompatible. Biocompatible polymers are generally polymers that do not cause significant cell death at reasonable concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment, such as within the body.

[0557] In some embodiments, the polymer can be protamine or a polyalkyleneimine, particularly protamine.

[0558] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and hydrolyzes to yield primarily arginine. In purified form, it is used in long-acting insulin preparations and to neutralize the anticoagulant effect of heparin.

[0559] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence (including fragments thereof) obtained or derived from natural or biological sources and multimeric forms of said amino acid sequence or fragments thereof, as well as (synthetic) polypeptides that are artificial, specifically designed for a specific purpose, and cannot be isolated from natural or biological sources.

[0560] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-10 2 ~10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0561] Preferred according to the invention are linear polyalkyleneimines, such as linear polyethyleneimine (PEI).

[0562] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In some embodiments, cationic polymers contemplated for use herein include any cationic polymer that can bind to nucleic acids, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is entrapped or encapsulated.

[0563] The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0564] Lipids and lipid-like substances The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are generally poorly soluble in water. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and various phases. One of these phases consists of a lipid bilayer, as they exist in aqueous environments as vesicles, multilamellar / unilamellar liposomes, or membranes. Hydrophobicity is conferred by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups are polar and / or charged groups and may include carbohydrate, phosphate, carboxylate, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other similar groups.

[0565] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion can have a formal positive charge or a formal negative charge. Alternatively, the polar portion can have both a formal positive and negative charge and can be a zwitterion or an inner salt. For purposes of this disclosure, an amphiphilic compound can be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0566] The term "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refers to a substance that is structurally and / or functionally related to lipids but cannot be considered a lipid in the strict sense. For example, this term includes compounds that can form amphiphilic layers by existing in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, and that contain surfactants or synthetic compounds in both the hydrophilic and hydrophobic portions. Generally speaking, this term refers to molecules that contain hydrophilic and hydrophobic portions of various structural configurations that may or may not resemble lipids. As used herein, the term "lipid" is considered to encompass both lipids and lipid-like substances, unless otherwise specified or clearly contradicted by the context.

[0567] Specific examples of amphiphilic compounds that may be included in the amphiphilic layer may include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0568] In some embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0569] Fatty acids or fatty acid residues are a diverse group of molecules consisting of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, generally 4 to 24 carbons long, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains double bonds, it can exhibit either cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis-double bonds cause the fatty acid chain to bend, and the more double bonds in the chain, the more complex the effects. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0570] Glycerolipids are mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are generally each esterified with a different fatty acid. A further subclass of glycerolipids is glycosylglycerol, characterized by the presence of one or more sugar residues attached to glycerol by glycosidic bonds.

[0571] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached to two fatty acid-derived "tails" by ester bonds and to one "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids), are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0572] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are generally saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The predominant phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily possess ceramide phosphoethanolamine, and fungi possess phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues attached by glycosidic bonds to a sphingoid base. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0573] Sterol lipids, such as cholesterol and its derivatives or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0574] Saccharolipids are compounds in which fatty acids are directly attached to a sugar backbone, forming a structure compatible with membrane bilayers. In saccharolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The most well-known saccharolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth of Escherichia coli is Kdo2-lipid A, a hexa-acylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0575] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by iterative and multimodular enzymes that share mechanistic properties with classical enzymes as well as fatty acid synthases. They represent a large proportion of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and represent a large amount of structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0576] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0577] Cationic or cationic ionizable lipids or lipid-like substances The nucleic acid particles described herein can include at least one cationic or cationically ionizable lipid or lipid-like substance as a particle-forming agent. The cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein includes any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acid. In some embodiments, the cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein can bind to nucleic acid, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or enclosed.

[0578] As used herein, "cationic lipid" or "cationic lipid-like substance" refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or higher acyl chain, and a lipid head group that generally has a positive charge.

[0579] In some embodiments, cationic lipid or lipid-like substance only has net positive charge at a certain pH, particularly acidic pH, while at a different, preferably high pH, ​​such as physiological pH, it preferably does not have net positive charge, preferably has no charge, i.e., is neutral.This ionizable behavior is thought to enhance efficacy and reduce toxicity by helping endosomal escape compared to particles that remain cationic at physiological pH.

