RSV vaccine

Abstract

A vaccine against RSV is provided herein. The vaccine comprises mRNA encoding a stabilized pre-fusion RSV F protein immunogen linked to a lumazine synthase-based scaffold.

Description

Technical Field

[0001] mRNA molecules for RSV vaccines and RSV mRNA vaccines containing the mRNA molecules are provided herein.

Background Art

[0002] Respiratory syncytial virus (RSV) is a non-segmented negative-strand RNA virus with an envelope of the genus Pneumovirus in the family Paramyxoviridae. This seasonal virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age. RSV also causes recurrent infections, including severe lower respiratory tract diseases that can occur at any age, particularly in the elderly or in those with impaired cardiac, pulmonary, or immune systems. RSV has two antigenic subtypes: RSV A and RSV B, both of which circulate during epidemics, and their relative morbidity varies by season. Currently, there is no targeted treatment for RSV infection, and a vaccine that provides protection against RSV infection and disease is needed.

[0003] The RSV F protein is a trimer surface glycoprotein fixed to the viral envelope, mediating viral fusion and facilitating viral entry into target cells. The protein is initially expressed as a single polypeptide precursor called F0. F0 trimers within the endoplasmic reticulum and is processed intracellularly by frin proteases at two conserved sites, generating F1, F2, and Pep27 polypeptides. The Pep27 polypeptide is cleaved and does not form part of the mature F protein. The F2 polypeptide originates from the N-terminal portion of the F0 precursor and ligates to the F1 polypeptide via two disulfide bonds. The F1 polypeptide fixes the mature F protein to the viral envelope via a transmembrane domain attached to its approximately 24-amino acid cytoplasmic tail. The three protomers of the F1-F2 heterodimer assemble to form the mature F protein, which adopts a metastable "pre-fusion" conformation that undergoes spontaneous conformational changes to a "post-fusion" conformation that fuses the viral membrane and the target cell membrane. The "pre-fusion" conformation is known as "Pre-F," and the "post-fusion" confirmation is known as "Post-F."

[0004] Several neutralizing epitopes are unavailable in the post-fusion F protein, and it has been found that the level of pre-F specific antibodies determines the magnitude of RSV neutralizing activity in human serum (Ngwuta et al., Science Translational Medicine 7(309):309ra162, 2015). Therefore, in the context of vaccines, stabilization of the RSV F protein in the pre-F conformation is advantageous, and RSV F proteins stabilized in the pre-fusion conformation have been identified that result in an enhanced neutralizing immune response in animal models compared to the native protein.

[0005] An early example of a stabilized pre-F RSV F protein is the DS-Cav1 protein (McLellan et al., Science 342:592-598, 2013). DS-Cav1 contains mutations 155C, 290C, 190F, and 207L, with introduced cysteine ​​residues forming disulfide bonds and other mutations filling cavities in the pre-F structure. The trimer structure was stabilized with a Foldon trimerizing motif. To date, two RSV vaccines have been approved: GSK's Arexvy and Pfizer's Abrysvo, both approved for adults aged 60 and older. At least one of these is a protein subunit vaccine based on the DS-Cav1 protein.

[0006] Research is ongoing to develop improved RSV antigens, including those based on DS-Cav1. One approach being attempted utilizes nanoparticles formed from 24 subunits of a fusion protein containing Helicobacter pylori ferritin ligated to the C-terminus of a modified single-strand DS-Cav1, in which the furin cleavage site and p27 region are deleted, the cysteine ​​mutation is reversed, and a glycan moiety is added to mask non-neutralizing and non-neutralizing epitopes (Swanson et al., Science Immunology 5:eaba6466, 2020). Another approach is based on iterative cycles of structure-based design, which have yielded multiple candidate antigens, including various additional mutations and single-strand DS-Cav1 derivatives (Joyce et al., Nature Structural and Molecular Biology 23(9):811-820, 2016; International Publication No. 2017 / 172890).

[0007] In recent years, messenger RNA (mRNA) vaccine technology has attracted attention, particularly with the use of the Moderna and Pfizer / BioNTech Covid-19 vaccines in vaccination campaigns worldwide. Instead of directly injecting antigens into patients, mRNA vaccines administer mRNA molecules that encode antigens to the patient. The antigen is expressed from the mRNA in a small number of cells in the patient's body, and as a result, the antigen is presented to the immune system, which initiates an immune response. [Overview of the project]

[0008] The inventors have developed an mRNA RSV vaccine utilizing a second-generation pre-FF protein antigen based on DS-Cav1. The antigen is provided in the form of a fusion protein further comprising a lumazine synthase scaffold. The fusion protein constitutes nanoparticle subunits that multimerize to form nanoparticles (and the terms “fusion protein” and “nanoparticle subunits” are used interchangeably herein). In animal models, as shown in the following example, this vaccine yields high neutralizing antibody titers against both RSV A and RSV B.

[0009] Accordingly, in a first embodiment, mRNA molecules encoding nanoparticle subunits are provided herein, the nanoparticle subunit comprising an immunogen and a scaffold linked by a linker, the linker being 9-13 amino acid length, the scaffold comprising lumazine synthase, the immunogen comprising a deletion at positions 104-144 of the RSV F protein, a GS peptide linker between positions 103 and 145 of the RSV F protein, and a modified RSV F protein comprising the following mutations: S155C, N183GC, S190F, V207L, S290C, L373R and N428C.

[0010] When expressed by cells, the nanoparticle subunits multimerize via a lumazine synthase scaffold to form protein nanoparticles, whose immunogen forms an exposed outer layer. In certain cases, the protein nanoparticles contain 60 self-assembling nanoparticle subunits (i.e., they are 60-mers).

[0011] In certain cases, the modified RSV F protein is the modified RSV F protein of SEQ ID NO: 1 or SEQ ID NO: 2 described below, or a variant of SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] In a second embodiment, an RSV vaccine (specifically an RSV mRNA vaccine) comprising the mRNA molecule of the first embodiment is provided herein.

[0013] In a third aspect, an mRNA molecule of the first aspect or a vaccine of the second aspect is provided herein for use as a therapeutic or prophylactic agent.

[0014] In a fourth aspect, a first-aspect mRNA molecule or a second-aspect vaccine for use in inducing an immune response in a subject is provided herein. In connection therewith, this aspect provides a method for inducing an immune response in a subject, comprising administering the subject the first-aspect mRNA molecule or the second-aspect vaccine. Also in connection therewith, this aspect provides the use of the first-aspect mRNA molecule or the second-aspect vaccine in the manufacture of a pharmaceutical product for use in a method for inducing an immune response in a subject.

[0015] The vaccines provided herein have been found useful in preventing RSV infection and RSV infection-related illnesses, as well as in preventing serious illnesses associated with RSV infection. Accordingly, in a fifth aspect, an RSV vaccine of the second aspect or an mRNA molecule of the first aspect is provided herein for use in a manner that prevents or attenuates illness caused by RSV infection in a subject. As will be discussed further below, illness caused by RSV infection may be substituted herein for “RSV infection.”

[0016] In connection therewith, this embodiment provides a method for preventing or attenuating RSV infection-related disease in a subject, comprising administering to the subject an RSV vaccine of the second embodiment or an mRNA molecule of the first embodiment.

[0017] This embodiment also provides the use of a second embodiment of an RSV vaccine or a first embodiment of an mRNA molecule in the manufacture of a pharmaceutical product for use in preventing or reducing disease caused by RSV infection.

[0018] In this fifth aspect, the disease caused by RSV infection may be a lower respiratory tract disease (LRTD), such as bronchiolitis, bronchitis, or pneumonia.

[0019] In a sixth aspect, an expression vector comprising an expression cassette encoding the mRNA molecule of the first aspect is provided herein.

[0020] In the seventh aspect, cells containing the expression vector of the sixth aspect are provided herein. Such cells may be referred to as “host cells”.

[0021] In an eighth aspect, a method for producing an mRNA molecule of the first aspect is provided herein, comprising expressing mRNA from an expression vector of the sixth aspect. In some examples, the mRNA is expressed by in vitro transcription.

[0022] In a ninth aspect, protein nanoparticles comprising a polymer of nanoparticle subunits as defined in the first aspect are provided herein. That is, this aspect provides protein nanoparticles comprising a plurality of copies of a nanoparticle subunit comprising an immunogen and a scaffold linked by a linker, wherein the linker is 9 to 13 amino acid long, the scaffold comprises a lumazine synthase, and the immunogen comprises a modified RSV F protein including a deletion at positions 104 to 144 of the RSV F protein, a GS peptide linker between positions 103 and 145 of the RSV F protein, and the following mutations: S155C, N183GC, S190F, V207L, S290C, L373R and N428C. The polymer of nanoparticle subunits is generally a 60-mer (i.e., a polymer containing 60 copies of the subunit). [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows the presentation of DS-CAV1 trimers and antigenic sites of DS-CAV-1 on alternative nanoparticle (NP) scaffolds. Multiple capture antibodies specific for epitopes specific for only the pre-fusion conformation (1G7, AM14 or MPE8), specific for only the post-fusion conformation (MP43), or binding to both (Pali- vizumab (Pali) and MP43) were assayed. [Figure 2] Figure 2 shows the RSV-A neutralizing antibody titers from mice immunized with mRNA encoding DS-CAV1 trimers, DS2-2 trimers or recombinant DS-CAV1 protein trimers. [Figure 3-1] Figure 3A shows the vaccination schedule of mice that received a prime / boost regimen of either recombinant DS-CAV1 trimers (Ds-Cav1 foldon), or mRNA encoding DS-CAV1 trimers (Ds-Cav1 foldon), or Ds-Cav1 or DS2-2 nanoparticle subunits fused to either ferritin, β-annulus (Bann) or lumazine synthase (LuS). Figure 3B shows the RSV-A neutralizing titers on day 14 from the vaccination regimens described in Figure 3A. [Figure 3-2] Figure 3C shows the RSV-A neutralizing titers on day 42 from the vaccination regimens described in Figure 3A. Figure 3D shows the RSV-B neutralizing titers from the vaccination regimens described in Figure 3A. [Figure 3-3] Figure 3E shows the pre-fusion / post-fusion antibody ELISA responses for all vaccination regimens on day 14 (left panel) and day 42 (right panel). [Figure 4-1]Figure 4A shows the vaccination regimen of RSV-exposed mice. The mice were nasally exposed to RSV on day 0 before receiving vaccination ("boost") on day 80. Figure 4B shows the RSV-A neutralizing titers from the vaccines tested in the regimen described in Figure 4A. The mouse groups were administered either PBS (negative control), RSV-A live virus, DS-CAV1 trimer (DS-CAV1 soluble), or mRNA encoding DS-CAV1 transmembrane, DS2-2 trimer (DS2 foldon), DS2-2 LuS (DS2 lumazine synthase), and DS2-2 ferritin (DS2 ferritin). [Figure 4-2] Figure 4C shows the RSV-B neutralizing titers against the same vaccine candidates described in Figure 4B. [Figure 5] Figure 5 is a bar graph comparing the expression of eGFP-encoding mRNA (in A549 cells) with a constant 3'UTR region (albumin) and candidate 5'UTRs (UTR-11, -37, -52, -53 (identified in the examples)). The black bars represent vectors with the control 5'UTR region (HSD17B4). The selected 5'UTRs increase the fluorescence intensity of eGFP in A549 cells. The candidate 5'UTRs show the ability to increase eGFP expression in A549 cells compared to the control 5'UTR (HSD17B4). The data are representative of three independent experiments, with 3-4 technical replicates in each experiment. [Figure 6] Figure 6 is a bar graph comparing the expression of eGFP-encoding mRNA (in HeLa cells) with a constant 3'UTR region (albumin) and candidate 5'UTRs (UTR-11, -37, -52, -53). The black bars represent vectors with the control 3'UTR (albumin) and control 5'UTR (HSD17B4). The candidate 5'UTRs show the ability to increase eGFP expression in HeLa cells compared to the control 5'UTR (HSD17B4). The data are representative of three independent experiments, with 3-4 technical replicates in each experiment. [Figure 7]Figure 7 is a bar graph comparing eGFP mRNA expression (in A549 cells) with candidate 3'UTR regions (UTR-3, -4, -36 (identified in the example)) and the control 5'UTR region (HSD17B4). The black bars represent the control 3'UTR (albumin) and 5'UTR (HSD17B4). The candidate 3'UTRs can increase eGFP mRNA expression in A549 cells compared to the control 3'UTRs. The data are representative of two independent experiments, with three technical replicates in each experiment. [Figure 8] Figure 8 is a bar graph comparing eGFP mRNA expression (in HeLa cells) with candidate 3'UTRs (UTR-3, -4, -36) and a control 5'UTR (HSD17B4). The candidate 3'UTRs were able to increase eGFP mRNA expression in HeLa cells compared to the control 3'UTR. The black bars represent the control 3'UTR (albumin) and control 5'UTR (HSD17B4). The data are representative of two independent experiments, each with three technical replicates. [Figure 9] Figure 9 is a bar graph comparing eGFP mRNA expression (in A549 cells) with candidate 3'UTR (CHIT-1, CS) and 5'UTR (GOT1, PRKACB, CHIT1) combinations to a control consisting of a control 3'UTR (albumin) and a control 5'UTR (HSD17B4). The black bars represent eGFP mRNA expression in the control. Combining the candidate 3'UTR with the candidate 5'UTR can increase eGFP mRNA expression in A549 cells. The data are representative of three independent experiments, each with three technical replicates. [Figure 10]Figure 10 is a bar graph showing the expression of eGFP-coding modified mRNA (in A549 cells) with candidate 5'UTR (GOT1, PRKACB, CHIT) and 3'UTR (CHIT, CS) combinations. The black bars represent the expression of eGFP-coding modified mRNA with the control 5' (HSD17B4) and 3'UTR (albumin) regions. The candidate 5'UTR and candidate 3'UTR combinations increase the expression of eGFP-coding modified mRNA compared to expression with the control 5' and 3'UTRs. The data are representative of three independent experiments, with three technical replicates in each experiment. [Figure 11] Figure 11 shows antibody expression in A549 cells containing scFv-Fc coding mRNA with candidate or control 5' and 3' UTR combinations. Black bars represent scFv-Fc coding mRNA expression with control 5' UTR (HSD17B4) and control 3' UTR (albumin). The PRKACB(5'UTR) / CHIT(3'UTR) or CHIT(5'UTR) / CS(3'UTR) combinations result in higher scFv-Fc coding mRNA expression than with control 5' and 3' UTR regions. Data are representative of three independent experiments, with three technical replicates in each experiment. [Figure 12] Figure 12 shows antibody expression in A549 cells containing scFv-Fc coding modified mRNA (in A549 cells) with candidate or control 5' and 3'UTR combinations. The modified mRNA contains modified uridine (5-methoxyuridine). Black bars represent the expression of scFv-Fc coding mRNA with the control 5'UTR (HSD17B4) and control 3'UTR (albumin). The combinations PRKACB(5'UTR) / CHIT(3'UTR), PRKACB(5'UTR) / CS(3'UTR), CHIT(5'UTR) / CHIT(3'UTR), and CHIT(5'UTR) / CS(3'UTR) result in higher scFv-Fc coding mRNA expression than with the control 5' and 3'UTR regions. The data are representative of three independent experiments, with three technical replicates in each experiment. [Figure 13]Figure 13A shows that mRNAs using 5'UTR CHIT1 and 3'UTR CS UTR produced the highest levels of eGFP expression in BHK-21 cells compared to two competing mRNA molecules. mRNA_AZ, mRNA comp A, and mRNA comp B all contain 100% pseudo-U. mRNA_AZ with 0% pseudo-U was included for comparison. Figure 13B shows that mRNAs using 5'UTR CHIT1 and 3'UTR CS UTR produced the highest levels of eGFP expression in HEK293 cells compared to two competing mRNA molecules. mRNA_AZ, mRNA comp A, and mRNA comp B all contain 100% pseudo-U. mRNA_AZ with 0% pseudo-U was included for comparison. [Figure 14] Figure 14 shows that the RSV mRNA-VLP vaccine induced high-titer neutralizing antibodies in non-human primates that had experienced RSV. A group of non-human primates (NHPs) that had experienced RSV were immunized with either a benchmark RSV F protein vaccine (DS-CAV1 + adjuvant) or two different RSV mRNA vaccines encoding pre-fusion-stabilized F protein, and immunogenicity was evaluated by ELISA binding titer and viral neutralization assay. [Figure 15] Figure 15 shows that the RSV mRNA-VLP vaccine protected infected cotton rats from viral replication in lung and nasal tissue. Naive cotton rats were immunized with either a benchmark RSV F protein vaccine (DS-CAV1 + adjuvant) or two different RSV mRNA vaccines encoding pre-fusion-stabilized F protein to evaluate vaccine efficacy in a viral challenge model. [Modes for carrying out the invention]

[0024] Over the past century or so, vaccines based on attenuated live pathogens, inactivated pathogens, and toxoids have transformed public health, eliminating or nearly eliminating dreadful diseases such as smallpox, polio, and tetanus. However, it has not been proven that it is possible to develop effective vaccines against all targeted infections using these traditional vaccine technologies. One such target is respiratory syncytial virus (RSV). RSV is a very common seasonal virus that causes respiratory tract infections. In healthy adults and older children, RSV infection generally causes mild illnesses such as the common cold, but in infants, the elderly, and immunocompromised individuals, it can cause more severe lower respiratory tract illnesses, particularly bronchiolitis in infants and pneumonia in adults.

[0025] Formalin-inactivated RSV virus was developed in the 1960s, but it was found to cause antibody-enhancing disease (ADE) in children and bonnet monkeys (Ponnuraj et al., Journal of Infectious Diseases 187(8):1257-1263, 2003). Since then, new vaccine technologies have been developed, one of which is nanoparticle-based vaccines. One type of nanoparticle of interest is a protein nanoparticle containing a protein nanocage formed by the self-assembly of protein subunits into a symmetrical multimeric structure. The protein nanocage contains a high density of repeating regions on its surface that can be recognized and crosslinked by B cell receptors, driving a larger immune response than that obtained by basic protein subunits (Curley & Putnam, Frontiers in Bioengineering and Biotechnology 10:867119, 2022). Another new vaccine technology that has recently become prominent is mRNA vaccines.

[0026] An mRNA molecule for use as an mRNA vaccine against RSV is provided herein. The mRNA encodes a fusion protein containing an RSV immunogen ligated to a scaffold based on lumazine synthase. The lumazine synthase polymerizes to form protein nanoparticles, and thus the encoded fusion protein constitutes protein nanoparticle subunits (referred to herein simply as “nanoparticle subunits”).

[0027] mRNA molecule The term “mRNA molecule” is used herein to refer to an RNA (ribonucleic acid) molecule that codes for a polypeptide and can be translated to produce the coded polypeptide. The polypeptide may be of any length and may be naturally occurring or synthetic; however, in the context of the mRNA molecule provided herein, the polypeptide is a synthetic fusion protein as described below. The mRNA molecule can be translated in any context, for example, in vitro, in vivo, or ex vivo, to produce the coded polypeptide.

[0028] mRNA molecules may encode multiple polypeptides or only a single polypeptide. When an mRNA molecule encodes multiple polypeptides, this can be in the context of a single open reading frame (ORF), for example, utilizing a 2A skip sequence to divide them, or in the context of multiple open reading frames. If an mRNA molecule contains multiple open reading frames, translation may begin at an internal ribosome entry site (IRES). However, generally, the mRNA molecules provided herein contain a single open reading frame encoding a single polypeptide (the fusion protein described above).

[0029] The basic components of an mRNA molecule typically include at least one coding region (as shown above), a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a polyA tail. The mRNA molecules provided herein may have the structure of naturally occurring mRNA or may be distinguishable from wild-type mRNA in features of their functional and / or structural design.

[0030] In some cases, the mRNA molecule is a self-amplifying mRNA (saRNA) molecule. Unlike traditional mRNA molecules, saRNA molecules replicate themselves after entering a cell. In addition to the basic mRNA components mentioned above, saRNA molecules contain a second open reading frame (ORF) that is expressed in the cell and encodes RNA-dependent RNA polymerase (along with its helper protein) that replicates the mRNA molecule. This allows for the use of smaller doses of mRNA compared to vaccines using non-amplifying mRNA molecules, but substantially increases the size of the delivered mRNA molecule.

[0031] In some cases, mRNA molecules are circular RNA (circRNA) molecules. CircRNA synthesis can be achieved by producing traditional linear mRNA molecules and then ligating the 5' and 3' ends to create a circular structure (as described, e.g., Obi & Chen, Methods 196:85-103, 2021). Because circRNA can be more stable than linear mRNA due to the lack of ends and inability to access exonucleases, the use of circRNA is another means of reducing the required mRNA dosage.

[0032] mRNA structure The mRNA molecules provided herein may contain one or more modified nucleotides or nucleosides. Such modified nucleotides may be modified adenosine (A), guanosine (G), uridine (U), or cytidine (C). In this specification, “modification” means that the structure of the nucleotide is altered compared to its native structure. For any given nucleotide, all examples of the nucleotide in the mRNA may be modified, not all examples may be modified (i.e., having the native structure of the nucleotide), or a certain percentage of the nucleotides in the mRNA may be modified, for example, about 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the examples of nucleotides in the mRNA may be modified. If mRNA contains modified nucleotides, the percentage of nucleotides in the mRNA that can be modified is, for example, 5-20%, 5-25%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90%, 5-95%, 10-20%, 10-25%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-95%, 10-100%, 20-25%, 20 It may be ~50%, 20~60%, 20~70%, 20~80%, 20~90%, 20~95%, 20~100%, 50~60%, 50~70%, 50~80%, 50~90%, 50~95%, 50~100%, 70~80%, 70~90%, 70~95%, 70~100%, 80~90%, 80~95%, 80~100%, 90~95%, 90~100%, or 95~100%. Modified nucleotides may include modified nucleic acid bases (modified adenine, guanine, cytosine, or uracil), modified ribose sugars, or modified phosphate groups.

[0033] If some specific nucleotides (e.g., adenosine) are modified, but not all nucleotides in the mRNA are modified, the modified nucleotides may be uniformly or randomly distributed throughout the mRNA, or they may be localized in particular to selected sequence regions. For example, modified nucleotides may be available in the coding region but not in the UTR.

[0034] mRNA molecules may contain modified versions of 1, 2, 3, or 4 of the natural nucleosides, such as modified adenosine and modified cytidine; modified adenosine and modified uridine; modified adenosine and modified guanosine; modified cytidine and modified uridine; modified cytidine and modified guanosine; modified uridine and modified guanosine; modified adenosine, cytidine, and guanosine; modified adenosine, cytidine, and uridine; modified adenosine, guanosine, and uridine; or modified cytidine, guanosine, and uridine.

[0035] If an mRNA molecule contains one or more modified nucleotides, the total percentage of nucleotides in the molecule that are modified can be any percentage up to 100%. For example, mRNA may contain approximately 10, 20, 30, 40, 50, 60, 70, 80, or 90% modified nucleotides. For example, mRNA is divided into 1-10%, 1-20%, 1-25%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90%, 1-95%, 1-100%, 5-10%, 5-20%, 5-25%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90%, 5-95%, 10-20%, 10-25%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-95%, and 10-1 This may include 00%, 20-25%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%, or 95-100%.

[0036] In some cases, mRNA does not contain modified nucleotides, i.e., it contains only the natural adenosine, cytidine, guanosine, and uridine structures.

[0037] The mRNA molecules provided herein may be modified mRNA molecules that include modified nucleoside bonds, and may include, for example, modifications to binding phosphates, phosphodiester bonds, or phosphodiester backbones.

[0038] Modified mRNA molecules (i.e., mRNA molecules containing one or more modified nucleotides and / or modified nucleoside bonds) can be synthesized using modified nucleotides or modified after synthesis.

[0039] As used herein, “nucleoside” refers to a compound containing a sugar molecule or its derivative (in the context of RNA, ribose sugar or its derivative) in combination with an organic base (e.g., purine or pyrimidine) or its derivative (also referred herein as “nucleic acid base”). The nucleic acid bases found in mRNA molecules provided herein are generally standard RNA nucleic acid bases, namely adenine, cytosine, guanine, and uracil. “Nucleotide” refers to a nucleoside containing a phosphate group. Modified mRNA molecules may be synthesized by any preferred method, e.g., chemically, enzymatically, or recombinantly, and may contain one or more modifications or non-natural nucleotide or nucleoside bonds.

[0040] When a modified nucleotide contains a modified ribose moiety, the 2'-hydroxyl group (OH) may be modified or replaced with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2'-hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar moiety); polyethylene glycol (PEG); "locked" nucleic acids (LNA) in which the 2'-hydroxyl is bonded to the 4' carbon of the same ribose sugar, for example, by a methylene crosslink; and amino groups (-O-amino, where the amino group may be, for example, alkylamino, dialkylamino, heterocyclylamino, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, polyamino, or aminoalkoxy groups).

[0041] Examples of "deoxy" modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), or the amino group may be attached to the sugar via a linker, the linker comprising one or more atoms C, N, and O.

[0042] The ribose moiety may, alternatively or further, include any additional suitable or desired modifications at any other position of the 2'OH group or sugar.

[0043] If a modified nucleotide contains a modified nucleic acid base, the nucleic acid base may be modified at any position by any suitable or desired modification, for example, by the addition of an amino group, a thiol group, an alkyl group, or a halo group.

[0044] Modified nucleosides that may be used in mRNA molecules provided herein include: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine; 2-methyl Denosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1- Methyladenosine; N6-cis-hydroxyisopentenyladenosine; α-thio-adenosine; 2-aminoadenosine; 2-(aminopropyl)adenosine; 2-propyladenosine; 2'-amino-2'-deoxyadenosine; 2'-azido-2'-deoxyadenosine; 8-aminoadenosine; 8-hydroxyadenosine; 8-thioadenosine; 8-azidoadenosine; 8-azaadenosine; 7-deaza-8-azaadenosine; 7-methyladenosine; 1-deazaadenosine; 2'-fluoro-N6-benzoyl-deoxyadenosine; 2 '-O-methyl-2-amino-adenosine; 2'-O-methyl-N6-benzoyl-deoxyadenosine; 2-ethynyladenosine; 2'-O-trifluoromethyladenosine; 2-azidoadenosine; 2'-ethynyladenosine; 2-bromoadenosine; 2-trifluoromethyladenosine; 2-chloroadenosine; 2'-deoxy-2,2'-difluoroadenosine; 2'-deoxy-2'-mercaptoadenosine; 2'-deoxy-2'-aminoadenosine; 2'-deoxy-2'-azidoadenosine; 2'-deoxy-2'-bromoadenosine;2'-Deoxy-2'-Chloroadenosine; 2'-Deoxy-2'-Fluoroadenosine; 2'-Deoxy-2'-Iodoadenosine; 2-Fluoroadenosine; 2-Iodoadenosine; 2-Mercaptoadenosine; 2-Methoxyadenosine; 2-Methylthioadenosine; 3-Deaza-3-Bromoadenosine; 3-Deaza-3-Chloroadenosine; 3-Deaza-3-Fluoroadenosine; 3-Deaza-3-Iodoadenosine; 3-Deazaadenosine; 4'-Azidoadenosine; 8-Bromoadenosine; 8-Trifluoromethyladenosine; 9-Deaza Denosine; 2-thiocytidine; 3-methylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 5-formyl-2'-O-methylcytidine; lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-O-methylcytidine; 4-methylcytidine; 5-azacytidine; pseudoisocytidine; α-thiocytidine; 2'-amino-2'-deoxycytidine; 2'-azide-2'-deoxycytidine N; 3-deaza-5-azacytidine; 5-propynylcytidine; 5-trifluoromethylcytidine; 5-bromocytidine; 5-iodocytidine; 6-azacytidine; pseudoisocytidine; 2'-O-methyl-5-methylcytidine; 2-thio-5-methylcytidine; 5-methylzebralin; zebralin; (E)-5-(2-bromovinyl)cytidine; N4-benzoyl-2'-fluoro-2'-deoxycytidine; N4-acetyl-2'-fluoro-2'-deoxycytidine; 2'-O-methyl-N4-acetylcytidine; N4-benzoyl-2' -O-methylcytidine; 2'-ethynylcytidine; 5-trifluoromethyl-2'-deoxycytidine; 2'-deoxy-2',2'-difluorocytidine; 2'-deoxy-2'-mercaptocytidine; 5-bromo-2'-deoxycytidine; 2'-chloro-2'-deoxycytidine; 2'-deoxy-2'-fluorocytidine; 5-iodo-2'-deoxycytidine; 5-(l-propynyl)-2'-O-methylcytidine; 3'-C-ethynylcytidine; 4'-azidocytidine; 5-aminoallylcytidine; cyanocytidine; 5-ethynylcytidine;5-Methoxycytidine; N4-Aminocytidine; N4-Benzoylcytidine; 7-Methylguanosine; N2,2'-O-Dimethylguanosine; N2-Methylguanosine; Uiosin; I,2'-O-Dimethylguanosine; 1-Methylguanosine; 2'-O-Methylguanosine; 7-Aminomethyl-7-Deazaguanosine; 7-Cyano-7-Deazaguanosine; Archiosin; Methyluosin; N2,7-Dimethylguanosine; N2,N2,2'-O-Trimethylguanosine; N2,N2,7-Trimethylguanosine; N2,N2-Dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thioguanosine; 7-deazaguanosine; 8-oxoguanosine; α-thioguanosine; 2'-amino-2'-deoxyguanosine; 2'-azido-2'-deoxyguanosine; 6-O-methylguanosine; 8-aminoguanosine; 8-hydroxyguanosine; 8-thioguanosine; 8-azaguanosine; 7-methyl-6-thioguanosine; 6-thio-7-methylguanosine; 7-deaza-8-azaguanosine; 7-methyl-8-oxoguanosine; N2-isobutyryl-2'-O-methylguanosine; 2'-Deoxy-2',2'-Difluoroguanosine; 2'-Deoxy-2'-Chloroguanosine; 2'-Deoxy-2'-Fluoroguanosine; 8-Bromoguanosine; 9-Deazaguanosine; 1-Methylinosine; Inosine; l,2'-O-Dimethylinosine; 2'-O-Methylinosine; 7-Methylinosine; Epoxycuosin; Galactosylcuosin; Mannosylcuosin; Cuosin; 2'-O-Methyluridine; 2-Thiouridine; 3-Methyluridine; 5-Carboxymethyluridine; 5-Hydroxyuridine; 5-Meth Luuridine; 5-Taurinomethyl-2-thiouridine; 5-Taurinomethyluridine; Dihydrouridine; Pseudouridine; 3-(3-amino-3-carboxypropyl)uridine; l-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine; 1-Methylpseudouridine; 2'-O-methylpseudouridine; 2-Thio-2'-O-methyluridine; N3-Methyl-2'-O-methyluridine; 3-Methylpseudouridine; 4-Thiouridine; 5-Carboxyhydroxymethyluridine; 5-Methyl-2'-O-methyluridine;5,6-Dihydrouridine; 5-Aminomethyl-2-thiouridine; 5-Carbamoylmethyl-2'-O-methyluridine; 5-Carbamoylmethyluridine; 5-Carboxymethylaminomethyl-2'-O-methyluridine; 5-Carboxymethylaminomethyl-2-thiouridine; 5-Carboxymethylaminomethyluridine; 5-Methoxycarbonylmethyl-2'-O-methyluridine; 5-Methoxycarbonylmethyl-2-thiouridine; 5-Methoxycarbonylmethyluridine; 5-Methoxyuridine; 5-Methyl-2-thiouridine; 5-Methylaminomethyl-2-selenouridine; 5-Methylaminomethyl-2-thiouridine; 5-Methylaminomethyluridine; 5-Methyldihydrouridine; Uridine- This includes 5-oxyacetic acid; methyluridine-5-oxyacetic acid; 5-(isopentenylaminomethyl)uridine; 5-propynyluridine; α-thiouridine; 2'-deoxyuridine; 2'-deoxy-2'-fluorouridine; 2'-amino-2'-deoxyuridine; 2'-azido-2'-deoxyuridine; 4-thiopsuduridine; 5-(aminopropyl)uridine; 5-methyl-4-thiouridine; 5-(trifluoromethyl)uridine; 5-(3-aminopropyl)uridine; 5-aminoallyluridine; 5-bromouridine; 5-iodouridine; 5-chlorouridine; 5-fluorouridine; 6-azauridine; 3-deazauridine; 2-thio-6-azauridine and 2-thiopsuduridine.

[0045] If an mRNA molecule contains modified versions of a particular nucleotide, it may contain one or more different modified versions of that nucleotide, for example, two, three, or four.

[0046] In certain cases, mRNA molecules contain modified uridine nucleotides, particularly pseudouridine nucleotides or modified pseudouridine nucleotides. Pseudouridine is an isomer of the natural uridine nucleoside in which uracil is bound to ribose via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Examples of modified pseudouridine nucleosides include those listed above. mRNA molecules may contain one or more species of pseudouridine and / or modified pseudouridine. mRNA molecules may contain exclusively pseudouridine nucleosides or modified pseudouridine nucleosides (i.e., all uridine residues may be replaced by pseudouridine residues or modified pseudouridine residues), or only some of the uridine residues may be replaced by pseudouridine residues or modified pseudouridine residues.

[0047] In certain cases, the mRNA molecule contains modified pseudouridine N1-methylpseudridine (also called 1-methylpseudridine). The structure of N1-methylpseudridine is shown in formula I below. [ka]

[0048] In certain cases, the mRNA molecule contains exclusively N1-methylpseudridine; that is, in these cases, all uridine residues are replaced with N1-pseudridine. In other cases, the mRNA molecule contains at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% N1-methylpseudridine (i.e., at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the uridine residues in the mRNA molecule may be replaced with N1-methylpseudridine).

[0049] When nucleoside bonds are modified, the phosphate group of the skeleton may be modified by substituting one or more oxygen atoms with different substituents. Furthermore, nucleotides may be modified by completely replacing the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups that may be used in mRNA molecules provided herein include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotryesters. In phosphorothioates, both unbound oxygen atoms are replaced with sulfur. Phosphate linkers may also be modified by replacing the bound oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), or carbon (bridged methylene phosphonate).

[0050] Naturally occurring mRNA contains a 5' cap structure. The mRNA molecules provided herein may similarly contain a 5' cap ligated to the 5' terminal nucleotide of the mRNA molecule. The cap may have any preferred structure. Typically, the 5' cap can be formed by a modified nucleotide, particularly a derivative of a guanine nucleotide. Generally, the 5' cap is ligated to the 5' end via a 5'-5'-triphosphate bond.

[0051] The cap may have a Cap0 structure, i.e., N7-methylguanosine attached to the 5' nucleotide of the mRNA molecule via a 5'-to-5' triphosphate bond. The Cap0 structure may be called m7G or m7GpppN cap (where N represents any nucleotide). The Cap0 structure is shown below in formula II (where the guanosine residue is shown as an example of the first residue of mRNA, although this can actually be any nucleotide). [ka]

[0052] Alternatively, the mRNA cap may have a Cap1 structure. The Cap1 structure differs from the Cap0 structure by adding a methyl group to the 2'O position of the first residue of the mRNA. The Cap1 structure may be called the m7GpppNm cap (where Nm refers to any 2'-O-methylated nucleotide). This structure may alternatively be presented as m7Gppp(2'Om)N. The Cap1 structure is shown in Equation III below (again, guanosine is shown as the exemplary first residue). [ka]

[0053] In certain examples, the mRNA molecules provided herein include a 5' cap having a Cap1 structure. The Cap1 structure may, in particular, have the structure m7GpppAm (i.e., the first nucleotide in the mRNA molecule is adenosine).

[0054] In other examples, mRNA molecules provided herein include a 5' cap having a modified Cap1 structure. A modified Cap1 structure is defined herein as a methylguanosine cap itself, or a Cap1 structure (i.e., including an m7GpppNm structure) having additional structural modifications to the 5' nucleotide of the mRNA molecule or the triphosphate linker between them. For example, the 5' cap may have a Cap1 structure in which the N7-methylguanosine cap has an additional methyl group at the 3'O position. Such a modified Cap1 may be called an m7(3'Om)GpppNm cap. In certain examples, mRNA molecules include a modified cap having the structure m7(3'Om)GpppAm (i.e., the first nucleotide residue of the mRNA molecule is adenosine). An exemplary structure of such a cap (showing, as an example, the guanosine residue at position 2 of the mRNA molecule) is shown in formula IV below. [ka]

[0055] Another example of a modified Cap1 structure is a cap having the structure m7(3'Om)Gpppm6(2'Om)A. Such a cap is based on the m7(3'Om)GpppAm cap shown in the figure above, and further includes an additional methyl group at position 6 of the adenosine residue at the 5' end of the mRNA molecule. An exemplary structure of such a cap (showing, as an example, the guanosine residue at position 2 of the mRNA molecule) is shown in formula V below. [ka]

[0056] Such caps can be applied to mRNA molecules by any suitable process, for example, chemically or enzymatically. For example, the Cap1 structure can be applied to mRNA using CleanCap® Reagent AG (TriLink, California, USA). Modified Cap1 structures of formulas IV and V can be applied to mRNA using CleanCap® Reagent AG (3'OMe) and CleanCap® Reagent M6, respectively.

[0057] Other suitable 5' cap structures include, for example, the Cap2 structure or ARCA cap analogues. If the mRNA molecule is a circular RNA molecule, no cap is present.

[0058] UTR and other array elements As described above, mRNA molecules naturally contain a 5'UTR and a 3'UTR. mRNA molecules provided herein may similarly contain a 5'UTR and / or a 3'UTR, or generally both. The term “3'UTR” refers to the portion of an mRNA molecule located at the 3' end (i.e., “downstream”) of the coding region that is not translated into a protein. Typically, the 3'UTR is the portion of mRNA located between the protein-coding region (coding region or coding sequence (CDS)) and the poly(A) sequence. If the mRNA contains multiple CDSs, the 3'UTR is located at the 3' end of the final (i.e., 3') CDS. The 3'UTR may begin immediately 3' to the stop codon of the CDS (or final CDS), or there may be intervening nucleotides or nucleotide sequences. The 3'UTR can be any preferred length, for example, at least 20, 30, 40, 50, or 60 nucleotides long, for example, 30-100, 40-90, 50-80, or 60-70 nucleotides long.

[0059] The term "5'UTR" refers to the portion of an mRNA molecule located at the 5' end (i.e., "upstream") of the coding region that is not translated into protein. Typically, for mRNA transcribed from DNA, the 5'UTR begins at the transcription start site and ends one nucleotide before the start codon of the coding region (or, if multiple coding regions exist, the first (5') coding region). The 5'UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites. The 5' nucleotides of the mRNA molecule are located within the 5'UTR, which, as described above, are capped. The 5'UTR can be of any preferred length, e.g., at least 10, 15, 20, 25, or 30 nucleotides long, e.g., 10–50, 20–40, or 30–35 nucleotides long.

[0060] Some 5'UTRs contain a 5'-terminal oligopyrimidine tract (TOP). This is typically a stretch of pyrimidine nucleotides located in the 5'UTR. The sequence generally begins with a cytidine, usually corresponding to the transcription start site, followed by a stretch of, for example, about 3 to 30 pyrimidine nucleotides. For example, a TOP may contain at least 3, 5, 10, 15, 20, 25, or 30 pyrimidine nucleotides. The pyrimidine stretch, and therefore the 5'TOP, ends with one nucleotide on the 5' side of the first purine nucleotide located downstream of the TOP.