[0580] For the purposes of the present invention, such "cationically ionizable" lipids or lipid-like substances are included within the term "cationic lipids or lipid-like substances," unless the context indicates otherwise.

[0581] In certain embodiments, the cationic or cationically ionizable lipid or lipid-like substance comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated.

[0582] Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane (DODAP); Dioctadecyldimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxy Propyl-3-dimethyl-hydroxyethylammonium bromide (DORIE) and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-methyl)-2-propanaminium trifluoroacetate (DOSPA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N'-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N'-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 2,3-di ...N-Dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleyl -4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy) -1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP ...2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

[0583] In certain embodiments, the cationic lipid may comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipid present in the particle.

[0584] Additional lipids or lipid-like substances The particles described herein may also contain lipids or lipid-like substances other than cationic or cationically ionizable lipids or lipid-like substances, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances). Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances. In addition to ionizable / cationic lipids or lipid-like substances, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipids can enhance particle stability and the effectiveness of nucleic acid delivery.

[0585] Additional lipids or lipid-like substances may be incorporated, which may or may not affect the overall charge of the nucleic acid particles. In some embodiments, the additional lipids or lipid-like substances are non-cationic lipids or lipid-like substances. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following: (1) a phospholipid; (2) cholesterol or a derivative thereof; or (3) a neutral lipid component of a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0586] Specific phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids are in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE) and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0587] In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.

[0588] In some embodiments, the nucleic acid particle comprises both a cationic lipid and an additional lipid.

[0589] In some embodiments, the particles described herein comprise a polymer-conjugated lipid, such as a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art.

[0590] Without wishing to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid can affect important nucleic acid particle characteristics such as nucleic acid charge, particle size, stability, tissue selectivity and biological activity.Therefore, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2 or about 3:1 to about 1:1.

[0591] In certain embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more of phospholipids and / or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the particle.

[0592] Lipoplex particles In certain embodiments of the present invention, the RNA described herein may be present in RNA lipoplex particles.

[0593] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle comprising lipids, particularly cationic lipids, and RNA. Electrostatic interaction between positively charged liposomes and negatively charged RNA leads to complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes are generally synthesized using cationic lipids such as DOTMA and additional lipids such as DOPE. In some embodiments, the RNA lipoplex particle is a nanoparticle.

[0594] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0595] In certain embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0596] The RNA lipoplex particles described herein, in certain embodiments, have an average diameter ranging from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In certain embodiments, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In other embodiments, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In exemplary embodiments, the RNA lipoplex particles have an average diameter of about 400 nm.

[0597] The RNA lipoplex particles and compositions comprising the RNA lipoplex particles described herein are useful for delivering RNA to target tissues after parenteral administration, particularly intravenous administration. RNA lipoplex particles can be prepared using liposomes, which can be obtained by injecting a lipid solution in ethanol into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase contains, for example, about 5 mM of acetic acid. Liposomes can be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In some embodiments, the liposomes and RNA lipoplex particles contain at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0598] Spleen-targeting RNA lipoplex particles are described in WO2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or splenocytes, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Therefore, the RNA lipoplex particles of the present invention can be used to express RNA in the spleen. In some embodiments, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression does not or essentially does not occur in the lung and / or liver. In some embodiments, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Therefore, the RNA lipoplex particles of the present invention can be used to express RNA in such antigen-presenting cells. In some embodiments, the antigen-presenting cells are dendritic cells and / or macrophages.

[0599] Lipid nanoparticles (LNPs) In some embodiments, nucleic acids, such as RNA, described herein are administered in the form of lipid nanoparticles (LNPs). LNPs can include any lipid capable of forming particles to which one or more nucleic acid molecules can be bound or encapsulated.

[0600] In certain embodiments, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and pegylated lipids.

[0601] In certain embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and RNA encapsulated within or attached to the lipid nanoparticle.