[0061] mRNA molecules may contain a polyadenosine (poly-A) tail at their 3' end. The poly-A tail is located on the 3' side of the 3'UTR. Alternatively, the poly-A tail may be seen as part of the 3'UTR. The poly-A tail may contain approximately 10 to 200 adenosine residues, generally about 40 to 100 adenosine residues, for example, about 40, 50, 60, 70, or 80 adenosine residues, for example, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 40 to 50, 50 to 100, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 60 to 100, 60 to 90, 60 to 80, 60 to 70, 70 to 100, 70 to 90, 70 to 80, 75 to 85, 75 to 80, or 80 to 100 adenosine residues.

[0062] Any preferred combination of UTRs may be used in the mRNA molecules provided herein. The 5' and 3' UTRs may be genetically derived or derived, or synthetic. The 5' and 3' UTRs may have the same or different origins; for example, the 5' UTR may be synthetic, and the 3' UTR may be genetically derived or derived, or vice versa. A genetically derived UTR refers to a UTR identical to a naturally occurring UTR in the context of a gene. A genetically derived UTR refers to a UTR based on a naturally occurring UTR, but modified compared to that natural (parental) UTR. A genetically derived UTR may, for example, have at least 70, 75, 80, 85, 90, or 95% sequence identity with the parental UTR and / or may contain additional sequence elements on the 5' and / or 3' sides of the parental UTR.

[0063] If UTR is caused by or originates from a gene, that gene is a eukaryotic gene, generally a mammalian gene, and especially a human gene.

[0064] Examples of such 5'UTR sequences that may be used in mRNA molecules provided herein include those shown in SEQ ID NOs. 15 and 20-23. Examples of such 3'UTR sequences that may be used in mRNA molecules provided herein include those shown in SEQ ID NOs. 16 and 24-26.

[0065] Therefore, the 5'UTR may contain or consist of the nucleotide sequence shown in any one of SEQ ID NOs. 15 and 20-23, or a variant of any one of SEQ ID NOs. 15 and 20-23 having at least 70, 75, 80, 85, 90, or 95% sequence identity with it. In a particular example, the 5'UTR may contain or consist of the nucleotide sequence of SEQ ID NOs. 15, or a variant thereof having at least 90 or 95% sequence identity with it. An extended UTR containing SEQ ID NOs. 15 with additional sequence elements at both ends is shown in SEQ ID NOs. 43. In a particular example, the 5'UTR may contain or consist of the nucleotide sequence of SEQ ID NOs. 43, or a variant thereof having at least 90 or 95% sequence identity with it.

[0066] Therefore, the 3'UTR sequence may contain or consist of the nucleotide sequence shown in any one of SEQ ID NOs. 16 and 24-26, or a variant of any one of SEQ ID NOs. 16 and 24-26 having at least 70, 75, 80, 85, 90, or 95% sequence identity with it. In a particular example, the 3'UTR contains or consists of the nucleotide sequence of SEQ ID NOs. 16, or a variant thereof having at least 90 or 95% sequence identity with it. An extended UTR containing SEQ ID NOs. 16 with additional sequence elements at both ends is shown in SEQ ID NOs. 44. In a particular example, the 3'UTR contains or consists of the nucleotide sequence of SEQ ID NOs. 44, or a variant thereof having at least 90 or 95% sequence identity with it.

[0067] The 5' and 3' UTR sequences shown above may be used in combination. Thus, an mRNA molecule may include (i) a 5' UTR containing or consisting of the nucleotide sequence shown in any one of SEQ ID NOs. 15 and 20-23, or a variant of any one of SEQ ID NOs. 15 and 20-23 having at least 70, 75, 80, 85, 90, or 95% sequence identity thereto, and (ii) a 3' UTR containing or consisting of the nucleotide sequence shown in any one of SEQ ID NOs. 16 and 24-26, or a variant of any one of SEQ ID NOs. 16 and 24-26 having at least 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0068] As shown in the following example, particularly high levels of protein expression can be obtained from mRNA molecules containing the 5'UTR of SEQ ID NO: 15 (derived from the 5'UTR of human CHIT1) and the 3'UTR of SEQ ID NO: 16 (derived from the 3'UTR of human CS). Therefore, in certain examples, the mRNA molecule contains (i) a 5'UTR containing or consisting of the nucleotide sequence shown in SEQ ID NO: 15, or a variant thereof having at least 90 or 95% identity with SEQ ID NO: 15, and (ii) a 3'UTR containing or consisting of the nucleotide sequence shown in SEQ ID NO: 16, or a variant thereof having at least 90 or 95% identity with SEQ ID NO: 16.

[0069] As shown below, particularly high levels of protein expression are obtained when the 5'UTR containing SEQ ID NO: 15 is the 5'UTR of SEQ ID NO: 43, and the 3'UTR containing SEQ ID NO: 16 is the 3'UTR of SEQ ID NO: 44. Therefore, in certain examples, the mRNA molecule contains (i) a 5'UTR containing or consisting of the nucleotide sequence shown in SEQ ID NO: 43, or a variant thereof having at least 90 or 95% identity with SEQ ID NO: 43, and (ii) a 3'UTR containing or consisting of the nucleotide sequence shown in SEQ ID NO: 44, or a variant thereof having at least 90 or 95% identity with SEQ ID NO: 44.

[0070] When a variant of a given UTR is used, the variant is generally at least functionally equivalent to the given UTR. “Functionally equivalent” in the context of a UTR sequence means that the variant UTR produces a level of protein expression equivalent to (or similar to) that of the given UTR sequence. “Similar” levels of protein expression may mean that the variant UTR produces a level of protein expression as high as, or at least 70, 75, 80, 85, 90, or 95% of, that of the given unmodified UTR sequence. “At least functionally equivalent” to the given sequence means that the variant sequence may be superior to the given unmodified UTR sequence, i.e., it may produce a higher level of protein expression than the given unmodified UTR sequence.

[0071] Nanoparticle subunit The mRNA molecules provided herein encode fusion proteins that, when expressed, form subunits of protein nanoparticles. That is, the mRNA molecules contain nucleotide sequences encoding such fusion proteins or "nanoparticle subunits." The nanoparticle subunits include an immunogen and a scaffold linked by a linker. When the mRNA molecules are administered to a target, the nanoparticle subunits are expressed by the target cells, triggering an immune response to the immunogen.

[0072] In some examples, the nanoparticle subunit consists of an immunogen, a scaffold, and a linker. In other examples, the nanoparticle subunit includes one or more additional elements, such as a signal peptide (although in some examples the signal peptide may be included as part of the immunogen).

[0073] The components of the nanoparticle subunit may be arranged in any order; that is, the immunogen may be located at the N-terminus or the C-terminus. Therefore, in some examples, the nanoparticle subunit includes (or consists of) the immunogen, linker, and scaffold, from the N-terminus to the C-terminus. In other examples, the nanoparticle subunit includes (or consists of) the scaffold, linker, and immunogen, from the N-terminus to the C-terminus. As described above, the immunogen and scaffold are linked by a linker, and therefore the linker is located between them. Generally, the immunogen and scaffold are directly adjacent to the linker; that is, there are no additional sequence elements between the linker and the immunogen or scaffold.

[0074] Protein nanoparticles Upon expression, multiple copies of the nanoparticle subunits assemble to form protein nanoparticles. Protein nanoparticles are nanoparticles formed by the polymerization of one or more proteins, such as the nanoparticle subunits disclosed herein.

[0075] Protein nanoparticles can be homomultimers (i.e., formed from multiple copies of the same subunit protein) or heteromultimers (i.e., containing at least two different subunit proteins). Generally, protein nanoparticles formed from subunits encoded by mRNA molecules are homomultimers formed from the multimerization of the encoded subunits. The encoded subunits multimerize via the protein scaffold region, which will be discussed further below.

[0076] Nanoparticles formed by subunits encoded by mRNA molecules may have diameters of approximately 1–100 nm, for example, 1–80, 1–60, 1–50, 1–40, 1–35, 1–30, 1–25, 1–20, 5–100, 5–80, 5–60, 5–50, 5–40, 5–35, 5–30, 5–25, 5–20, 10–100, 10–80, 10–60, 10–50, 10–40, 10–35, 10–30, 10–25, or 10–20 nm. In general, nanoparticles may have any shape, for example, spherical (or nearly spherical) or icosahedral. If the shape does not have a single uniform diameter, the term “diameter” as used herein refers to the longest internal dimension of the nanoparticle. The diameter is measured between the outer surfaces of the nanoparticle.

[0077] Protein nanoparticles formed by subunits encoded by mRNA molecules may contain, for example, at least 5, 10, 20, 30, 40, 50, 60 or more subunits. In certain examples, protein nanoparticles may contain 60 subunits (i.e., they may be 60-mers), and may be homomeric nanoparticles containing 60 subunits encoded by mRNA molecules in particular. In some examples, protein nanoparticles may be homomeric 60-mers.

[0078] Protein nanoparticles formed by subunits encoded by mRNA molecules may have molecular weights in the range of 650kDa to 15mDa, e.g., 650kDa to 12, 10, 9, 8, 7, 6, or 5mDa, e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 15, 2 to 12, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 3 to 15, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 12, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5mDa.

[0079] scaffold As described above, the scaffold essentially functions as a polymerization domain for the encoded nanoparticle subunits, through which the individual subunits interact to form nanoparticles.

[0080] The scaffold is either a lumazine synthase protein or contains a lumazine synthase protein. Lumazine synthase is an enzyme found across all domains of life that catalyzes the second-to-last step in the biosynthesis of riboflavin (vitamin B2). All lumazine synthase proteins multimerize to form complexes of various sizes. In particular, some bacterial species and plants possess lumazine synthase that forms a 60-mer icosahedral capsid. The structure of such a capsid is described in Ladenstein & Morgunova, Biotechnology Reports 27:e00494, 2020. Lumazine synthase capsids can be described as protein cage nanoparticles. The capsid is a homomeric 60-mer containing 60 identical lumazine synthase subunits. The 60-mer structure can be thought of as a pentameric dodecamer, with particularly strong interactions formed between the subunits within each pentamer and weaker interactions formed between the pentamers that assemble to form the capsid.

[0081] In 60-mer lumazine synthase capsids, both the N-terminus and C-terminus of each subunit are exposed to the outer surface. Therefore, lumazine synthase capsids can be used as nanoparticles for antigen presentation by fusing a target antigen to either the N-terminus or C-terminus of the lumazine synthase enzyme.

[0082] The lumazine synthase used as a scaffold for the fusion protein described herein may be a lumazine synthase that multimerizes to form a 60-mer, i.e., a lumazine synthase that multimerizes to form a 60-mer icosahedral capsid or 60-mer nanoparticle. The lumazine synthase may be derived from or originate from any species that produces such enzymes, such as plants (e.g., spinach (Spinacia oleracea), tobacco (e.g., Nicotiana tabacum or Nicotiana rustica) or Arabidopsis thaliana) or bacteria.

[0083] For example, rumazine synthase may be derived from or originate from any bacterium that produces a 60-mer version of the enzyme. Suitable bacteria include those of the Bacillaceae family, e.g., Bacillus subtilis; those of the Enterobacteriaceae family, e.g., Escherichia coli; and those of the Aquificaceae family, e.g., Aquifex aeolicus.

[0084] As mentioned above regarding UTR, lumazine synthases originating from a particular species have the native sequence of the enzyme from that species. Lumazine synthases originating from a species are modified versions of the native enzyme. When a modified version of a naturally occurring lumazine synthase is used, the modified version retains the ability to form a multimeric complex, fusing the immunogen to the capsid and presenting it on the capsid surface, with at least one of the N-terminus and C-terminus remaining exposed on the outer surface of the capsid.

[0085] Modified versions of lumazine synthases that naturally form 60-mer structures may retain the ability to form 60-mer structures or may form different structures with more or fewer than 60 subunits. Modified bacterial lumazine synthases that form 180-mer or 360-mer nanoparticles are known (Ladenstein & Morgunova, cited above), and such modified lumazine synthases may be used in fusion proteins encoded by mRNA molecules provided herein.

[0086] Alternatively, the lumazine synthases used herein may naturally form smaller polymers containing, for example, 5 or 10 subunits. Modified versions of such lumazine synthases may also be used, which may form larger polymers than the natural enzymes.

[0087] If the lumazine synthase used is a variant of a natural enzyme, the enzyme may be inactive. Since the enzyme is simply used as a scaffold, it is not important whether it retains its synthase activity.

[0088] In certain examples, the lumazine synthase is A. aeolicus lumazine synthase or a derivative thereof. A. aeolicus lumazine synthase has the amino acid sequence shown in SEQ ID NO: 3 (UniProt entry O66529). Therefore, the scaffolds used in the nanoparticle subunits herein may include or consist of the amino acid sequence shown in SEQ ID NO: 3, or variants thereof having at least 70, 75, 80, 85, 90, or 95% identity with SEQ ID NO: 3. In certain examples, the scaffolds include or consist of the amino acid sequence shown in SEQ ID NO: 3.

[0089] When the variant of Sequence ID No. 3 is used, the variant retains the ability to form multimeric protein nanoparticles. In particular, the variant may retain the ability to form the 60-mer complex described above. The assembly of the A. aeolicus lumazine synthase pentamer into an icosahedral capsid has been found to depend on eight amino acid residues in each subunit that form hydrophobic interactions (L121 and I125), electrostatic interactions (E5, R21, D36, R40 and E145), and hydrogen bonds (H41) between adjacent pentamers. Disruption of these interactions (especially hydrogen bonds) by point mutations has been found to prevent dodecamerization of the pentameric capsid building block, so that the enzyme forms only the pentameric structure (Hickman et al., Proteins 83:1733-1741, 2015). To avoid the loss of the ability to form 60-mers or other larger structures, variants of the enzyme of SEQ ID NO: 3 may not be modified at these positions, or may contain conservative substitutions at these positions that do not disrupt the inter-subunit interactions.

[0090] Linker The inventors have found that an enhanced immune response is obtained when the immunogen is fused to lumazine synthase via a linker rather than directly. While not bound by theory, it is hypothesized that when a linker is not used, access to key neutralizing epitopes is hindered by the immunogen's proximity to the lumazine synthase multimer.

[0091] The linker used is long enough so that the immunogen is sufficiently far from the lumazine synthase polymer and its epitope is accessible to the antibody. For example, the linker may be at least 5, 6, 7, 8, 9, or 10 amino acids long. The linker may have a maximum length of 20, 18, 16, 14, or 12 amino acids. For the immunogen of SEQ ID NO: 1 or 2 (discussed further below), we have found that the linker is optimally about 9–13 amino acids long, for example, 10–12 amino acids long. In certain examples, the linker is 11 amino acids long.

[0092] The linker sequence is not particularly limited, as long as it is long enough to detach the scaffold from the immunogen. Various linker sequences for linking fusion protein components are known in the art and can be used in this fusion protein, including glycine-serine linkers (i.e., linkers consisting of glycine residues and serine residues), such as [G]n, [S]n, [A]n, [GS]n, [GGS]n, [GGGS]n (SEQ ID NO: 27), [GGGGS]n (SEQ ID NO: 28), [GGSG]n (SEQ ID NO: 29), [GSGG]n (SEQ ID NO: 30), [SGGG]n (SEQ ID NO: 31), [SSGG]n (SEQ ID NO: 32), [SSSG]n (SEQ ID NO: 33), [GG]n, [GGG]n, [SA]n, [SAGS]n (SEQ ID NO: 34), or [GGGSG]n (SEQ ID NO: 35) (wherein "n" is between 1 and 20 (a suitable value for "n" depends on the length of the repeating unit, and a suitable value for "n" can be selected to obtain a linker of a length that falls within the above range)).

[0093] For example, suitable linker sequences include TGGGGSGGGGS (sequence number 36), GGGGSGGGGS (sequence number 37), and SGGSSGSSGGS (sequence number 4).

[0094] In certain cases, the linker may contain or consist of the amino acid sequence shown in SEQ ID NO: 4, or a variant thereof that includes up to three amino acid insertions, deletions, or substitutions compared to SEQ ID NO: 4. For example, a variant of SEQ ID NO: 4 may include up to two amino acid insertions, deletions, or substitutions compared to SEQ ID NO: 4, or a single amino acid insertion, deletion, or substitution compared to SEQ ID NO: 4.

[0095] In certain cases, the linker may consist of or comprise the amino acid sequence shown in SEQ ID NO: 4, or a variant thereof containing up to three amino acid substitutions compared to SEQ ID NO: 4, i.e., one, two, or three amino acid substitutions compared to SEQ ID NO: 4. Such a linker may be an 11-amino acid linker, i.e., consisting of SEQ ID NO: 4 or its substituted variants.

[0096] Linker - Scaffolding A combination of a linker and a scaffold is referred to herein as a "linker-scaffold." A linker-scaffold may include any scaffold and any linker sequence in either orientation (i.e., the linker or scaffold may be at the N-terminus and the other at the C-terminus), as described above.

[0097] In certain cases, the linker scaffold includes or consists of the amino acid sequence shown in SEQ ID NO: 5, or a variant thereof having at least 70, 75, 80, 85, 90, or 95% identity with SEQ ID NO: 5. SEQ ID NO: 5 includes the scaffold of SEQ ID NO: 3, which has the linker of SEQ ID NO: 4 at its N-terminus.

[0098] immunogen The immunogen comprises a modified RSV F protein, which is desirable to generate an immune response to derive immunity against RSV. The immunogen used herein is an antigen, i.e., the modified RSV F protein generates an antibody response (at least an antibody response to the RSV F protein) when expressed in a subject.

[0099] In some cases, an immunogen may consist of multiple (i.e., two or more) proteins or protein fragments that are fused to one another. That is, an immunogen may consist of a modified RSV F protein (as further described below) and at least one additional protein or protein fragment. Generally, an immunogen is (i.e., consists of) a modified RSV F protein. The RSV F protein is known to be the primary target of neutralizing antibodies during RSV infection. The modified RSV F protein may originate from any RSV strain, for example, from an RSV A strain (subtype A1 or A2, etc.) or an RSV B strain (subtype B1 or B2, etc.).

[0100] The modified RSV F protein is stabilized in a pre-fusion conformation (i.e., a pre-F stabilized F protein). It contains both the F1 and F2 polypeptides in a single chain with a linker between them. Specifically, the modified RSV F protein includes a deletion at positions 104-144 of the RSV F protein and a GS peptide linker between positions 103 and 145 of the RSV F protein, as well as the following mutations: S155C, N183GC, S190F, V207L, S290C, L373R, and N428C. Thus, the C-terminus of the F2 polypeptide is linked to the N-terminus of the F1 polypeptide by the GS linker.

[0101] The above amino acid numbering is based on the full-length wild-type RSV F protein. This may be due to the wild-type RSV F protein on which the modified F protein is based, or any other wild-type RSV F protein. The full-length F protein is the primary F protein sequence prior to any post-translational modifications or processing, including the signal sequence. In a specific example, the amino acid position numbering is based on the exemplary RSV F protein of SEQ ID NO: 19. This full-length RSV F protein originates from RSV strain A2 and has UniProt accession number P03420 (sequence version 1).

[0102] In SEQ ID NO: 19, amino acids 1-25 constitute the signal sequence (SEQ ID NO: 41), and amino acids 26-574 constitute the F0 chain (SEQ ID NO: 38). Within this chain, amino acids 26-109 (numbered based on the precursor protein of SEQ ID NO: 19) constitute the F2 chain (shown in SEQ ID NO: 40), amino acids 110-136 constitute the p27 peptide (SEQ ID NO: 42), and amino acids 137-574 constitute the F1 chain (shown in SEQ ID NO: 39). The F1 polypeptide contained in the modified F protein may be a variant of SEQ ID NO: 39, or the polypeptide corresponding to the F1 chain of SEQ ID NO: 39, or a variant of the polypeptide corresponding to SEQ ID NO: 39. Similarly, the F2 polypeptide contained in the modified F protein may be a variant of SEQ ID NO: 40, or the polypeptide corresponding to the F2 chain of SEQ ID NO: 40, or a variant of the polypeptide corresponding to SEQ ID NO: 40.