[0602] In some embodiments, the LNP comprises 40-55 mol percent, 40-50 mol percent, 41-49 mol percent, 41-48 mol percent, 42-48 mol percent, 43-48 mol percent, 44-48 mol percent, 45-48 mol percent, 46-48 mol percent, 47-48 mol percent, or 47.2-47.8 mol percent of the cationic lipid. In some embodiments, the LNP comprises about 47.0 mol percent, 47.1 mol percent, 47.2 mol percent, 47.3 mol percent, 47.4 mol percent, 47.5 mol percent, 47.6 mol percent, 47.7 mol percent, 47.8 mol percent, 47.9 mol percent, or 48.0 mol percent of the cationic lipid.

[0603] In some embodiments, the neutral lipids are present at a concentration ranging from 5 to 15 molar percent, 7 to 13 molar percent, or 9 to 11 molar percent, hi some embodiments, the neutral lipids are present at a concentration of about 9.5 molar percent, 10 molar percent, or 10.5 molar percent.

[0604] In some embodiments, the steroid is present at a concentration ranging from 30 to 50 molar percent, 35 to 45 molar percent, or 38 to 43 molar percent, hi some embodiments, the steroid is present at a concentration of about 40 molar percent, 41 molar percent, 42 molar percent, 43 molar percent, 44 molar percent, 45 molar percent, or 46 molar percent.

[0605] In certain embodiments, the LNPs comprise 1 to 10 mole percent, 1 to 5 mole percent, or 1 to 2.5 mole percent of the polymer-conjugated lipid.

[0606] In some embodiments, the LNP comprises 40-50 mole percent cationic lipid; 5-15 mole percent neutral lipid; 35-45 mole percent steroid; 1-10 mole percent polymer-conjugated lipid; and RNA encapsulated or bound within the lipid nanoparticle.

[0607] In some embodiments, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle.

[0608] In some embodiments, neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE and SM.In some embodiments, neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.In some embodiments, neutral lipid is DSPC.

[0609] In some embodiments, the steroid is cholesterol.

[0610] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid. In some embodiments, the PEGylated lipid has the following structure:

[0611] [ka] [In the formula, R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has an average value of 30 to 60. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In certain embodiments, R 12 and R 13 are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 are each independently a linear, saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0612] In some embodiments, the pegylated lipid is DMG-PEG 2000, for example, having the structure:

[0613] [ka]

[0614] In certain embodiments, the cationic lipid component of the LNP has the formula (III):

[0615] [ka] [During the ceremony, L 1 or L 2 One of the is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0616] In some of the above embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB):

[0617] [ka] [During the ceremony, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is, in each occurrence, independently H, OH, or C-C 24 is alkyl; n is an integer ranging from 1 to 15. The first embodiment has one of the following:

[0618] In some of the above embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0619] In other embodiments of Formula (III), the lipid has structure (IIIC) or (IIID):

[0620] [ka] (wherein y and z each independently represent an integer ranging from 1 to 12.) It has one of the following.

[0621] In any of the above embodiments of formula (III), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0622] In certain different embodiments of formula (III), the lipid has structure (IIIE) or (IIIF):

[0623] [ka] It has one of the following.

[0624] In some of the above embodiments of formula (III), the lipid has the structure (IIIG), (IIIH), (IIII) or (IIIJ)

[0625] [ka] It has one of the following.

[0626] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0627] In certain other of the above embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in certain embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0628] In some of the above embodiments of formula (III), R 6 is H. In other of the above embodiments, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0629] In certain embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0630] In certain other such embodiments of Formula (III), R 1 or R 2 Or both are C6-C 24 For example, in one embodiment, R 1 and R 2 are each independently of the following structure:

[0631] [ka] [In the formula, R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; and a is an integer from 2 to 12, where R 7a , R 7b and a is R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0632] In some of the above embodiments of formula (III), at least one occurrence of R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In another different embodiment of the above, at least one occurrence of R 7b is C1-C8 alkyl. For example, in certain embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0633] In various embodiments of formula (III), R 1 or R 2 or both, structure

[0634] [ka] It has one of the following.

[0635] In some of the above embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0636] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

[0637] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0638] In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

[0639] In some embodiments, the cationic lipid is present in the LPN in an amount of about 40 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LPN in an amount of about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LPN in an amount of about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LPN in an amount of about 1 to about 10 mole percent.