[0103] A polypeptide that "corresponds" to the F1 or F2 chain of SEQ ID NO: 39 or 40 means a polypeptide derived from the RSV F protein that, when its F protein is aligned with the F protein of SEQ ID NO: 19, aligns with SEQ ID NO: 39 or SEQ ID NO: 40. A variant of SEQ ID NO: 39 may have at least 70, 75, 80, 85, 90, or 95% sequence identity with SEQ ID NO: 39. Similarly, a variant of SEQ ID NO: 40 may have at least 70, 75, 80, 85, 90, or 95% sequence identity with SEQ ID NO: 40.

[0104] As described above, the modified F protein used herein contains several mutations compared to the wild-type sequence. If the amino acid position numbering of these mutations is based on SEQ ID NO: 19, the mutated amino acid positions correspond to the same numbered positions in SEQ ID NO: 19. The corresponding amino acid positions can be identified by sequence alignment; that is, the position in the target F protein sequence corresponding to position 155 of SEQ ID NO: 19 is the position that corresponds to (or aligns with) position 155 of SEQ ID NO: 19 when the target F protein sequence is aligned to SEQ ID NO: 19.

[0105] As described above, at least the F1 chain of the modified F protein contains mutations compared to the wild-type F protein sequence. These mutations may act to stabilize the protein in a pre-F conformation. They may also, or alternatively, enhance the immune response to the protein, and in particular, enhance the pre-F specific immune response to the protein. These features of modified F proteins are known in the art and include, for example, techniques including the level of recognition by pre-F specific antibodies, as described in Joyce et al. (cited above), which are incorporated herein by reference.

[0106] As is clear from the above paragraph, the F2 protein is shortened at its C-terminus compared to SEQ ID NO: 40, and the F1 protein is shortened at its N-terminus compared to SEQ ID NO: 39. Specifically, the F2 protein has a 6-amino acid C-terminal shortening corresponding to the deletion of amino acids 104-109 in SEQ ID NO: 19, and the F1 protein has an 8-amino acid N-terminal shortening corresponding to the deletion of amino acids 137-144 in SEQ ID NO: 19. The modified RSV F protein does not contain the p27 peptide, which, in combination, results in the aforementioned deletion at the position corresponding to 104-144 in SEQ ID NO: 19. Taking this deletion into account, the amino acid at the position corresponding to 103 in SEQ ID NO: 19 is fused with the amino acid at the position corresponding to 145 in SEQ ID NO: 19. A glycine-serine (GS) linker is used to link the two positions. The modified RSV F protein containing this deletion and linker is described by Joyce et al. (mentioned above) and is called the sc9-10 variant of DS-Cav1.

[0107] Of the mutations present in the modified F protein described above, the S190F and V207L ​​mutations are cavity-filling substitutions, while the S155C and S290C mutations provide additional disulfide bonds. These four mutations are DS-Cav1 mutations that stabilize the protein in its pre-fusion conformation. The pre-fusion F protein ectodomain contains a cavity that collapses during transition to the post-fusion conformation. Filling the cavity with bulky amino acid side chains acts to counteract the transition to the post-fusion conformation. Additional disulfide bonds are also provided by cysteine ​​residues introduced by N183GC and N428C substitutions, further stabilizing the pre-fusion conformation. On the other hand, residue L373 naturally interacts with L141 and L142, but their deletions leave L373 exposed. The L373R mutation introduces a hydrophilic side chain at this position, which also improves stability.

[0108] If a modified F protein is based on an F protein other than the one in SEQ ID NO: 19, but its amino acid numbering is based on SEQ ID NO: 19, the modified F protein will have the specified amino acid residue at the specified position. For example, a modified F protein with the S155C mutation based on the numbering of SEQ ID NO: 19 will have a cysteine ​​residue at the amino acid position corresponding to position 155 in SEQ ID NO: 19. It is not important whether this cysteine ​​residue was introduced as a result of a Ser=>Cys mutation, a different mutation, or whether it is naturally located at that position in the wild-type F protein.

[0109] Modified RSV F proteins may also contain one or more additional substitutions in the F1 and / or F2 chains compared to SEQ ID NO: 19, in addition to the S155C, N183GC, S190F, V207L, S290C, L373R, and N428C mutations specified above. In particular, modified RSV F proteins may further contain one or more substitutions at the S46, E92, and S215 positions. For example, modified RSV F proteins may contain one or more of the following substitutions: S46G, E92D, and S215P. For example, modified RSV F proteins may contain one, two, or three of these substitutions.

[0110] In certain examples, modified RSV F proteins contain only the S155C, N183GC, S190F, V207L, S290C, L373R, and N428C substitutions. In other examples, modified RSV F proteins contain the S46G, E92D, S155C, N183GC, S190F, V207L, S215P, S290C, L373R, and N428C substitutions.

[0111] Modified RSV F proteins may also contain other mutations. For example, in some cases, modified RSV F proteins may contain further A149C and Y458C substitutions, resulting in additional disulfide bonds. In some cases, modified RSV F proteins may contain further T369C and T455C substitutions, resulting in additional disulfide bonds. In some cases, modified RSV F proteins may contain further cysteine ​​substitutions at one of the positions 98-100 and one of the positions 361-362, e.g., Q98C and Q361C substitutions, S99C and Q361C substitutions, or T100C and S362C substitutions (or any other combination thereof), resulting in additional disulfide bonds.

[0112] Other mutations that can be introduced in any combination include N67I, L95M, I217P, I221M, R429K, and K465Q. In some examples, the modified F protein contains the L95M, I221M, R429K, and optionally the I217P mutation. In some examples, the modified F protein contains the N67I and K465Q mutations. Such mutations may increase the stability and / or expression of the modified F protein.

[0113] The pre-fusion conformation, in its native form, contains an antigenic site located at its distal membrane apex, called oe. The antigenic site oe contains RSV F residues 62-69 and 196-209, but is lost from the post-fusion conformation. Several pre-fusion specific antibodies bind to this site. Stabilization of the F protein in its pre-fusion domain preserves or stabilizes the antigenic site oe, and therefore, the pre-fusion stabilized F protein can be identified as one that is recognized by pre-fusion specific antibodies such as AM22, D25, and 5C4 (McLellan et al., previously cited). Typically, the pre-fusion stabilized RSV F protein has approximately 10 -6 Less than M, for example, about 10 -7 M, 10 -8 M or 10 -9 With a dissociation constant of less than M, it specifically binds to pre-fusion specific antibodies such as these.

[0114] Pre-fusion stabilized RSV F protein may retain specific binding to pre-fusion specific antibodies (such as those mentioned above) after incubation in phosphate-buffered saline (PBS) at 70°C for 30 minutes or 1 hour. For example, pre-fusion stabilized RSV F protein may retain at least 50, 60, or 70% binding to pre-fusion specific antibodies after such incubation.

[0115] Alternatively or additionally, pre-fusion stabilized RSV F protein may retain specific binding to pre-fusion specific antibodies (such as those mentioned above) after incubation for 1, 2, 3, 4, 5, or 6 months in PBS at 4°C. For example, pre-fusion stabilized RSV F protein may retain at least 50, 60, 70, 80, or 90% binding to pre-fusion specific antibodies after such incubation.

[0116] Therefore, in the pre-fusion stabilized RSV F protein, one or more antigenic epitopes are accessible on the protein surface. In particular, one or more neutralizing epitopes are accessible on the protein surface. Accessible epitopes may be pre-fusion specific epitopes, i.e., epitopes that are accessible in the pre-fusion conformation of the F protein but not in the post-fusion conformation. For example, the antigen site oe may be accessible. In this specification, "accessible" means that the epitope is accessible to an antibody so that it can be bound by the antibody.

[0117] The F2 chain of a modified RSV F protein may have an N-terminal position corresponding to one of the following RSV F positions 20–30, e.g., 25–30, e.g., 25, 26, 27, 28, 29, or 30. In certain cases, the F2 chain has an N-terminal position corresponding to position 26 of SEQ ID NO: 19 (i.e., the N-terminal residue of the F2 chain derived from the natural SEQ ID NO: 19 F protein). The F1 chain of a modified RSV F protein may have a C-terminal position corresponding to one of the following RSV F positions 510–525, e.g., 510–520 or 510–515 of RSV F in SEQ ID NO: 19, e.g., 510, 511, 512, 513, 514, or 515. In certain cases, the F1 chain has a C-terminal position corresponding to position 513 of SEQ ID NO: 19.

[0118] In some cases, the F1 chain contains the amino acid sequence shown in SEQ ID NO: 45, or a variant thereof having at least 80, 85, 90, or 95% identity thereto, provided that the variant contains the S155C, N183GC, S190F, V207L, S290C, L373R, and N428C substitutions (numbered according to SEQ ID NO: 19). SEQ ID NO: 45 corresponds to amino acids 145-513 of SEQ ID NO: 19, having the S155C, N183GC, S190F, V207L, S290C, L373R, and N428C substitutions. In some cases, the variant of SEQ ID NO: 45 is SEQ ID NO: 46, which corresponds to SEQ ID NO: 45 and further includes the S215P substitution. In some cases, the F1 chain contains the amino acid sequence shown in SEQ ID NO: 46, or a variant thereof having at least 80, 85, 90, or 95% identity thereto, provided that the variant includes the S155C, N183GC, S190F, V207L, S215P, S290C, L373R, and N428C substitutions (numbered according to SEQ ID NO: 19).

[0119] In some cases, the F2 chain contains the amino acid sequence shown in SEQ ID NO: 47, or a variant thereof that is at least 80, 85, 90, or 95% identical thereto. SEQ ID NO: 47 corresponds to amino acids 26-103 of SEQ ID NO: 19. In some cases, the variant of SEQ ID NO: 47 is SEQ ID NO: 48, which corresponds to SEQ ID NO: 47 and further includes the S46G and E92D substitutions. In some cases, the F2 chain contains the amino acid sequence shown in SEQ ID NO: 48, or a variant thereof that is at least 80, 85, 90, or 95% identical thereto, however the variant includes the S46G and E92D substitutions.

[0120] The modified F proteins encoded by the mRNA molecules provided herein may include an F1 chain comprising or consisting of the amino acid sequence or variant thereof shown in SEQ ID NO: 45 or 46, and an F2 chain comprising or consisting of the amino acid sequence or variant thereof shown in SEQ ID NO: 47 or 48, particularly the F1 chain of SEQ ID NO: 45 or 46 and the F2 chain of SEQ ID NO: 47 or 48.

[0121] For example, a modified F protein may include an F1 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 45, and an F2 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 47, in particular the F1 chain of SEQ ID NO: 45 and the F2 chain of SEQ ID NO: 47. Alternatively, a modified F protein may include an F1 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 46, and an F2 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 48, in particular the F1 chain of SEQ ID NO: 46 and the F2 chain of SEQ ID NO: 48.

[0122] Alternatively, the modified F protein may include an F1 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 45, and an F2 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 48, in particular the F1 chain of SEQ ID NO: 45 and the F2 chain of SEQ ID NO: 48. Or, the modified F protein may include an F1 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 46, and an F2 chain containing or consisting of the amino acid sequence or variant shown in SEQ ID NO: 47, in particular the F1 chain of SEQ ID NO: 46 and the F2 chain of SEQ ID NO: 47.

[0123] The modified F protein may further contain an N-terminal signal sequence to direct its secretion from the cell on which it is expressed. Any signal sequence may be used, but for convenience, the signal sequence of SEQ ID NO: 41 may be used, or a variant thereof having at least 80, 85, 90, or 95% identity with SEQ ID NO: 41 may be used.

[0124] The modified RSV F protein, consisting of the signal peptide of SEQ ID NO: 41, the F1 chain of SEQ ID NO: 45, and the F2 chain of SEQ ID NO: 47, has the amino acid sequence shown in SEQ ID NO: 2.

[0125] The modified RSV F protein, consisting of the signal peptide of SEQ ID NO: 41, the F1 chain of SEQ ID NO: 46, and the F2 chain of SEQ ID NO: 48, has the amino acid sequence shown in SEQ ID NO: 1. This modified RSV F protein is described by Joyce et al. (mentioned above) and is called sc9-10 DS-Cav1 N183GC N428C S46G S215P E92D.

[0126] The modified RSV F protein of SEQ ID NO: 1 may be referred to herein as DS2-2, and the modified RSV F protein of SEQ ID NO: 2 may be referred to as DS2-2(A).

[0127] In certain cases, modified RSV F proteins include: (i) The amino acid sequence shown in Sequence ID No. 1, or (ii) Variants of Sequence ID No. 1 that are at least 80, 85, 90, or 95% identical thereto, but include the following: a) The glycine residue at the position corresponding to position 46 of Sequence ID No. 1, b) The aspartic acid residue at position 92 of Sequence ID No. 1, c) The cysteine ​​residue at position 116 of sequence number 1, d) The glycine residue at the position corresponding to position 144 of sequence number 1, e) The cysteine ​​residue at position 145 of sequence number 1, f) The phenylalanine residue at position 152 of Sequence ID No. 1, g) The leucine residue at the position corresponding to position 169 of Sequence ID No. 1, h) The proline residue at position 177 of sequence number 1, i) The cysteine ​​residue at position 252 of sequence number 1, j) The arginine residue at position 335 of Sequence ID No. 1, and k) The cysteine ​​residue at position 390 of sequence number 1.

[0128] Other specific examples of modified RSV F proteins include: (i) The amino acid sequence shown in Sequence ID No. 2, or (ii) Variants of Sequence ID No. 2 that are at least 80, 85, 90, or 95% identical thereto, but include the following: a) The serine residue at the position corresponding to position 46 of Sequence ID No. 2, b) The glutamic acid residue at position 92 of Sequence ID No. 2, c) The cysteine ​​residue at position 116 of sequence number 2, d) The glycine residue at the position corresponding to position 144 of sequence number 2, e) The cysteine ​​residue at position 145 of sequence number 2, f) The phenylalanine residue at position 152 of Sequence ID No. 2, g) The leucine residue at the position corresponding to position 169 of Sequence ID No. 2, h) The serine residue at position 177 of Sequence ID No. 2, i) The cysteine ​​residue at position 252 of Sequence ID No. 2, j) The arginine residue at position 335 of Sequence ID No. 2, and k) The cysteine ​​residue at position 390 of sequence number 2.

[0129] Note that positions 46, 92, 116, 144, 145, 152, 169, 177, 252, 335, and 390 of sequence numbers 1 and 2 correspond to positions 46, 92, 155, 183, 190, 207, 215, 290, 373, and 428 of the reference sequence for sequence number 19, respectively (both positions 144 and 145 of sequence numbers 1 and 2 correspond to position 183 of sequence number 19).

[0130] The amino acid position of a variant of SEQ ID NO: 1 or 2 that corresponds to a specific position in SEQ ID NO: 1 or 2 is the position of the variant that aligns to the relevant position in SEQ ID NO: 1 or 2, if the variant's sequence is aligned with SEQ ID NO: 1 or 2. Therefore, for example, if a variant of SEQ ID NO: 1 is aligned with SEQ ID NO: 1, the position of the variant corresponding to position 46 of SEQ ID NO: 1 is the position of the variant that aligns to position 46 of SEQ ID NO: 1. The position corresponding to position 46 of SEQ ID NO: 1 may be at position 46 of the variant, but may be at a different position if the variant contains an insertion or deletion mutation compared to the position preceding SEQ ID NO: 1.

[0131] In certain cases, the immunogen contains or consists of the amino acid sequence shown in SEQ ID NO: 1. The following example demonstrates that the use of SEQ ID NO: 1 as an antigen in an animal model of RSV vaccination yields particularly high levels of neutralizing antibodies.

[0132] In other specific cases, the immunogen contains or consists of the amino acid sequence shown in Sequence ID No. 2.

[0133] If the immunogen is a variant of SEQ ID NO: 1 or SEQ ID NO: 2, at least one of the accessible epitope sequences (particularly the neutralizing epitope sequence) is either unchanged compared to SEQ ID NO: 1 or SEQ ID NO: 2, or not sufficiently altered to induce an antibody (or neutralizing antibody) titer at least similar to that of the wild-type epitope sequence upon administration to a subject, for example, at least 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120% or higher of the titer obtained with the specific F protein antigen of SEQ ID NO: 1 and 2. The variant of SEQ ID NO: 1 or SEQ ID NO: 2 induces the production of antibodies that recognize the wild-type RSV F protein; that is, the amino acid mutations compared to SEQ ID NO: 1 or 2 do not reduce the affinity of the antibodies produced against the native F protein.

[0134] In certain cases, the fusion protein forming the nanoparticle subunit contains or consists of the amino acid sequence shown in SEQ ID NO: 6, or a variant thereof having at least 80, 85, 90, or 95% identity with SEQ ID NO: 6. SEQ ID NO: 6 is a fusion protein containing the modified RSV F protein of SEQ ID NO: 1, the linker of SEQ ID NO: 4, and the lumazine synthase scaffold of SEQ ID NO: 3, from the N-terminus to the C-terminus. In certain cases, the fusion protein contains or consists of SEQ ID NO: 6.

[0135] In certain cases, the fusion protein contains or consists of the amino acid sequence shown in SEQ ID NO: 7, or a variant thereof having at least 80, 85, 90, or 95% identity with SEQ ID NO: 7. SEQ ID NO: 7 is a fusion protein containing the modified RSV F protein of SEQ ID NO: 2, the linker of SEQ ID NO: 4, and the lumazine synthase scaffold of SEQ ID NO: 3, from the N-terminus to the C-terminus. In certain cases, the fusion protein contains or consists of SEQ ID NO: 7.

[0136] The nucleotide sequences used to encode immunogens and scaffolds may be native sequences (or, in the case of modified F proteins, modified compared to the native sequence only when necessary for amino acid sequence modification compared to the native F protein). Alternatively, the nucleotide sequences may be codon-optimized for human expression compared to the native sequence (particularly in the case of sequences encoding rumazine synthases derived from non-human species).

[0137] The RNA nucleotide sequences encoding the immunogens of SEQ ID NOs: 1 and 2 are shown in SEQ ID NOs: 8 and 9, respectively. The RNA nucleotide sequence of the lumazine synthase of SEQ ID NOs: 3 is shown in SEQ ID NOs: 10, and the RNA sequence of the linker of SEQ ID NOs: 4 is shown in SEQ ID NOs: 11. In the mRNA molecules provided herein, the immunogen may be encoded by the nucleotide sequence shown in SEQ ID NOs: 8 or 9, or the nucleotide sequence degenerated in SEQ ID NOs: 8 or 9, or a nucleotide sequence encoding the modified F protein described above and having at least 90 or 95% identity with SEQ ID NOs: 8 or 9; the scaffold may be encoded by the nucleotide sequence shown in SEQ ID NOs: 10, or the nucleotide sequence degenerated in SEQ ID NOs: 10, or a nucleotide sequence encoding the lumazine synthase described above and having at least 90 or 95% identity with SEQ ID NOs: 10; and the linker may be encoded by the nucleotide sequence shown in SEQ ID NOs: 11, or the nucleotide sequence degenerated in SEQ ID NOs: 11, or a nucleotide sequence encoding a linker of a length suitable for use herein and having at least 90 or 95% identity with SEQ ID NOs: 11.

[0138] A nucleotide sequence in an mRNA molecule encoding an immunogen may be called an immunogen coding sequence. Similarly, a nucleotide sequence encoding a linker may be called a linker coding sequence, and a nucleotide sequence encoding a scaffold may be called a scaffold coding sequence. As described above, an mRNA molecule may include an immunogen coding sequence comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, the degenerate nucleotide sequence in SEQ ID NO: 8 or SEQ ID NO: 9, or a nucleotide sequence encoding the modified F protein described above and having at least 90 or 95% identity with SEQ ID NO: 8 or 9; a scaffold coding sequence comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10, the degenerate nucleotide sequence in SEQ ID NO: 10, or a nucleotide sequence encoding a lumazine synthase as described above and having at least 90 or 95% identity with SEQ ID NO: 10; and a linker coding sequence comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 11, the degenerate nucleotide sequence in SEQ ID NO: 11, or a nucleotide sequence encoding a linker of a length suitable for use herein and having at least 90 or 95% identity with SEQ ID NO: 11.