[0640] In some embodiments, the LNPs comprise compound III-3 in an amount of about 40 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 10 mole percent.

[0641] In certain embodiments, the LNPs comprise compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

[0642] In various different embodiments, the cationic lipid has one of the structures shown in the table below.

[0643] [Table 8]

[0644] In some embodiments, LNP comprises the cationic lipid shown in the table above, for example, the cationic lipid of formula (B) or formula (D), particularly the cationic lipid of formula (D), RNA, a neutral lipid, a steroid and a PEGylated lipid.In some embodiments, the neutral lipid is DSPC.In some embodiments, the steroid is cholesterol.In some embodiments, the PEGylated lipid is DMG-PEG 2000.

[0645] In some embodiments, the LNPs comprise a cationic lipid that is an ionizable lipidoid. In some embodiments, the cationic lipid has the following structure:

[0646] [ka]

[0647] The N / P value is preferably at least about 4. In certain embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In certain embodiments, the N / P value is about 6.

[0648] The LNPs described herein can, in certain embodiments, have an average diameter ranging from about 30 nm to about 200 nm or from about 60 nm to about 120 nm.

[0649] RNA targeting Certain embodiments of the present invention include targeted delivery of the RNAs disclosed herein (eg, RNAs encoding vaccine antigens and / or immunostimulatory agents).

[0650] In some embodiments, the present invention includes pulmonary targeting.Pulmonary targeting is particularly preferred if the administered RNA is RNA encoding a vaccine antigen.RNA can be delivered to the lungs by inhalation administration, for example, by administering RNA, which can be formulated as a particle, for example, a lipid particle, as described herein.

[0651] In one embodiment, the present invention involves targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. Targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen, is particularly preferred if the administered RNA is RNA encoding a vaccine antigen.

[0652] In some embodiments, the target cells are splenocytes. In some embodiments, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen.

[0653] The "lymphatic system" is a part of the circulatory system and an important part of the immune system, including the network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, the conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including the lymph nodes and spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0654] RNA can be delivered to the spleen using so-called lipoplex formulations, in which RNA is bound to liposomes containing cationic lipids and, optionally, additional or helper lipids to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a lipid solution in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeting RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or splenocytes, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Therefore, the RNA lipoplex particles of the present invention can be used for RNA expression in the spleen. In certain embodiments, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the lung and / or liver does not or essentially does not occur. In some embodiments, after administration of RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen.Therefore, the RNA lipoplex particles of the present invention can be used to express RNA in such antigen-presenting cells.In some embodiments, the antigen-presenting cells are dendritic cells and / or macrophages.

[0655] The charge of the RNA lipoplex particles of the present invention is the sum of the charge present on at least one cationic lipid and the charge present on the RNA. The charge ratio is the ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA. The charge ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA is calculated by the following formula: Charge ratio = [(cationic lipid concentration (mol)) x (total number of positive charges on the cationic lipid)] / [(RNA concentration (mol)) x (total number of negative charges on the RNA)].

[0656] The spleen-targeting RNA lipoplex particles described herein preferably have a net negative charge, such as a positive to negative charge ratio of about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2, at physiological pH. In certain embodiments, the positive to negative charge ratio of the RNA lipoplex particles is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0, at physiological pH.

[0657] The immunostimulatory agent can be administered to a subject by administering RNA encoding the immunostimulatory agent to the subject in a formulation for preferential delivery of the RNA to the liver or liver tissue. In particular, delivery of RNA to such target organs or tissues is preferred when large amounts of the immunostimulatory agent are desired and / or when the systemic presence of the immunostimulatory agent in particularly significant amounts is desired or necessary.

[0658] RNA delivery systems have a unique preference for the liver. This is associated with lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles, such as liposomes, nanomicelles, and lipophilic ligands of bioconjugates. Liver accumulation is due to the discontinuous nature of the liver vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0659] For in vivo RNA delivery to the liver, a drug delivery system can be used to transport RNA to the liver while preventing its degradation. For example, polyplex nanomicelles, consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core, are useful systems because the nanomicelles provide excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface, consisting of a dense PEG palisade, allow them to efficiently evade host immune defenses.