[0139] The nucleotide sequence encoding the fusion protein of SEQ ID NO: 6 is provided as SEQ ID NO: 13, and the nucleotide sequence encoding the fusion protein of SEQ ID NO: 7 is provided as SEQ ID NO: 14. In the mRNA molecules provided herein, the fusion protein may be encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, a nucleotide sequence that is degenerate in SEQ ID NO: 13 or SEQ ID NO: 14, or a nucleotide sequence that encodes the fusion protein described herein and has at least 90 or 95% identity with SEQ ID NO: 13 or SEQ ID NO: 14. In certain examples, the fusion protein is encoded by SEQ ID NO: 13 or SEQ ID NO: 14. The nucleotide sequence encoding the fusion protein in an mRNA molecule may be called a fusion protein coding sequence or a nanoparticle subunit coding sequence. Thus, the mRNA molecules provided herein may contain or consist of a nucleotide sequence that encodes the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, a nucleotide sequence that is degenerate in SEQ ID NO: 13 or SEQ ID NO: 14, or a nanoparticle subunit coding sequence that encodes the nanoparticle subunit described herein and has at least 90 or 95% identity with SEQ ID NO: 13 or SEQ ID NO: 14.

[0140] In certain examples, the mRNA molecules provided herein contain or consist of a nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18, or a nucleotide sequence having at least 90 or 95% identity thereto, which encodes the fusion protein described above. SEQ ID NO: 17 contains the 5'UTR of SEQ ID NO: 15, the fusion protein gene of SEQ ID NO: 13, and the 3'UTR of SEQ ID NO: 16, from 5' to 3'. SEQ ID NO: 18 contains the 5'UTR of SEQ ID NO: 15, the fusion protein gene of SEQ ID NO: 14, and the 3'UTR of SEQ ID NO: 16, from 5' to 3'.

[0141] In certain examples, the mRNA molecules provided herein contain or consist of the nucleotide sequence shown in SEQ ID NO: 17.

[0142] In another specific example, the mRNA molecule provided herein contains or consists of the nucleotide sequence shown in Sequence ID No. 18.

[0143] vaccine Vaccines containing the above mRNA molecules, specifically RSV vaccines (i.e., RSV mRNA vaccines), are further provided herein.

[0144] In vaccines, mRNA molecules can be formulated into any suitable delivery vehicle, particularly lipid-based delivery systems such as lipid nanoparticles, liposomes, or lipoplexes. Lipid nanoparticles may be particularly preferred.

[0145] Lipid nanoparticles In the vaccines provided herein, mRNA molecules are generally formulated into lipid nanoparticles. Lipid nanoparticles are essentially spherical particles having a surface containing a lipid layer surrounding a core. The lipid layer may be monolayer or bilayer. The core may be lipophilic, such as a solid lipid matrix, and may be stabilized by a surfactant.

[0146] The characteristics and in vivo behavior of lipid nanoparticles (LNPs) can be modified by adding a hydrophilic polymer coating, such as polyethylene glycol (PEG), to their surface. Furthermore, LNPs can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, or carbohydrates) to their surface or to the ends of the bound PEG chains (Front Pharmacol. 2015 Dec 1;6:286).

[0147] mRNA molecules formulated in lipid nanoparticles are generally encapsulated within the lipid nanoparticles; that is, the mRNA molecule is located in the core of the nanoparticle, completely surrounded by lipids and unable to access the external environment. Within the core, cationic and / or ionizable lipids can organize into inverse micelles around the encapsulated negatively charged mRNA molecule.

[0148] Lipid nanoparticles (LNPs) as used herein may comprise cationic lipids and flocculation reducers (such as PEGylated lipids, also known herein as polyethylene glycol (PEG)-modified lipids or PEG-lipids), as well as optionally non-cationic lipids (such as neutral lipids) and / or sterols. Generally, LNPs comprise cationic lipids, non-cationic lipids, PEG-lipids, and sterols.

[0149] LNPs may contain any cationic lipid suitable for forming lipid nanoparticles. Cationic lipids may have a net positive charge at physiological pH. Cationic lipids may contain fatty acid chains of any suitable length, e.g., C10, C12, or C14-C22, or C24. Fatty acid chains may be saturated, monounsaturated, or polyunsaturated (as used herein to mean containing two or more carbon-carbon double bonds).

[0150] Cationic lipids can be aminolipids. As used herein, the term "aminolipid" means a lipid having one or two fatty acids or fatty alkyl chains and an amino head group (including an alkylamino group or dialkylamino group) that can be protonated at physiological pH to form a cationic lipid.

[0151] Suitable aminolipids include those having alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). Generally, aminolipids with fewer saturated acyl chains are easier to size, especially when the complex must be less than approximately 0.3 microns in size for filter sterilization purposes. Aminolipids containing unsaturated fatty acids with carbon chain lengths in the C14-C22 range may also be used. Other scaffolds can also be used to separate the amino groups from the fatty acid or fatty alkyl moieties of the aminolipids.

[0152] In some cases, amino or cationic lipids have at least one protonable or deprotonable group (sometimes called ionizable lipids) such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, the addition or removal of protons as a function of pH is an equilibrium process, and references to charged or neutral lipids refer to the properties of the main species, and it will be understood that not all lipids are required to exist in either a charged or neutral form. Lipids having two or more protonable or deprotonable groups, or being zwitterionic, are not excluded from use in this invention.

[0153] In some examples, protonable lipids have a pKa of protonable groups ranging from about 4 to about 11, for example, about 5 to about 7.

[0154] LNPs may contain two or more cationic lipids. Cationic lipids may be selected to contribute to different advantageous properties. For example, cationic lipids with different properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in LNPs. In particular, cationic lipids may be selected so that the properties of the mixed LNP are more desirable than the properties of the individual lipids in a single LNP. In some examples, the ratio of cationic lipids to nucleic acids in LNPs is about 3 to about 15, for example, about 5 to about 13 or about 7 to about 11.

[0155] Noncationic lipids may be neutral lipids, anionic lipids, or amphiphilic lipids. Neutral lipids may be any of several lipid species that, if present, exist in an uncharged or neutral zwitterionic form at physiological pH. The selection of neutral lipids for use in the particles described herein is generally guided by considering, for example, LNP size and the stability of LNPs in blood flow.

[0156] Neutral lipids may contain fatty acid chains of any preferred length, for example, C10, C12, or C14-C22 or C24. The fatty acid chains may be saturated, monounsaturated, or polyunsaturated. In some examples, neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10-C20. In other examples, neutral lipids containing monounsaturated or diunsaturated fatty acids with carbon chain lengths in the range of C10-C20 are used. Furthermore, neutral lipids containing a mixture of saturated and unsaturated fatty acid chains can be used.

[0157] Amphiphilic lipids are lipids in which the hydrophobic portion of the lipid material is oriented towards the hydrophobic phase and the hydrophilic portion is oriented towards the aqueous phase. Examples of such compounds include, but are not limited to, phospholipids, aminolipids, sphingolipids, sphingoglycolipids, diacylglycerols, and β-acyloxy acids.

[0158] Sterols are generally cholesterol.

[0159] Aggregation reducers can be lipids that can reduce aggregation. Examples of such lipids include, but are not limited to, polyethylene glycol (PEG) modified lipids, monosialogangliosides Gm1, and polyamide oligomers (PAOs). Other compounds having uncharged hydrophilic steric barrier moieties that prevent aggregation in formulations, such as PEG, Gm1, or ATTA, can also be coupled to lipids.

[0160] The composition of LNPs can be influenced, in particular, by biophysical parameters such as the selection of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and their sizes.

[0161] In some examples, LNPs include cationic lipids, non-cationic lipids, PEG-lipids, and cholesterol.

[0162] In some cases, LNPs may contain approximately 35–45%, 40–50%, 40–60%, 50–60%, or 55–65% cationic lipids on a molar basis (i.e., mol%). In some cases, cationic lipids are present in a ratio of approximately 50 mol% to approximately 60, 65, 70, 75, 80, 85, 90, or 95 mol% of the total lipids present in the LNP, for example, approximately 60 mol% to approximately 70, 75, 80, 85, 90, or 95 mol% of the total lipids present in the LNP, or approximately 70 mol% to approximately 80, 85, 90, or 95 mol% of the total lipids present in the LNP. Cationic lipids may be present in a ratio of approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mol% of the total lipids present in the LNP.

[0163] In some examples, LNPs may contain approximately 1–25%, 1–20%, 1–15%, 1–10%, 3–25%, 3–20%, 3–15%, 3–10%, 5–25%, 5–20%, 5–15%, or 5–10% noncationic lipids on a molar basis (i.e., mol%). In some examples, noncationic lipids may be present in a ratio of approximately 5 mol%–90 mol%, 50 mol%, 40 mol%, 30 mol%, 20 mol%, 15 mol%, or approximately 10 mol% of the total lipids present in the LNP, for example, approximately 10 mol%–90 mol%, 50 mol%, 40 mol%, 30 mol%, or 20 mol% of the total lipids present in the LNP. Noncationic lipids may be present in a ratio of approximately 5, 10, 15, 20, 25, 30, 35, or 40 mol% of the total lipids present in the LNP.

[0164] In some examples, the molar or weight ratio of lipids to mRNA in nanoparticles ranges from approximately 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, or at least 30:1.

[0165] In some examples, LNPs may contain approximately 0.1–5% of an anti-aggregation agent (e.g., PEG-lipid) on a molar basis (i.e., mol%), such as 0.1–4 mol%, 0.1–3 mol%, 0.1–2 mol%, 0.1–1 mol%, 0.5–5 mol%, 0.5–4 mol%, 0.5–3 mol%, 0.5–2 mol%, 0.5–1 mol%, 1–5 mol%, 1–4 mol%, 1–3 mol%, or 1–2 mol% of an anti-aggregation agent.

[0166] The average molecular weight of the PEG portion in PEG-modified lipids can range from approximately 1000 to 8000 daltons (e.g., approximately 1000 to 4000 daltons). In some examples, the average molecular weight of the PEG portion is approximately 2000 daltons.

[0167] In some cases, LNPs may contain approximately 10–70 mol% sterols (e.g., cholesterol), or, for example, approximately 20–60 mol% or 30–50 mol% sterols.

[0168] In some examples, LNPs may contain, by weight, approximately 40–60% cationic lipids, 5–15% non-cationic lipids, 1–2% PEG-lipids, and 30–50% cholesterol.

[0169] In some examples, LNPs have median diameter sizes of approximately 50-300 nm, for example, approximately 50-250 nm, 50-200 nm, 50-150 nm, 100-300 nm, 100-250 nm, 100-200 nm, or 150-300 nm.

[0170] Alternatively, nucleic acids are approximately 1-100 nm, for example, 1-10 nm, 1-20 nm, 1-30 nm, 1-40 nm, 1-50 nm, 1-60 nm, 1-70 nm, 1-80 nm, 1-90 nm, 5-100 nm, 5-10 nm, 5-20 nm, 5-30 nm, 5-40 nm, 5-50 nm, 5-60 nm, 5-70 nm, 5-80 nm, 5-90 nm, 10-50 nm, 20-50 nm, 30-50 nm, 40-50 nm, They can be delivered using smaller LNPs that may include median diameters of 20-60nm, 30-60nm, 40-60nm, 20-70nm, 30-70nm, 40-70nm, 50-70nm, 60-70nm, 20-80nm, 30-80nm, 40-80nm, 50-80nm, 60-80nm, 20-90nm, 30-90nm, 40-90nm, 50-90nm, 60-90nm and / or 70-90nm.

[0171] Alternatively, nucleic acids can be delivered using larger LNPs having median diameters greater than 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm.

[0172] In other examples, LNPs have a single-mode particle size distribution (i.e., they are not bimodal or polymodal).

[0173] In addition to the above, LNPs may further contain one or more lipids and / or other components. Other lipids may be included in the LNP composition for various purposes, such as preventing lipid oxidation or binding ligands to the LNP surface. Any of several lipids, including amphiphilic lipids, neutral lipids, cationic lipids, and anionic lipids, may be present in the LNP. Such lipids may be used alone or in combination. Additional components that may be present in the LNP include polyamide oligomers (see, for example, U.S. Patent No. 6,320,017, which incorporates the whole by reference), peptides, proteins, and bilayer stabilizing components such as detergents.

[0174] Liposomes mRNA molecules can be formulated into liposomes in vaccines. Cationic lipid-based liposomes can be complexed with negatively charged nucleic acids (e.g., mRNA) via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical applications. The liposomes can fuse with the plasma membrane for uptake, and once inside the cell, the liposomes are processed via the endocytosis pathway, after which the nucleic acids are released from the endosome / carrier into the cytoplasm.

[0175] Liposomes typically consist of a lipid bilayer that may be composed of cationic, anionic, or neutral (phospho) lipids and cholesterol, encapsulating an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. Liposomes may have one or more lipid membranes. Liposomes can be monolayered (called unilamellae) or multilayered (called multilamellae).

[0176] The characteristics and in vivo behavior of liposomes can be modified by adding hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the liposome surface to confer steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the bound PEG chains.

[0177] Liposomes can be of various types and sizes, ranging from small unicellular vesicles (SUVs), which may have a diameter of less than 100 nm, to giant unicellular vesicles (GUVs), which may have a diameter exceeding a micrometer. Large unilamellar vesicles (LUVs), with a diameter of 100–1000 nm, may also be used. Other suitable species of liposomes include multilamellar vesicles (MLVs), which may have a diameter exceeding several hundred nanometers or 1 micron. MLVs contain a series of concentric bilayers separated by narrow aqueous compartments. Multivesicular liposomes (MVLs) are those in which multiple smaller non-concentric vesicles are encapsulated within a single large vesicle, which may have a diameter exceeding several hundred nanometers or 1 micron. Liposome designs may contain opsonins or ligands to improve the binding of liposomes to specific tissues or to activate events such as endocytosis (but are not limited to these). Liposomes may contain low or high pH to improve the delivery of pharmaceutical formulations.

[0178] Lipoplex Alternatively, mRNA molecules can be formulated as lipoplexes, i.e., cationic lipid bilayers sandwiched between nucleic acid layers. Such cationic lipids can form complexes or lipoplexes with negatively charged nucleic acids to create nanoparticles through electrostatic interactions.

[0179] Vaccine compositions and their administration The vaccines provided herein may be provided in liquid form or in lyophilized form for reconstitution in liquid. The vaccine components (e.g., mRNA molecules formulated optionally with the lipid nanoparticles mentioned above) may be composed (or reconstituted) in an aqueous solution of a suitable buffer, particularly one suitable for injection, such as Ringer's lactate solution, Ringer's solution, or phosphate buffer.

[0180] Buffers suitable for injection can be used as a carrier in a vaccine or simply to resuspend the vaccine or vaccine components. Such buffers suitable for injection may contain salts selected from, for example, sodium chloride (NaCl), calcium chloride (CaCl), or potassium chloride (KCl), with additional anions present in addition to the chlorides. The injection buffer may be hypertonic, isotonic, or hypotonic with respect to the relevant body fluid, such as blood, lymph, or cytoplasm. A suitable concentration may be selected so as not to cause cell damage due to osmosis or other concentration effects.

[0181] Furthermore, the liquid vaccine composition may contain one or more of the following: polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetates, citrates or phosphates; and agents for adjusting tonicity such as dextrose. Parenteral formulations may be sealed in glass or plastic ampoules, disposable syringes or multi-dose vials. The vaccine composition is preferably sterile.

[0182] The selection of a pharmaceutically acceptable carrier or diluent for a vaccine is, in principle, determined by the mode of administration. Vaccines can be administered systemically or topically, generally topically. Routes for systemic administration include, for example, transdermal, oral, and parenteral routes, including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injections and / or intranasal routes. Routes for topical administration include, for example, topical, intradermal, transdermal, subcutaneous, and intramuscular injections. In certain cases, vaccines are administered intramuscularly. Intramuscular administration is generally by injection, which may be via a needle or needleless.

[0183] Vaccine compositions may include adjuvants. Adjuvants may be used to enhance the immunostimulatory properties of the vaccines provided herein. In this context, an adjuvant can be understood as any compound that, when administered to a subject, initiates or increases an immune response by the innate immune system, i.e., a nonspecific immune response. In other words, the vaccines provided herein, when administered, initiate an adaptive immune response to an immunogen encoded by an mRNA molecule. Furthermore, vaccines may generate a (supportive) innate immune response by adding adjuvants as defined herein to the vaccine.

[0184] In other examples, vaccines may not contain adjuvants or dedicated adjuvant components; for instance, a vaccine may contain an mRNA molecule carrier that also functions as an adjuvant. For example, since lipid nanoparticles can have immunostimulatory or adjuvant effects, a vaccine containing mRNA molecules formulated on LNPs may utilize the intrinsic adjuvant activity of the LNPs and may not contain additional adjuvant components.

[0185] Treatment method The mRNA molecules or vaccines provided herein may be used in therapeutic, particularly in the form of vaccination. Therefore, the above mRNA molecules or vaccines for use as therapeutic agents are provided herein. In particular, mRNA molecules or vaccines are provided for use as prophylactic agents. “Therapeutic agents” as used herein are agents used to treat or prevent a disease or condition, and “prophylactic agents” as used herein are agents used in medicine for the prevention of a disease or condition. Specifically, the mRNA molecules or vaccines provided herein may be used to prevent diseases caused by RSV infection.

[0186] Prevention of disease caused by RSV infection is broadly defined herein as reducing the likelihood that an individual will develop disease caused by RSV infection, or, if an individual develops disease caused by RSV infection, reducing (i.e., attenuating) the severity of that disease. Disease caused by RSV infection may be less severe if, for example, the individual is less likely to die, be hospitalized, require intensive care, or require mechanical ventilation; if the individual has milder symptoms with reduced impact on the individual's daily life, for example, if the individual is less likely to be bedridden and has a high capacity to care for themselves or others, work, etc.; or if the individual recovers more quickly than they would have if they were unvaccinated and / or compared to an equivalent unvaccinated individual, for example, requiring a shorter hospital stay, intensive care, mechanical ventilation, etc. An equivalent unvaccinated individual is an individual of similar size and age, of the same sex, and in similar overall health.

[0187] When used herein, prophylactic agents may be administered to individuals at particular risk of developing disease caused by RSV infection, or more generally, to individuals who are severely ill with disease caused by RSV infection. For example, an individual may be at high risk of exposure to RSV or at risk of developing severe disease if infected with RSV due to, for example, age and / or other health conditions. For example, prophylactic agents may be administered as part of a routine or regular schedule of immunization for individuals of certain ages, for example, older adults over 50, 60, or 70 years of age. Prophylactic agents may be administered repeatedly, for example, annually, to enhance protection against RSV. For example, RSV vaccines may be administered annually or every two, three, four, or five years or more, as needed, to individuals at risk, such as the elderly. Such a reduction in the risk of developing disease caused by RSV infection, or severely ill with disease caused by RSV infection, may be, for example, at least 50, 60, 70, 80, or 90% compared to an unvaccinated individual.

[0188] In particular, mRNA molecules or vaccines for use in a manner that prevents and / or attenuates RSV infection in subjects are provided herein. “RSV infection” is the disease caused by RSV infection. mRNA molecules or vaccines for use according to this embodiment may prevent RSV infection. Alternatively, mRNA molecules or vaccines may not prevent RSV infection but may prevent symptomatic disease in the event of infection, or reduce the severity of the disease if symptomatic disease occurs. That is, mRNA molecules or vaccines may reduce the likelihood of subjects becoming infected with RSV, for example, in certain contexts where there is a risk of exposure to the virus, or in daily life, compared to unvaccinated subjects. Similarly, mRNA molecules or vaccines may reduce the risk of subjects developing symptomatic disease caused by RSV, or the risk of subjects developing severe disease (e.g., disease requiring hospitalization) caused by RSV, compared to unvaccinated subjects. Such a reduction in risk may be, for example, at least 50, 60, 70, 80, or 90% compared to unvaccinated subjects.

[0189] In particular, RSV infections that can be prevented or attenuated using mRNA molecules or vaccines provided herein may be lower respiratory tract diseases (LRTDs) caused by RSV. By standard practice in the art, the lower respiratory tract can be defined as the airways below the vocal cords, including the trachea, lungs, bronchi, and bronchioles. Examples of LRTDs caused by RSV include bronchiolitis, bronchitis, and pneumonia, particularly bronchiolitis and pneumonia.

[0190] Those who may be prevented or attenuated from RSV infection, such as LRTD, may be those who are administered mRNA molecules or vaccines. These subjects are generally human patients, e.g., those over 1, 2, 5, 12, 16, 18, or 21 years of age. In particular, subjects may be adults, generally older individuals, e.g., those over 50, 55, 60, 65, or 70 years of age. Alternatively or additionally, subjects may have health conditions that increase their risk of serious illness from RSV infection, such as cancer, immunodeficiency, or respiratory diseases like COPD. In adults and older children (e.g., over 5 years of age or over 2 years of age), the LRTD that may be prevented or attenuated may, in certain cases, be pneumonia.

[0191] In other cases, the subjects may be young children, such as infants, babies, or toddlers, for example, children in the age range of 6 months, 12 months, 18 months, or 24 months from birth. In young children, the LRTD to be prevented or reduced may be bronchiolitis in certain cases.

[0192] Alternatively, the target population for which RSV infection, e.g., LRTD, is prevented or attenuated may not be the same population to which the mRNA molecule or vaccine is administered. In particular, the target population protected by the vaccine (i.e., for which RSV infection is prevented or attenuated) may be the infant of a woman who was administered the mRNA molecule or vaccine before giving birth. In particular, the target population to which the mRNA molecule or vaccine is administered may be women, e.g., 20–38 weeks of gestation, e.g., 22–36 weeks, 24–36 weeks, 24–34 weeks, 24–32 weeks, 26–36 weeks, 26–34 weeks, or 26–32 weeks of gestation. When pregnant women are vaccinated in this way, anti-RSV antibodies may be passed from the mother to the child before birth through the placenta, providing passive protection against RSV infection and disease during the first few months of the child's life. Such protection may be effective for at least, for example, 1, 2, 3, 4, 5, or 6 months after the child's birth.

[0193] The embodiments described above may be considered, alternatively, to provide a method for preventing or attenuating RSV infection, in particular LRTD caused by RSV, in subjects. As detailed above, such a method may include administering the mRNA molecule or vaccine provided herein to the subject. Alternatively, if the subject is an infant, the method may include administering the mRNA molecule or vaccine to the mother of the infant during pregnancy, as detailed above. Similarly, the embodiments described above may be considered, alternatively, to provide the use of the above mRNA molecule or vaccine in the manufacture of a pharmaceutical product for use in preventing or attenuating disease caused by RSV infection, in particular LRTD, in subjects. The subjects may be as described above.

[0194] More broadly, the foregoing provides a method for inducing an immune response in a subject, comprising administering the above-mentioned mRNA molecule or vaccine to the subject. This aspect may be considered, alternatively, as providing the above-mentioned mRNA molecule or vaccine for use in a method for inducing an immune response in a subject, or providing the use of the above-mentioned mRNA molecule or vaccine in the manufacture of a pharmaceutical product for inducing an immune response in a subject. In this aspect, the immune response is an immune response to RSV, specifically to the RSV F protein, and in particular to the pre-fusion conformation of the F protein.

[0195] Such specific immune responses may involve cellular (i.e., T cell) responses and / or humoral (i.e., antibody) responses. Generally, the immune response includes at least an antibody response against the RSV F protein; that is, the subject is induced to produce antibodies against the RSV F protein, particularly against pre-F RSV F. The antibodies produced by the subject may include neutralizing antibodies against the RSV F protein.

[0196] Antibody responses induced by mRNA molecules or vaccines can be identified and / or measured by ELISA (enzyme-linked immunosorbent assay). The size of the antibody response can be measured, for example, by ELISA, based on antibody titer. Antibody titer is a measure of the amount of antibody in a subject, e.g., antibody specific to a particular antigen (e.g., RSV F protein) or an epitope of the antigen. Antibody titer is typically expressed as the reciprocal of the maximum dilution that yields a positive result.

[0197] In some cases, the induction of an immune response results in an antibody titer in a subject that is at least an order of magnitude higher than the titer before vaccination (i.e., compared to the titer in the same individual before vaccination). For example, the titer may increase by at least 1.5, 2, 2.5, or 3 orders of magnitude compared to the titer before vaccination. In other cases, the induction of an immune response results in an antibody titer in a subject that is at least twofold higher than the titer before vaccination. For example, the titer may increase by at least 3, 4, 5, 6, 7, 8, or 9fold compared to the titer before vaccination.

[0198] Antibody titers may measure the total amount of all antibodies against a specific antigen in a subject, or they may measure only a specific type of antibody, such as a neutralizing antibody. In some cases, an increase in titer may be an increase in the titer of a neutralizing antibody against RSV. The titer of a neutralizing antibody against viruses such as RSV can be determined by a neutralization assay. For example, the titer of a neutralizing antibody against RSV can be determined by an RSV neutralization assay. A reliable neutralization assay is the plaque reduction neutralization assay (PRNT), in which a heat-inactivated serum of interest is applied to human cells along with the live virus. Viral infection of cells causes plaque formation in a cell culture dish, and the neutralizing antibody titer can be calculated by the reduction of the plaque. The RSV neutralization assay is described in detail in Raghunandan et al., Vaccine 39(33):4591-4597, 2021, which is incorporated herein by reference.

[0199] The induced immune response may be long-lasting; for example, increased antibody titers or neutralizing antibody titers may persist for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 18 months after vaccination.

[0200] The induced immune response may include a memory response, i.e., a memory B cell response to RSV may be generated. Memory B cells can be identified, for example, by staining PBMCs with a fluorescently labeled antigen (e.g., RSV F protein) and then identifying antigen-specific memory B cells by flow cytometry based on the binding of the fluorescently labeled antigen and the expression of a memory B cell marker (e.g., CD27).

[0201] Vectors and cells Vectors encoding mRNA molecules provided herein are also provided herein. Such vectors are DNA vectors and may be, for example, cloning vectors or expression vectors.

[0202] As used herein, “expression vector” is a DNA molecule used for the expression of foreign genetic material in cells or in vitro systems. Any suitable vector known in the art may be used. Suitable vectors include DNA plasmids, binary vectors, viral vectors, and artificial chromosomes (e.g., yeast artificial chromosomes). Expression vectors may be circular or linear. An expression vector comprises an expression cassette on which the mRNA molecule provided herein can be expressed.

[0203] As used herein, an expression cassette is a polynucleotide sequence capable of transcribing an expression product. Typically, an expression cassette includes a promoter operably ligated to a sequence encoding an mRNA molecule. In this context, the term "operably ligated" means that the mRNA molecule sequence and the promoter are covalently bonded so that the expression of the mRNA molecule is under the influence or control of the promoter. Therefore, a promoter is operably ligated to an mRNA sequence if the promoter is capable of transcribing the mRNA sequence to produce the mRNA molecule as described above.

[0204] Any suitable promoter known in the art may be used in the expression cassette to provide its function in the cell type being used. For example, if the cell is a mammalian cell, the promoter may be a cytomegalovirus (CMV) promoter. If the expression cassette is expressed in vitro, the promoter may be a phage promoter, such as an SP6, T7, or T3 promoter. The promoters used in the expression cassettes provided herein may be constitutive or inductive promoters.

[0205] The expression cassette may include additional elements useful for regulating transcription, such as one or more enhancer sequences and one or more transcription termination (terminator) sequences.

[0206] The vector may further contain other standard sequence elements, such as selection markers that encode, for example, antibiotic resistance genes.

[0207] Cells containing the vectors provided herein are also provided herein. Such cells may, for example, be cloning hosts. Suitable cells include prokaryotic cells, such as bacterial cells, and eukaryotic cells, such as mammalian cells. Suitable bacterial cells include, for example, Escherichia coli (E. coli) cells or Bacillus subtilis (B. subtilis) cells.

[0208] mRNA production The mRNA molecules provided herein can be prepared by any preferred method. mRNA can be expressed in cells, such as mammalian cells, particularly human cells, and purified from them. Generally, mRNA molecules are prepared in vitro, particularly by in vitro transcription. mRNA molecules can be transcribed from the expression vectors described above.

[0209] Therefore, a method for producing mRNA molecules provided herein, comprising expressing mRNA from an expression vector provided herein, is provided herein. Generally, mRNA is expressed by in vitro transcription.

[0210] Expression vectors used for in vitro transcription are generally linear, such as linearized plasmids. Transcription can be carried out using phage RNA polymerases, such as T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase. The expression vector contains a promoter recognized by the selected polymerase. In vitro transcription can be carried out using commercially available kits, such as the HiScribe® kit from New England Biolabs (NEB, USA) or the RiboMAX® kit from Promega (USA), according to the manufacturer's instructions.

[0211] Protein nanoparticles Protein nanoparticles containing the above-described nanoparticle subunits are also provided herein. Protein nanoparticles may be as described above in the context of nanoparticle subunits encoded by mRNA molecules.

[0212] Nanoparticle subunits can be expressed from expression vectors, as described above. Such expression vectors can be introduced into host-producing cells, such as mammalian cells like human cells, and the proteins expressed therefrom. Nanoparticle subunits can be expressed with signal peptides so that they are released from cells and multimers form extracellularly; in this case, the protein nanoparticles can be isolated from the culture supernatant. Otherwise, multimers can form intracellularly; in this case, the cells can be lysed to allow for the isolation of the protein nanoparticles. Cell lysis can be carried out by any method known in the art, such as mechanical lysis using a French press, sonication, or chemical lysis using a solvent.

[0213] The isolation and purification of protein nanoparticles can be carried out using standard methods in the art, for example, fusion proteins may be expressed with affinity tags (such as His tags) to enable affinity purification.

[0214] The protein nanoparticles provided herein may be administered to a target as a protein vaccine as described above.

[0215] Therefore, in some examples, the present disclosure provides recombinant protein nanoparticles comprising 60 nanoparticle subunits described herein that self-assemble into nanoparticles. In some examples, when self-assembled, the nanoparticle subunits do not contain signal peptides.

[0216] Sequence identity and variants Sequence identity is generally defined with reference to the GAP algorithm (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP aligns two complete sequences using the Needleman and Wunsch algorithms to maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap generation penalty of 12 and a gap expansion penalty of 4. While the use of GAP may be preferred, other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990)), FASTA (using the method of Pearson and Lipman (1988)), or the Smith-Waterman algorithm (Smith and Waterman (1981)), the aforementioned TBLASTN program by Altschul et al. (1990), or Emboss Needle (Madeira et al, Nucleic Acids Research 50(W1):W276-W279), generally using default parameters.

[0217] Where this disclosure refers to a particular amino acid or nucleotide sequence having at least 90% sequence identity with a reference amino acid or nucleotide sequence, this includes amino acid or nucleotide sequences (rounded to the nearest integer percentage) having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a reference amino acid or nucleotide sequence.

[0218] If an amino acid or nucleotide sequence has less than 100% identity with a reference sequence (e.g., about 90 or 95% identity), then that amino acid or nucleotide sequence contains one or more sequence changes compared to the reference sequence. As used herein, the term “sequence change” includes substitutions, deletions, and / or insertions of amino acid residues or nucleotides.

[0219] Therefore, a protein containing one or more amino acid sequence changes compared to a reference sequence contains one or more substitutions, one or more deletions, and / or one or more insertions of amino acid residues compared to the reference sequence. With respect to peptide or polypeptide sequences, the term “amino acid mutation” is used herein interchangeably with “sequence change” unless otherwise clearly specified in the context.

[0220] In some cases where one or more amino acids are substituted for other amino acids, the substitutions may be conservative substitutions, for example, according to the table below. In some cases, amino acids in the same block in the middle column are substituted, i.e., a nonpolar amino acid is substituted for another nonpolar amino acid. In some cases, amino acids in the same row in the rightmost column are substituted, i.e., G is substituted for A or P.

[0221] TIFF2026524691000006.tif5994

[0222] In some cases, substitutions may be functionally conserved; that is, in some cases, substitutions may not affect (or substantially affect) one or more functional properties of the protein containing the substitution compared to an equivalent non-substituted protein.

[0223] Similarly, a nucleic acid molecule (e.g., an RNA molecule) containing one or more nucleotide sequence changes compared to a reference sequence contains one or more nucleotide substitutions, one or more deletions, and / or one or more nucleotide insertions compared to the reference sequence. With respect to nucleic acid sequences, the term “nucleotide mutation” is used herein interchangeably with “sequence change” unless otherwise clearly specified in the context.

[0224] If a nucleic acid (e.g., RNA) sequence contains one or more sequence changes, these changes may not (or substantially) affect the functional properties of the nucleic acid. If the nucleic acid sequence is a protein-coding sequence, whether the functional properties of the sequence are affected by the changes depends on whether the function or expression of the encoded protein is affected. If the nucleic acid sequence is a non-coding nucleotide sequence, the effect of the sequence changes on the function of the non-coding sequence is evaluated.

[0225] Nucleotide sequences can also be defined by degeneracy with respect to a reference sequence. As a result of genetic code degeneracy, there are many nucleotide sequences that can encode any given amino acid sequence. Specifically, a degenerate nucleotide sequence refers to two (or more) nucleotide sequences that encode the same peptide or polypeptide (or amino acid sequence) in the open reading frame of a reference nucleotide sequence starting at position 1 (i.e., codon 1 of the coding sequence corresponds to positions 1-3 of the reference nucleotide sequence). Therefore, for example, the nucleotide sequence that degenerates at SEQ ID NO: 8 is different from SEQ ID NO: 8, but due to genetic code degeneracy, it encodes the same protein sequence as SEQ ID NO: 8 (i.e., the modified RSV F protein of SEQ ID NO: 1). ***

[0226] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, are expressed in their specific forms, or in relation to means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, and may be used separately or in any combination of such features as needed.

[0227] While illustrative examples are provided above and below, many equivalent modifications and variations will be apparent to those skilled in the art. Therefore, the illustrative examples provided above and below are illustrative and not limiting. Various modifications to the examples described may be made without departing from the spirit and scope of this disclosure.

[0228] To avoid misunderstanding, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0229] Any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0230] Throughout this Spec., including the following claims, unless otherwise required by context, the words “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” are understood to mean that they include the integer or process or group of integers or processes described, but not any other integer or process or group of integers or processes.

[0231] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value to and / or “about” another particular value. Where such ranges are expressed, another example includes one particular value to and / or another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that a particular value forms another example. The term “about” with respect to numbers is arbitrary and means, for example, + / - 10%. [Examples]

[0232] The objective of the experiments outlined below was to develop an mRNA vaccine against respiratory syncytial virus (RSV) by targeting the fusion (F) protein. Several preclinical and clinical trials have highlighted the importance of inducing an antibody response to the pre-fusion conformation of the F protein, specifically to pre-fusion specific sites 0 and 5. To achieve this, various pre-fusion-stabilized fusion protein constructs presented on self-assembling protein nanoparticles ("nanoparticles") were constructed. Several nanoparticle scaffolds were evaluated, including ferritin, a 24-mer derived from Helicobacter pylori; lumazine synthase (LuS), a 60-mer derived from Aquifex aeolicus; and β-annulus (Bann), a 180-mer derived from tomato bushy stunt virus. It was hypothesized that increased antigen density could occlude the antigenic site near the center of the F trimer, while still exposing the apex of the trimer where site 0 resides.

[0233] Example 1: Preparation of plasmid DNA expressing pre-RSV fusion F, which is presented on protein nanoparticles and expressed in HEK293 cells. method Pre-fusion F cloning Several pre-fusion F sequences (sequence numbers in Table 1) were cloned into pCAG DNA expression vectors. The protein sequences of the pre-fusion stabilized F constructs (Table 2) were codon-optimized and genetically linked to a panel of self-assembling scaffolds at their C-terminuses. The sequences used to link pre-fusion F to the scaffolds are listed in Table 3. Cloning was performed using Gibson assembly.

[0234] [Table 1]

[0235] [Table 2]

[0236] [Table 3]

[0237] HEK293 cell transfection HEK293 cells were grown in T175 flasks in complete medium (DMEM supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using trypsin at 37°C for 5 minutes and seeded at 300,000 cells / well in 24-well plates on day 1. On day 0, cells were transfected with pDNA encoding the RSV-F linker scaffold antigen (500 ng / well) using Lipofectamine 2000 according to the manufacturer's protocol (Thermo Fisher Scientific, USA). On day 3, the supernatant was collected and evaluated by ELISA for RSV-F nanoparticle production.

[0238] Sandwich ELISA The supernatant from transfected cells was analyzed by sandwich ELISA. Briefly, 384-well ELISA plates were coated overnight at 4°C with a panel of 3 μg / ml monoclonal antibodies (1G7, AM14, MPE8, palivizumab, 101F, and M43). The plates were washed and incubated with diluted cell supernatant at room temperature for 1 hour. The plates were then washed and nanoparticles were detected with mAB 133-1H, a mouse antibody that recognizes both pre- and post-fusion conformations of the F protein. The plates were washed and incubated with anti-mouse IgG-HRP antibody. The plates were washed and incubated with TMB substrate for 5 minutes, the color development was stopped with 2N H2SO4, and the plates were read at a wavelength of 450 nm using Envision.

[0239] result To characterize the antigen sites maintained on the nanoparticles, HEK293 cells were transfected, and the supernatants were evaluated by sandwich ELISA using a panel of capture antibodies that recognize various antigen sites on RSV-F. Supernatants from cells transfected with DS-CAV1 presented on ferritin, lumazine synthase (LuS), and β-annulus (Bann) all produced nanoparticles captured by pre-fusion specific mAB 1G7, indicating that site 0 was maintained and that the RSV-F protein presented on VLPs was in the pre-fusion conformation (Figure 1). Furthermore, both DS-CAV1-ferritin and DS-CAV1-LuS generated nanoparticles that could be captured using AM14 and MPE8, which recognize quaternary structure epitopes spanning multiple protomers. These results suggest that the nanoparticles form trimers that are properly folded in the pre-fusion conformation. The supernatant from cells transfected with DS-CAV1-Bann did not produce particles that could be bound by AM14, but it did produce particles that could bind to MPE8 (Figure 1). Antigen density was highest with Bann VLP (180-mer).

[0240] DS-CAV1-ferritin and DS-CAV1-LuS particles could be captured by palivizumab but not by mAB 101F (Figure 1). These results suggest that the binding site of 101F can be occluded on these particles as well. Similarly, DS-CAV1-Bann nanoparticles could not bind to 101F and had very low binding to palivizumab. Post-fusion specific mAB M43 could bind to DS-CAV1-ferritin and DS-CAV1-LuS particles but not to DS-CAV1-Bann nanoparticles. Overall, these results indicate that RSV-F can be presented on nanoparticles, and that antigen density on the surface can occlude specific antigen sites, but all nanoparticles maintained accessibility to site 0.

[0241] Example 2: DS2 immunogen induces high-titer neutralizing antibodies in naive mice. Mice were immunized with a panel of mRNAs encoding pre-fusion stabilization constructs linked to different scaffolds, and immunogenicity was evaluated by ELISA binding titer and neutralization assay.