[0660] Examples of suitable immunostimulatory agents for liver targeting are cytokines involved in T cell proliferation and / or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0661] In other embodiments, RNA encoding an immunostimulatory agent may be administered in a formulation that delivers the RNA preferentially to the lymphatic system, particularly to secondary lymphatic organs, more particularly to the spleen. Delivery of the immunostimulatory agent to such target tissues is preferred, particularly when the presence of the immunostimulatory agent in this organ or tissue is desired (e.g., to induce an immune response, particularly when an immunostimulatory agent such as a cytokine is required during T cell priming or activation of resident immune cells), but the systemic presence of the immunostimulatory agent is undesirable, especially in significant amounts (e.g., because the immunostimulatory agent has systemic toxicity).

[0662] Examples of suitable immunostimulatory agents are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0663] immune stimulants In certain embodiments, the RNA encoding the vaccine antigen may be non-immunogenic. In this and other embodiments, the RNA encoding the vaccine antigen may be co-administered with an immunostimulatory agent or an RNA encoding an immunostimulatory agent. The methods and agents described herein are particularly effective if the immunostimulatory agent is linked to a pharmacokinetic-modifying group (hereinafter referred to as an "extended pharmacokinetics (PK)" immunostimulatory agent). The methods and agents described herein are particularly effective if the immunostimulatory agent is administered in the form of an RNA encoding the immunostimulatory agent. In certain embodiments, the RNA is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and can produce large amounts of protein.

[0664] An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components of the immune system, particularly immune effector cells. Immunostimulants may be pro-inflammatory.

[0665] In some embodiments, the immunostimulatory agent is a cytokine or a variant thereof. Examples of cytokines include interferons such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins such as IL2, IL7, IL12, IL15, and IL23, colony-stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factors. In other embodiments, the immunostimulatory agent comprises an adjuvant-type immunostimulatory agent such as an APC toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of toll-like receptor agonists include costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0666] Cytokines are a category of small proteins (approximately 5–20 kDa) important in cell signaling. Their release affects the behavior of surrounding cells. Cytokines act as immunoregulators and participate in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in the terminology). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one cell type. Cytokines act through receptors and are particularly important to the immune system; they regulate the balance between humoral and cell-based immune responses and control the maturation, proliferation, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0667] According to the present disclosure, the cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, particularly any mammal. One particularly preferred cytokine is interferon-α.

[0668] interferon Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, virus-infected cells release interferons, which mount the antiviral defenses of neighboring cells.

[0669] Depending on the type of receptor through which they signal, interferons are generally divided into three classes: type I interferons, type II interferons, and type III interferons.

[0670] All type I interferons bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

[0671] The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, leading to the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0672] IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs) and are involved in innate immunity, primarily against viral infections. The genes responsible for their synthesis fall into 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

[0673] IFNβ protein is produced in large amounts by fibroblasts. It has antiviral activity and is primarily involved in the innate immune response. Two types of IFNβ have been described: IFNβ1 and IFNβ3. Natural and recombinant forms of IFNβ1 have antiviral, antibacterial, and anticancer properties.

[0674] Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12. Additionally, type II interferons are released by cytotoxic T cells and T helper cells.

[0675] Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information indicates the importance of type III IFNs in certain types of viral or fungal infections.

[0676] In general, type I and type II interferons are responsible for regulating and activating the immune response.

[0677] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0678] According to the present disclosure, the interferon can be a naturally occurring interferon or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal.

[0679] Interleukin Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on distinct structural characteristics. However, their amino acid sequence similarity is rather weak (generally 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

[0680] According to the present disclosure, the interleukin may be a naturally occu...