[0242] method mRNA synthesis using in vitro transcription (IVT) Sequences encoding soluble DS-CAV1, membrane DS-Cav1, and DS2 nanoparticle subunits were cloned into mRNA vectors encoding the T7 promoter, 5' and 3' UTR, and poly(A) tail. Template plasmids were linearized by digesting pDNA with BSPQ1 (NEB, USA) at 50°C for 4 hours. mRNA was synthesized by in vitro transcription using NEB T7 HighScribe, as well as N1-methyl-pseuduridine and CleanCap-Ag (Trilink). The IVT reaction mixture was incubated at 37°C for 5 hours and then digested with RNase-free DNase. mRNA was then purified using LiCl precipitation for RNA purification. All mRNA was formulated into AZ lipid nanoparticles (LNPs) in 20 mM Tris / Tris-HCl, pH 7.4, 8% w / v sucrose, and then stored at -80°C.

[0243] Mouse immunization The mouse study was approved by the Animal Experimentation Committee of AstraZeneca. A group of six female BalB / c mice, 5–7 weeks old, were immunized with mRNA vaccine. The LNP preparation was diluted in PBS according to the indicated dose. Mice were subcutaneously immunized with 50 μl of diluted LNP on days 0 and 28. Blood samples were collected from the mice on days 14 and 42 to evaluate the immune response.

[0244] Binding ELISA ELISA was performed to evaluate pre- and post-fusion antibody binding titers in immunized mice. 384-well plates were coated overnight at 4°C with 3 μg / ml purified recombinant pre- or post-fusion F protein. The plates were then washed with PBS-T and blocked at room temperature for 1 hour in PBS supplemented with 5% milk powder and 3% BSA. Mouse serum was then diluted with blocking buffer and incubated with the coated plates at room temperature for 1 hour. The plates were then washed and incubated with HRP-conjugated anti-mouse IgG secondary antibody for 1 hour. The plates were washed and incubated with TMB substrate for 5 minutes, stopping the color development with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0245] RSV neutralization assay Serum derived from immunized mice was analyzed for neutralizing activity at the indicated time points. Serum was heat-inactivated and serially diluted in 96-well plates. The diluted serum was then incubated with the indicated virus at 37°C for 1 hour at an MOI of 0.04. Next, 20,000 Hep2 cells were added to the virus / serum mixture, and the plates were incubated at 37°C for 5 days. Cells were fixed with acetone to visualize infection. The cells were then washed and stained with biotinylated mAB 133-1H. The cells were then washed and incubated with strep-HRP antibody. The plates were washed and incubated with TMB substrate for 7 minutes, and color development was stopped with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0246] result DS2 immunogen induces enhanced neutralizing activity. We have previously demonstrated that stabilizing mutations in DS-CAV1 can significantly improve immunogenicity compared to immunization with wild-type fusion protein. Next, we wanted to determine whether further stabilization of the fusion protein could improve immunogenicity. To test this, we immunized mice with the DS2-2 immunogen expressed as mRNA encoding the previously described DS-CAV1 trimer or as the trimer. As a control, mice were also immunized with the pre-fusion stabilizing construct or recombinant DS-CAV1 protein + sigma adjuvant system (SAS). Mice immunized with DS2-2 induced the best RSV-A neutralizing activity, which was increased 11-fold compared to DS-CAV1 mRNA (p-value < 0.05) (Figure 2). Therefore, we then sought to evaluate DS2-2 presented in our panel of nanoparticle scaffolds to determine whether further immunogenicity could be increased.

[0247] DS2 presentation on lumazine synthase improves neutralization activity. mRNA vaccines encoding the DS2-2 immunogen were generated on ferritin, LuS, or Bann VLPs. To evaluate whether additional stabilization modifications present in DS2-2 along with the nanoparticle scaffold could improve immunogenicity, mice were immunized with 2 μg of mRNA on day 0 and day 28. On day 14 after initial immunization, DS2-2-LuS induced a significant increase in RSV-A neutralizing activity compared to DS-Cav1-Fordon (trimer) immunized mice (Figure 3B). By day 42 (two weeks after boost), all groups had similar RSV-A neutralizing titers, with DS2-2-LuS tending toward the highest activity (Figure 3C). However, mice immunized with mRNA-DS2-2-LuS induced a significant increase in RSV-B neutralizing activity compared to mRNA-DS-Cav1-Fordon (Figure 3D). Furthermore, DS2-2 presented on LuS induced superior neutralizing activity compared to DS2-2 presentation on ferritin or Bann (Figure 3D).

[0248] The results of the ELISA-binding antibodies showed a response similar to the neutralization activity (Figure 3E). Mice immunized with mRNA nanoparticles induced a favorable pre-fusion response on day 14. However, the pre-fusion versus post-fusion responses were similar on day 42 (Figure 3E).

[0249] Example 3: Immunization with DS2-2-LuS in animals that have experienced RSV enhances neutralization activity Method RSV infection in mice 5- to 7-week-old BalB / c mice were infected with RSV-A by intranasal inoculation. The mice were infected with 50 μl volume of 10 4 PFU of the virus. The mice were monitored throughout the study.

[0250] [[ID=1:6]]Mouse immunization The mouse study was approved by AstraZeneca's Animal Experimentation Committee. A group of 6 female BalB / c mice, 5- to 7-week-old, were immunized with the mRNA vaccine. According to the indicated dosages, the LNP formulation was diluted with PBS. On day 80, the mice were subcutaneously immunized with 50 μl volume of the diluted LNP. Two weeks later, blood was collected from the mice to evaluate the immune response.

[0251] Binding ELISA ELISA was performed to evaluate the pre-fusion and post-fusion antibody binding titers in immunized mice. 384-well plates were coated overnight at 4°C with 3 μg / ml of purified recombinant pre-fusion or post-fusion F protein. The plates were then washed with PBS-T and blocked for 1 hour at room temperature in PBS supplemented with 5% milk powder and 3% BSA. Serum from the mice was then diluted with the blocking buffer and incubated with the coated plates for 1 hour at room temperature. The plates were then washed and incubated for 1 hour with an HRP-conjugated anti-mouse IgG secondary antibody. The plates were washed and incubated for 5 minutes with TMB substrate, and the color development was stopped with 2N H2SO4. The plates were read on an Envision at a wavelength of 450 nm.

[0252] RSV neutralization assay Serum derived from immunized mice was analyzed for neutralizing activity at the indicated time points. Serum was heat-inactivated and serially diluted in 96-well plates. The diluted serum was then incubated with the indicated virus at 37°C for 1 hour at an MOI of 0.04. Next, 20,000 Hep2 cells were added to the virus / serum mixture, and the plates were incubated at 37°C for 5 days. Cells were fixed with acetone to visualize infection. The cells were then washed and stained with biotinylated mAB 133-1H. The cells were then washed and incubated with strep-HRP antibody. The plates were washed and incubated with TMB substrate for 7 minutes, and color development was stopped with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0253] result To mimic what might happen in patients, the inventors evaluated their mRNA-protein nanoparticle vaccine in mice previously exposed to RSV infection. The mice were given RSV-A(10) on day 0. 4 Mice were infected by intranasal inoculation of PFU and immunized with the inventors' vaccine construct on day 80 (Figure 4A). DS2-2-Bann was not included due to its low activity as measured in Figure 3. Control mRNA expressing transmembrane-bound DS-Cav1 was also included as a benchmark control. Serum was evaluated two weeks after immunization, and a three-fold increase in RSV-A neutralizing activity was observed in the DS2-2-LuS group compared to the transmembrane-bound DS-Cav1 group (Figure 4B). Similarly, a 3.6-fold increase in RSV-B neutralization was observed in mice immunized with DS2-2-LuS.

[0254] Example 4: Modification of the 5'UTR may affect eGFP mRNA expression in A549 cells and HeLa cells. Here, we describe 5'UTR and 3'UTR sequences that enhance the expression of coding sequences, including in the context of mRNA vaccine vectors.

[0255] method UTR cloning Candidate 5'UTR sequences (shown in Table 4 below) were cloned with eGFP and a reference 3'UTR sequence (e.g., albumin used in the CureVac vector described in European Patent No. 2831240). Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'UTR contained a T7 promoter sequence.

[0256] Preparation of in vitro transcription (IVT) templates using PCR IVT templates were prepared by PCR using Phusion PCR Mix (NEB). Upstream primers contained the T7 promoter sequence, and downstream primers contained the T80 sequence along with the reverse complement of the 3'UTR end of each clone. The resulting PCR product contained sequences encoding the relevant sequence elements in the following order: T7 promoter-5'UTR-eGFP-3'UTR-A80. The PCR reaction was then treated with DpnI to digest the template DNA and purified using a PCR purification kit.

[0257] mRNA synthesis using in vitro transcription (IVT) mRNA was prepared using a NEB IVT kit with T7 RNA polymerase, using a template prepared for IVT by PCR. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporated CleanCap® (AG) at the start of each mRNA, giving a Cap1 structure at the 5' end. mRNA was produced using unmodified nucleotides or using modified uridine (5'methoxyuridine) at a ratio of 25% to unmodified uridine. After the reaction was complete, the DNA template was digested with RNase-free DNase. The mRNA transcript contained a 5'Cap1-5'UTR-eGFP coding sequence-3'UTR-A80 structure. The mRNA was then purified using a silica column and resuspended in water.

[0258] Cell transfection Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded. 100,000 cells in 100 μl of medium were seeded into each well (96W plate) the day before. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well.

[0259] HeLa cells were grown in T175 flasks in HeLa complete medium (minimum essential medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded on collagen-treated plates. 100,000 cells in 100 μl of medium were seeded into each well (96W plate, collagen-treated) the day before transfection. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well.

[0260] Quantification of eGFP expression eGFP fluorescence was detected using an incucyte instrument that acquires images from living cells. Fluorescence was measured in relative fluorescence units (RFU) from the images using Incucyte software. The data reported in the figure shows eGFP fluorescence 24 hours after transfection.

[0261] result Table 4 shows the expression levels for candidate 5'-UTRs.

[0262] [Table 4]

[0263] Including candidate 5'UTRs (Table 4) resulted in increased eGFP-coding mRNA expression in A549 cells compared to eGFP expression induced by both control 5'UTR (HSD17B4) and control 3'UTR (albumin) (Figure 5). Including GOT1, PRKACB, CHIT1, or GUSB 5'UTRs corresponded to 4-fold, 3-fold, 3-fold, and 3-fold increases in eGFP expression compared to expression induced by control 5'UTR (HSD17B4), respectively.

[0264] These results were demonstrated in HeLa cells, showing that replacing the control 5'UTR with a candidate 5'UTR (Table 4) increased eGFP-coding mRNA expression (Figure 6). Inclusion of GOT1, PRKACB, CHIT1, or GUSB 5'UTR corresponded to 13, 13, 7, and 14-fold increases in eGFP expression compared to expression with the control 5'UTR (HSD17B4), respectively.

[0265] Example 5: Modification of the 3'UTR may affect eGFP mRNA expression in A549 cells and HeLa cells. method UTR cloning The 3’UTR candidate sequences (shown in Table 5 below) were cloned with eGFP and a reference 5’UTR sequence (e.g., HSD17B4 used in the CureVac vector described in European Patent No. 2831240). Cloning was performed using restriction sites (RE) or seamlessly. The 5’ end of the 5’UTR contained a T7 promoter sequence.

[0266] Preparation of in vitro transcription (IVT) templates using PCR The IVT templates were prepared by PCR using Phusion PCR mix (NEB). The upstream primer contained the T7 promoter sequence, and the downstream primer contained the reverse complement of the end of the 3’UTR of each clone together with the T80 sequence. The resulting PCR products contained sequences encoding the relevant sequence elements in the following order: T7 promoter - 5’UTR - eGFP - 3’UTR - A80. The PCR reaction was then treated with DpnI to digest the template DNA and purified using a PCR purification kit.

[0267] mRNA synthesis using in vitro transcription (IVT) Using the templates prepared for IVT by PCR, mRNA was prepared with the NEB IVT kit using T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, giving a Cap1 structure at the 5’ end. mRNA was produced using unmodified nucleotides or modified uridine (5’-methoxyuridine) at a 25% ratio relative to unmodified uridine. After the reaction was complete, the DNA template was digested with RNase-free DNase. The mRNA transcript contained the 5’Cap1 - 5’UTR - eGFP - 3’UTR - A8 structur. The mRNA was then purified using a silica column and resuspended in water.

[0268] Cell transfection Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded. 100,000 cells in 100 μl of medium were seeded into each well (96W plate) the day before. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well.

[0269] HeLa cells were grown in T175 flasks in HeLa complete medium (minimum essential medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded on collagen-treated plates. 100,000 cells in 100 μl of medium were seeded into each well (96W plate, collagen-treated) the day before transfection. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well.

[0270] Quantification of eGFP expression eGFP fluorescence was detected using an incucyte instrument that acquires images from living cells. Fluorescence was measured in relative fluorescence units (RFU) from the images using Incucyte software. The data reported in the figure shows eGFP fluorescence 24 hours after transfection.

[0271] result Table 5 shows the expression levels for candidate 3'-UTRs.

[0272] [Table 5]

[0273] Addition of candidate 3'UTRs (Table 5) resulted in increased eGFP-coding mRNA expression in A549 cells compared to eGFP expression induced by both control 3'UTR (albumin) and control 5'UTR (HSD17B4) (Figure 7). Addition of CHIT1, CS, or PKLR 3'-UTRs resulted in 3-fold, 3-fold, and 2-fold increases in eGFP expression compared to expression induced by control 3'UTR (albumin), respectively.

[0274] These results were replicated in HeLa cells, showing that replacing the control 3'UTR (albumin) with the candidate 3'UTR (Table 5) increased eGFP expression (Figure 8). Therefore, the addition of CHIT1, CS, or PKLR 3'UTR corresponded to 7-fold, 4-fold, and 3-fold increases in eGFP expression compared to expression with the control 3'UTR (albumin), respectively.

[0275] Example 6: eGFP mRNA expression can be increased by combining a modified 5'UTR with a modified 3'UTR. The combinations of selected 5'UTRs (Table 4) and selected 3'UTRs (Table 5) were evaluated to determine whether the combination approach could further increase eGFP mRNA expression. The following combinations were assayed: GOT1 / CHIT1, GOT1 / CS, PRKACB / CHIT1, PRKACB / CS, CHIT1 / CHIT1, and CHIT1 / CS.

[0276] method UTR cloning The 5'UTR and 3'UTR were cloned as ORFs using eGFP. Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'UTR contained the T7 promoter sequence.

[0277] Preparation of in vitro transcription (IVT) templates using PCR IVT templates were prepared by PCR using Phusion PCR Mix (NEB). Upstream primers contained the T7 promoter sequence, and downstream primers contained the T80 sequence along with the reverse complement of the 3'UTR end of each clone. The resulting PCR product contained sequences encoding the relevant sequence elements in the following order: T7 promoter-5'UTR-ORF-3'UTR-A80. The PCR reaction was then treated with DpnI to digest the template DNA and purified using a PCR purification kit.

[0278] mRNA synthesis using in vitro transcription (IVT) mRNA was prepared using a NEB IVT kit with T7 RNA polymerase, using a template prepared for IVT by PCR. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporated CleanCap® (AG) at the start of each mRNA, giving a Cap1 structure at the 5' end. mRNA was produced using unmodified nucleotides or using modified uridine (5'methoxyuridine) at a ratio of 25% to unmodified uridine. After the reaction was complete, the DNA template was digested with RNase-free DNase. The mRNA transcript contained a 5'Cap-1-5'UTR-ORF-3'UTR-A80 structure. The mRNA was then purified using a silica column and resuspended in water.

[0279] Cell transfection Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded. 100,000 cells in 100 μl of medium were seeded into each well (96W plate) the day before. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well.

[0280] Quantification of eGFP expression eGFP fluorescence was detected using an incucyte instrument that acquires images from living cells. Fluorescence was measured in relative fluorescence units (RFU) from the images using Incucyte software. The data reported in the figure shows eGFP fluorescence 24 hours after transfection.

[0281] result It has been previously shown that eGFP expression can be increased by incorporating selected 5'UTR and 3'UTR with control 3'UTR and 5'UTR, respectively (see Examples 3 and 4).

[0282] eGFP mRNA expression with these 5'UTR and 3'UTR combinations was compared to expression with control 5'UTR (HSD17B4) and control 3'UTR (albumin). In all cases, eGFP mRNA expression with candidate 5'UTR and candidate 3'UTR combinations was increased in A549 cells compared to control (Figures 9 and 10). Interestingly, the general trend for each combination was conserved regardless of whether the mRNA contained modified bases (Figure 10) or not (Figure 9). In both cases, GOT / CS, PRKACB / CS, and CHIT / CS resulted in the greatest increase in GFP fluorescence compared to control mRNA (HSDB / Alb). Surprisingly, GOT / CS1 appeared to work best in the context of modified mRNA (Figure 10), while PRKACB / CS yielded the highest GFP fluorescence levels when using wild-type bases.

[0283] Example 7: By combining a modified 5'UTR with a modified 3'UTR, the expression of scFv-Fc encoding mRNA can be increased. To test whether UTR combinations function similarly to increase the expression level of the target gene independently, the combinations were tested for scFv expression. This was also done with both wild-type mRNA and modified mRNA.

[0284] method UTR cloning The 5'UTR and 3'UTR were cloned using the scFv-Fc coding sequence. Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'UTR contained the T7 promoter sequence.

[0285] Preparation of in vitro transcription (IVT) templates using PCR IVT templates were prepared by PCR using Phusion PCR Mix (NEB). Upstream primers contained the T7 promoter sequence, and downstream primers contained the T80 sequence along with the reverse complement of the 3'UTR end of each clone. The resulting PCR product contained sequences encoding the relevant sequence elements in the following order: T7 promoter-5'UTR-ORF-3'UTR-A80. The PCR reaction was then treated with DpnI to digest the template DNA and purified using a PCR purification kit.

[0286] mRNA synthesis using in vitro transcription (IVT) mRNA was prepared using a NEB IVT kit with T7 RNA polymerase, using a template prepared for IVT by PCR. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporated CleanCap® (AG) at the start of each mRNA, giving a Cap1 structure at the 5' end. mRNA was produced using unmodified nucleotides or using modified uridine (5'methoxyuridine) at a ratio of 25% to unmodified uridine. After the reaction was complete, the DNA template was digested with RNase-free DNase. The mRNA transcript contained a 5'Cap-1-5'UTR-ORF-3'UTR-A80 structure. The mRNA was then purified using a silica column and resuspended in water.

[0287] Cell transfection Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and re-seeded. 100,000 cells in 100 μl of medium were seeded into each well (96W plate) the day before. On the day of transfection, the old medium was aspirated and 140 μl of fresh medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine were then vortexed, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of mRNA / Lipofectamine mix was added to each well. 100 μl of the supernatant was collected at the specified time.

[0288] SCFV-FC quantitative After 24 hours, the cell supernatant (sup) was collected from cells transfected with scFv-Fc mRNA. The sup was frozen at -80°C until quantification. A cis-bio kit for Fc quantification was used for quantification. The principle of quantification is based on a competitive immunoassay using HTRF technology. hFc-tagged proteins (or antibodies) can replace the binding between d2-labeled IgG and cryptotate-labeled PAb anti-human Fc. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of human Fc in the sample or standard. A standard curve was created from known concentrations of scFv-Fc, and the amount of scFv-Fc present in the sup was quantified by interpolating the signal from the sup of scFv-Fc-transfected cells using this standard curve. The concentration of scFv-Fc was measured in ng / mL. The data shown in the figure report the scFv-Fc levels 24 hours after transfection.

[0289] result Again, scFv mRNA expression with these 5'UTR and 3'UTR combinations was compared to expression with the control 5'UTR (HSD17B4) and control 3'UTR (albumin). Interestingly, similar to the eGFP fluorescence levels in Example 3, PRKACB / CHIT yielded the highest expression levels when using wild-type mRNA (Figure 11). However, apart from CHIT / CS, most of the other UTR combinations did not affect the detectable scFv expression levels compared to the control (HSBD / Alb). In contrast, CHIT / CS and PRKACB / CHIT performed best compared to the control when using modified mRNA as the expression substrate (Figure 12).

[0290] Therefore, among all the novel UTRs tested, the CHIT / CS and PRKACB / CHIT combinations, whether alone or in combination, resulted in the greatest increase in expression levels, regardless of the gene of interest or whether the mRNA contained modified U or wild-type U.