Claims

1. a) identifying amino acid positions in a parent viral protein that are modified compared to the corresponding amino acid positions of two or more viral protein variants; and b) providing a recombinant polyspecific amino acid sequence, or a recombinant nucleotide sequence encoding the modified amino acid sequence, comprising at least one fragment of a parent viral protein, wherein an amino acid position in at least one fragment of the parent viral protein has been modified to comprise an amino acid found in the corresponding amino acid position of two or more viral protein variants; The method includes:

2. i) the method comprises repeating step b) to provide two or more of the modified amino acid sequences, or two or more of the nucleotide sequences encoding the two or more of the modified amino acid sequences, optionally wherein the two or more modified amino acid sequences are based on the same parent viral protein, and / or optionally the amino acid modifications in the two or more modified amino acid sequences are at least partially different, and / or ii) providing a nucleotide sequence, b') replacing codons of a nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a recombinant mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in at least one fragment of the parent viral protein is modified to contain an amino acid found in the corresponding amino acid position of two or more viral protein variants. and optionally repeating step b') to provide two or more of the recombinant mutated nucleotide sequences encoding two or more of the modified amino acid sequences, further optionally wherein the two or more modified amino acid sequences are based on the same parent viral protein and / or further optionally wherein the amino acid modifications in the two or more modified amino acid sequences are at least partially different. The method of claim 1.

3. a) identifying amino acid positions in a parent viral protein that are modified compared to the corresponding amino acid positions of two or more viral protein variants; b) replacing codons of a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a first recombinant mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in at least one fragment of the parent viral protein is modified to contain an amino acid found in the corresponding amino acid position of two or more viral protein variants; and c) substituting codons of the second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein with other codons to obtain a second recombinant mutated nucleotide sequence encoding a modified amino acid sequence, wherein an amino acid position in at least one fragment of the parent viral protein is modified to contain an amino acid found in the corresponding amino acid position of two or more viral protein variants. Including, The amino acid modification in b) is at least partially different from the amino acid modification in c).

4. i) at least one fragment of a parent viral protein contained in the amino acid sequence encoded by the first nucleotide sequence is identical to at least one fragment of a parent viral protein contained in the amino acid sequence encoded by the second nucleotide sequence; ii) the amino acid sequence encoded by the first nucleotide sequence and the amino acid sequence encoded by the second nucleotide sequence are identical; iii) the first nucleotide sequence and the second nucleotide sequence are identical; iv) one or more of the modified amino acid positions in the modified amino acid sequence encoded by the first recombinant mutated nucleotide sequence are different from the modified amino acid positions in the modified amino acid sequence encoded by the second recombinant mutated nucleotide sequence; and / or v) one or more amino acids in the modified amino acid positions in the modified amino acid sequence encoded by the first recombinant mutated nucleotide sequence and in the modified amino acid sequence encoded by the second recombinant mutated nucleotide sequence are different from each other; The method according to claim 3.

5. i) the amino acid sequence comprising at least one fragment of a parent viral protein comprises the amino acid sequence of a full-length viral protein; ii) the method comprises providing a recombinant nucleic acid, optionally an RNA, comprising a nucleotide sequence encoding the modified amino acid sequence; iii) the method further comprises providing a vaccine comprising a nucleic acid, optionally RNA, comprising a nucleotide sequence encoding the modified amino acid sequence, optionally wherein the vaccine is an RNA vaccine and / or the vaccine has a reduced risk of immune evasion, and / or iv) the method is for producing a vaccine, optionally wherein the vaccine is an RNA vaccine and / or the vaccine has a reduced risk of immune evasion; The method according to claim 1 or 3.

6. the modified amino acid positions are those at which the amino acid sequences of the two or more viral protein variants differ from the amino acid sequence of the parent viral protein, and / or the modified amino acid positions are those at which the amino acid sequences of the two or more viral protein variants differ from the amino acid sequence of the wild-type viral protein. The method according to claim 1 or 3.

7. 4. The method of claim 1 or 3, wherein the modified amino acid position is a potential site of viral escape mutation.

8. The method described in claim 7, wherein the escape mutant of the virus is an antibody escape mutant of the virus.

9. The method described in claim 7, wherein the escape mutant of the virus is resistant to neutralization by antibodies against the viral proteins and / or the viral proteins of the escape mutant of the virus show reduced antibody binding.

10. 9. The method of claim 8, wherein the antibodies are used for the treatment of a patient infected with the virus or the antibodies are generated in a patient treated with a vaccine against infection with the virus.

11. The method of claim 1 or 3, wherein the parent viral protein is modified compared to the wild-type viral protein and / or in the modified amino acid sequence, the amino acid positions in the parent viral protein that are modified compared to the wild-type viral protein are not modified.

12. The parental viral protein is a viral protein of a parental viral strain, and optionally i) the parental virus strain is a natural isolate or the parental virus strain is a mutant of a natural isolate; ii) the parent virus strain is a circulating or rapidly spreading virus variant strain, and / or iii) the parental virus strain is a virus variant that is a variant of concern; The method according to claim 1 or 3.

13. i) two or more viral protein variants are modified compared to a wild-type viral protein; ii) two or more viral protein variants are modified compared to a parent viral protein; and / or iii) in the modified amino acid sequence, the amino acid positions in the two or more viral protein variants that are modified compared to the wild-type viral protein and / or the parent viral protein are modified; The method according to claim 1 or 3.

14. The method of claim 13, wherein one or more of the two or more viral protein variants are viral proteins of one or more viral strains, and optionally: i) one or more of the one or more virus strains is a natural isolate or one or more of the one or more virus strains is a mutant of a natural isolate; ii) one or more of the one or more virus strains is a circulating or rapidly spreading virus variant strain; iii) one or more of the one or more virus strains is a virus variant strain that is a variant of concern; iv) the parent virus strain and one or more virus strains are circulating or rapidly spreading virus variant strains; and / or v) the parent virus strain and one or more virus strains are virus variant strains that are variants of concern; The method according to claim 1 or 3.

15. The method of claim 1 or 3, wherein the parent viral protein and the two or more viral protein variants are modified compared to a wild-type viral protein and / or the two or more viral protein variants comprise viral protein variants of at least two viral strains.

16. 4. The method of claim 3, wherein the two or more viral protein variants in b) are different from the two or more viral protein variants in c) and / or the modified amino acid positions in b) and c) each comprise at least two amino acid positions.

17. The method of claim 17, wherein the amino acid modification in the parent viral protein compared to the wild-type viral protein in the modified amino acid sequence does not interfere with the amino acid modification at the modified amino acid position. ii) in the modified amino acid sequence, the amino acid modification in the parent viral protein compared to the wild-type viral protein is not in close spatial distance to the amino acid modification in the modified amino acid position; iii) in the modified amino acid sequence, the modification at the modified amino acid position does not result in a large structural rearrangement; iv) the amino acid at the modified amino acid position is surface exposed, and / or v) the modified amino acid positions include at least two amino acid positions; The method according to claim 1 or 3.

18. a) providing a recombinant nucleic acid, optionally RNA, comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parental viral protein, wherein an amino acid position in the at least one fragment of the parental viral protein has been modified to comprise an amino acid found at a corresponding amino acid position in two or more viral protein variants; and b) providing a recombinant nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parental viral protein, wherein an amino acid position in the at least one fragment of the parental viral protein is modified to comprise an amino acid found at a corresponding amino acid position in two or more viral protein variants; Including, The amino acid modification in b) is at least partially different from the amino acid modification in a).

19. A pharmaceutical formulation, optionally selected from a pharmaceutical composition, a vaccine or a kit, comprising: a) a recombinant nucleic acid, optionally an RNA, comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parental viral protein, wherein an amino acid position in at least one fragment of the parental viral protein has been modified to comprise an amino acid found in the corresponding amino acid position of two or more viral protein variants; b) a recombinant nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein an amino acid position in the at least one fragment of the parent viral protein is modified to comprise an amino acid found in a corresponding amino acid position of two or more viral protein variants; and optionally Instructions for use of the pharmaceutical preparation for vaccination against infection with said virus Including, The pharmaceutical preparation, wherein the amino acid modification in b) is at least partially different from the amino acid modification in a).

20. 20. The pharmaceutical preparation of claim 19, wherein the RNA is formulated in a lipid nanoparticle (LNP).

21. 21. A pharmaceutical formulation according to claim 19 or 20 for medical use, optionally including vaccination against infection with a virus.

22. 21. A method for inducing an immune response against a virus in a subject, comprising administering to the subject a pharmaceutical formulation according to claim 19 or 20, said method optionally being a method for prophylactic treatment against infection with said virus and / or a method for vaccination against infection with said virus.

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