[0291] Example 8 - In vitro enhancement of protein expression from mRNA vaccine vectors Next, using UTR sequences reported by Andrew Fire et al. of Stanford University via GitHub on April 14, 2021, we directly compared one of the optimal UTR combinations (CHIT / CS) for mRNAs containing putative UTR pairs from Moderna and Pfizer / BioNTech (mRNA Comp A and mRNA Comp B). In this study, the modified mRNAs contained 100% 5'-methoxyuridine (not 25% as in Examples 4-7).

[0292] eGFP construct cloning An mRNA construct was designed (mRNA_AZ) encoding the eGFP reporter and using the CHIT1 5'UTR (SEQ ID NO: 15) paired with the CS 3'UTR (SEQ ID NO: 16). In parallel, mRNA comparator A and mRNA comparator B constructs were designed to encode eGFP flanked by putative UTR sequences derived from each of the above COVID vaccines. Cloning was performed by Gibson-based assembly. Each plasmid had a T7 promoter sequence upstream of each 5'UTR and an 80-base-pair poly-A track downstream of the 3'UTR with a single BspQI site for subsequent linearization. All eGFP coding sequences were identical. The complete 5'UTR and 3'UTR sequences, including sequences derived from CHIT1 and CS UTR, are shown in SEQ ID NOs: 43 and 44, respectively.

[0293] Fabrication of in vitro transfer (IVT) molds IVT templates were prepared after plasmid purification from bacterial cells in the same manner as outlined in Examples 4-7.

[0294] mRNA synthesis using in vitro transcription (IVT) mRNA was prepared using a NEB IVT kit with T7 RNA polymerase, using a template prepared for IVT. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporated CleanCap® (AG) at the start of each mRNA, giving a Cap1 structure to the 5' end. mRNA was produced using unmodified nucleotides or 100% modified uridine (N1-methylpseudolidine). Both mRNA comp A and mRNA comp B (mRNA containing putative UTRs derived from the GitHub database) contained 100% modified U. After the reaction was complete, the DNA template was digested with RNase-free DNase. The mRNA was then purified using a silica column and resuspended in water.

[0295] Quantification of eGFP expression Following the manufacturer's protocol, purified mRNA was transfected into either BHK-21 or HEK293 cells using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher). eGFP fluorescence was detected using an IncuCyte instrument to acquire images from live RNA-transfected cells over 96 hours. Fluorescence was measured from the images as relative fluorescence units (arbitrary units) using IncuCyte software.

[0296] result mRNA using the 5'UTR CHIT1 (SEQ ID NO: 15) and 3'UTR CS (SEQ ID NO: 16) UTR sets resulted in the highest levels of eGFP expression in both BHK-21 cells (Figure 13A) and HEK293 cells (Figure 13B) compared to two competing mRNA molecules (mRNA Comp A and mRNA Comp B). Maximum expression from mRNA relied on the incorporation of modified nucleotides such as N1-methylpseudridine (pseudoU) to evade the host cell antiviral response in HEK293 cells (Figure 13B).

[0297] Example 9 - The RSV mRNA-VLP vaccine induces high-titer neutralizing antibodies in non-human primates that have experienced RSV. Non-human primates (NHPs) that had experienced RSV were immunized with either a benchmark RSV F protein vaccine (DS-CAV1 + adjuvant) or one of two different RSV mRNA vaccines encoding a pre-fusion-stabilized F protein, and immunogenicity was evaluated by ELISA binding titer and viral neutralization assay.

[0298] method mRNA synthesis using in vitro transcription (IVT) Sequences encoding the membrane DS-CAV1 F protein and DS-2-2 F protein-nanoparticle subunits were cloned into mRNA vectors encoding the T7 promoter, 5' and 3' UTR, and poly(A) tail. Template plasmids were linearized by digesting pDNA with BSPQ1 (NEB, USA) at 50°C for 4 hours. mRNA was synthesized by in vitro transcription using NEB T7 HighScribe supplemented with N1-methyl-pseudridine and CleanCap-AG (TriLink). The IVT reaction mixture was incubated at 37°C for 5 hours and then digested with RNase-free DNase. mRNA was then purified using LiCl precipitation. All mRNA was formulated into lipid nanoparticles (LNPs) in 20 mM Tris / Tris-HCl, pH 7.4, 8% w / v sucrose and stored at -80°C.

[0299] NHP immunization The animal study was approved by the AstraZeneca Animal Research Committee (IACUC) and conducted at BIOQUAL, Inc. (Rockville, Maryland). Twenty-four cynomolgus monkeys (Macaca fascicularis) were divided into three groups. Each animal received a 1 mL intramuscular (IM) injection of either a protein or mRNA vaccine on day 1 of the study. Serum samples were taken 7 days before and 14 days after immunization (days 7 and 14 of the study, respectively) to assess baseline and post-immunization antibody levels.

[0300] Binding ELISA ELISA was performed to evaluate the binding titer of pre-fusion F antibody in immunized animals. 384-well plates were coated overnight at 4°C with 3 μg / ml purified recombinant pre-fusion F protein. The plates were then washed with PBS-T and blocked at room temperature for 1 hour in PBS supplemented with 5% milk powder and 3% BSA. Animal serum was then diluted with blocking buffer and incubated with the coated plates at room temperature for 1 hour. The plates were then washed and incubated with HRP-conjugated anti-NHP IgG secondary antibody for 1 hour. The plates were washed and incubated with TMB substrate for 5 minutes, stopping the color development with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0301] RSV neutralization assay Serum derived from immunized animals was analyzed for neutralizing activity at the indicated time points. Serum was heat-inactivated and serially diluted in 96-well plates. The diluted serum was then incubated with the indicated virus at 0.04 MOI at 37°C for 1 hour. Next, 20,000 Hep2 cells were added to the virus / serum mixture, and the plates were incubated at 37°C for 5 days. To visualize infection, cells were fixed with acetone, washed, and then stained with biotinylated mAB 133-1H. Cells were washed and incubated with strep-HRP antibody. The plates were washed and incubated with TMB substrate for 7 minutes, and color development was stopped with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0302] result RSV mRNA-VLP expressing the DS-2-2 F protein on a lumazine synthase scaffold induced a potent virus-neutralizing antibody response comparable to that of benchmark proteins and mRNA vaccines administered at higher doses. Next, we wanted to compare the boosting capacity of our RSV mRNA-VLP vaccine in non-human primates (NHPs) that had experienced RSV. To test this, we immunized groups of NHPs with either 120 μg of recombinant DS-CAV1 protein + adjuvant, 50 μg of mRNA vaccine expressing DS-CAV1, or 15 μg of our mRNA-VLP vaccine expressing the DS-2-2 protein fused to a lumazine synthase (LS) scaffold. Prior to the start of the study (-7 days), all animals were profiled for baseline anti-RSV pre-fusion F antibody binding titer (Figure 14A) and virus-neutralizing antibody titer (Figure 14B), and a balance between groups was found. Fourteen days after immunization, animals in all groups achieved comparable virus-neutralizing antibody titers (Figure 14B). Importantly, 15 μg of mRNA-VLP vaccine induced a very similar response to protein and mRNA vaccines administered at 120 μg and 50 μg, respectively, further highlighting the immunogenic value of the VLP approach for antigen design and the potential for improved reactogenicity of mRNA-based vaccine approaches.

[0303] Example 10 - The RSV mRNA-VLP vaccine protects infected cotton rats from viral replication in lung and nasal tissue. Naive cotton rats were immunized with either a benchmark RSV F protein vaccine (DS-CAV1 + adjuvant) or one of two different RSV mRNA vaccines encoding a pre-fusion stabilized F protein to evaluate vaccine efficacy in a viral challenge model.

[0304] method mRNA synthesis using in vitro transcription (IVT) Sequences encoding the membrane DS-CAV1 F protein and DS-2-2 F protein-nanoparticle subunits were cloned into mRNA vectors encoding the T7 promoter, 5' and 3' UTR, and poly(A) tail. Template plasmids were linearized by digesting pDNA with BSPQ1 (NEB, USA) at 50°C for 4 hours. mRNA was synthesized by in vitro transcription using NEB T7 HighScribe supplemented with N1-methyl-pseudridine and CleanCap-AG (TriLink). The IVT reaction mixture was incubated at 37°C for 5 hours and then digested with RNase-free DNase. mRNA was then purified using LiCl precipitation. All mRNA was formulated into lipid nanoparticles (LNPs) in 20 mM Tris / Tris-HCl, pH 7.4, 8% w / v sucrose and stored at -80°C.

[0305] Cotton rat immunization and viral challenge The animal study was approved by the AstraZeneca Animal Research Committee (IACUC) and conducted at Sigmovir Biosystems, Inc. (Rockville, Maryland). Four groups of cotton rats (n=8 per group) received two intramuscular (IM) injections of either PBS control, protein vaccine, or mRNA vaccine on days 1 and 21 of the study. Serum samples were obtained 21 days after the second immunization (day 42 of the study) and immediately before RSV virus challenge. On day 42 of the study, all animals were administered live RSV intranasally and subsequently euthanized 5 days later (day 47 of the study). Lung and nasal tissues were collected at that time, and viral replication was evaluated by viral plaque assay.

[0306] RSV neutralization assay Serum derived from immunized animals was analyzed for neutralizing activity at the indicated time points. Serum was heat-inactivated and serially diluted in 96-well plates. The diluted serum was then incubated with the indicated virus at 0.04 MOI at 37°C for 1 hour. Next, 20,000 Hep2 cells were added to the virus / serum mixture, and the plates were incubated at 37°C for 5 days. To visualize infection, cells were fixed with acetone, washed, and then stained with biotinylated mAB 133-1H. Cells were washed and incubated with strep-HRP antibody. The plates were washed and incubated with TMB substrate for 7 minutes, and color development was stopped with 2N H2SO4. The plates were read at a wavelength of 450 nm using Envision.

[0307] result RSV mRNA-VLP expressing the DS-2-2 F protein on a lumazine synthase scaffold induced a potent virus-neutralizing antibody response, providing complete protection from viral challenge. Next, we wanted to compare the vaccine efficacy of our RSV mRNA-VLP vaccine in the gold-standard Cotton rat model of RSV virus challenge. To test this, on days 1 and 21 of the study, we immunized groups of Cotton rats with either control PBS, 20 μg of recombinant DS-CAV1 protein + adjuvant, 20 μg of mRNA vaccine expressing DS-CAV1, or 20 μg of our mRNA-VLP vaccine expressing the DS-2-2 protein fused to a lumazine synthase (LS) scaffold (Figure 15A). Before the live RSV virus challenge on day 42 of the study, all groups were profiled for virus-neutralizing antibody titers (Figure 15A). Both mRNA vaccine groups induced comparable neutralizing antibody titers, but higher than those measured in the protein vaccine group (Figure 15B).

[0308] Five days after intranasal viral challenge, all groups were euthanized, and lung and nasal tissues were collected to assess viral replication (Figure 15A). Among all vaccinated groups, the level of viral replication in the lungs (a measure of disease severity) was approximately 10 per gram of lung tissue. 5 The number of infected units was substantially reduced compared to the PBS control (Figure 15B). Interestingly, no detectable virus was recovered in the lungs of the mRNA-vaccinated group, but three animals in the protein-vaccinated group had detectable levels of viral replication, indicating a “breakthrough” infection that could potentially be attributed to lower neutralizing antibodies measured before the viral challenge (Figures 15B–15C).

[0309] Furthermore, substantial levels of viral replication in the nasal cavity were measured in most animals vaccinated with the protein vaccine, and these levels were, on average, approximately 10 times lower than those in the PBS control group (Figure 15D). The group receiving the mRNA vaccine expressing DS-CAV-1 had two animals exhibiting “breakthrough” infection. In contrast, there were no “breakthrough” infections in the mRNA-VLP vaccine group, suggesting bactericidal immunity. Given that both mRNA vaccines were administered at the same dose, these results further reinforce the value of VLP antigen design in maximizing the immune response and providing complete protection from viral challenge in the Cotton rat model.

[0310] Sequence List Sequences in uppercase letters are protein sequences. Sequences in lowercase letters are RNA sequences. The sequences show the basic amino acid / nucleotide sequence of the elements above. As shown above, standard nucleotides in RNA sequences can be replaced with their modified versions. Sequence ID 1-DS2-2 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTDLQLLMQSTPATGSGAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNYIDKQLLPILNKQSCSPNIETVIEFQQKNNRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMS NNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFDYPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SEQ ID NO:2-DS2-2(A) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATGSGAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLEITREFSVNAGVTTPVSTYMLTNSELLSLINMPITNDQKKLMS NNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFDYPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SEQ ID NO: 3-Aquifex aeolicus lumazine synthase MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR Sequence ID 4 - Linker 1 SGGSSGSSGGS Sequence ID 5 - Linker - Scaffolding SGGSSGSSGGSMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR Sequence ID 6-DS2-2-LuS MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNY IDKQLLPILNKQSCSPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFDYPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGGSS GSSGGSMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR sequence number 7-DS2-2(A)-LuS MELLILKANAITTILTAVTFFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNY IDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGGSS GSSGGSMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR Sequence ID 8-DS2-2 Sequence ID 9-DS2-2(A) SEQ ID NO: 10 - Aquifex aeolicus lumazine synthase augcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucg uucggcaugggggucgcgaggaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgcc aucggcguauugauucgcggggcuacccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucauuggaguugcgaaagccgauuacuuuuggaguca ucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugagg Sequence ID 11 - Linker 1 uccggcggaaguucuggcucuagugggggaagc Sequence ID 12 - Linker - Scaffolding uccggcggaaguucuggcucuagugggggaagcaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugagg SEQ ID NO: 13-DS2-2-LuS Sequence ID 14-DS2-2(A)-LuS Sequence ID 15 - Modified (m) CHIT1 5'UTR auugugcugcauc Sequence ID 16-CS 3'UTR aacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaa Sequence ID 17-DS2-2-LuS complete mRNA Sequence ID 18-DS2-2(A)-LuS complete mRNA Sequence ID 19-RSV AF protein (full length) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Sequence ID 20-PRKACB 5'UTR auucugcuguuugcuccuugccagguucaac Sequence ID 21-GOT1 5'UTR aaaaucucuugauuccuagucucucgau Sequence ID 22-GUSB 5'UTR auccucaaccaagcgccgcagacgguggccgagcgggggaccgggaagc Sequence ID 23-HSDB 5'UTR gucccgcagucggcguccagcggcucugcuuguucgugugugucguugcaggccuuauu Sequence ID 24-CHIT1 3'UTR gucgcuaaagcccuccagucccagcuuugaggcugggcccaggaucacucuacagccugccuccuggguuuucccuggggggccgcaaucuggcuccugcaggccuuucuguggucuuccuuuauccaggcuuucugcucucagccuugccuuccuuuuuuucugcgacuccugg cugccccuuucacuugcaaaauaaa Sequence ID 25-PKLR 3'UTR gacgccccucccucucuggagucuacguucuccagcccacaccccuccaaagccccaccuuuaaguccucuucucuauuccugacccucccuaccugaggccuaucugagacuauaacugucaucuagccccuucgagg uugccccuuccccaucuccauuucacacagguccugaaagucuguguccaauuaugcacuggccacccaacagcaccaauuguacauucccugcauccaaucugcucagcaggcccuaagaugccuugagucuuuaauccca Sequence ID 26 - Albumin 3'UTR gcaucacauuuaaaagcaucucagccuaccaugagaauaagagaaagaaaaugaagaucaauagcuuauucaucucuuuuucuuuucguugguguaaagccaacacccugucuaaaaaacauaaauuucuuuaaucauuuugccucuuuucucugugcuucaauuaauaaaaaauggaaagaaccu Sequence ID 27 - Linker 2 GGGS Sequence ID 28 - Linker 3 GGGGS Sequence ID 29 - Linker 4 GGSG Sequence ID 30 - Linker 5 GSGG Sequence ID 31 - Linker 6 SGGG Sequence ID 32 - Linker 7 SSGG Sequence ID 33 - Linker 8 SSSG Sequence ID 34 - Linker 9 SAGS Sequence ID 35 - Linker 10 GGGSG Sequence ID 36 - Linker 11 TGGGG SGGGGS Sequence ID 37 - Linker 12 GGGGSGGGGS Sequence ID 38-RSV F protein F0 chain QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEG EVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAY VVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFS NGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Sequence ID 39-RSV F protein F1 chain FLGFLLGVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Sequence ID 40-RSV F protein F2 chain QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARR SEQ ID NO: 41-RSV F Protein Signal Peptide MELLILKANAITTILTAVTFCFASG Sequence ID 42-RSV F protein P27 fragment ELPRFMNYTLNNAKKTNVTLSKKRKRR Sequence ID 43 - Extended mCHIT1 5'UTR aggauugugcugcaucaagcuugccgccacc Sequence ID 44 - Extended CS 3'UTR ugaugauaauaggacuaguggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccuagcguacguaaaaaauggaaagaaccuagcguac Sequence ID 45-DS2-2(A)F1 chain GSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL Array number 46 - DS2 - 2 F1 chain GSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSGCGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKCRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL Array number 47 - DS2 - 2(A) F2 chain QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAT Array number 48 - DS2 - 2 F2 chain QNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPAT Sequence ID 49-DS-Cav1 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL Sequence ID 50 - Foldon GYIPEAPRDGQAYVRKDGEWVLLSTFL Sequence ID 51-ferritin DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Sequence ID 52 - Ferritin with Bullfrog Leader ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHCFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Sequence ID 53-β-Annulus INHVGGTGGAIMAPVAVTRQLVGS Sequence ID 54 - DS-Cav1 with a transmembrane domain MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLG VGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

Claims

1. A respiratory syncytial virus (RSV) messenger RNA (mRNA) vaccine comprising an mRNA molecule encoding a nanoparticle subunit comprising an immunogen and a scaffold linked by a linker, wherein the linker is 9 to 13 amino acids long, the scaffold comprises lumazine synthase, and the immunogen comprises a modified RSV F protein including a deletion at positions 104 to 144 of the RSV F protein, a GS peptide linker between positions 103 and 145 of the RSV F protein, and the following mutations: S155C, N183GC, S190F, V207L, S290C, L373R and N428C.

2. The RSV mRNA vaccine according to claim 1, wherein when aggregated, the nanoparticles comprise approximately 60 nanoparticle subunits.

3. The RSV mRNA vaccine according to claim 1 or 2, wherein the lumazine synthase comprises the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof having at least 90% identity with SEQ ID NO:

3.

4. The RSV mRNA vaccine according to any one of claims 1 to 3, wherein the linker has a length of 11 amino acids.

5. The RSV mRNA vaccine according to claim 4, wherein the linker comprises the amino acid sequence shown in Sequence ID No. 4, or a sequence comprising up to three amino acid substitutions compared thereto.

6. The RSV mRNA vaccine according to any one of claims 1 to 5, wherein the nanoparticle subunit comprises the immunogen, the linker, and the scaffold from the N-terminus to the C-terminus.

7. The RSV mRNA vaccine according to claim 6, wherein the linker and scaffold together (linker-scaffold) comprise the amino acid sequence shown in SEQ ID NO:

5.

8. The RSV mRNA vaccine according to any one of claims 1 to 7, wherein the immunogen is a modified RSV F protein comprising an F1 polypeptide and an F2 polypeptide.

9. The RSV mRNA vaccine according to any one of claims 1 to 8, wherein the modified RSV F protein further comprises one or more of the following substitutions: S46G, E92D, and S215P.

10. The RSV mRNA vaccine according to claim 9, wherein the modified RSV-F protein further comprises S46G, E92D and S215P substitutions.

11. The RSV mRNA vaccine according to any one of claims 1 to 8, wherein the immunogen comprises the amino acid sequence shown in Sequence ID No.

1.

12. The RSV mRNA vaccine according to any one of claims 1 to 11, wherein the nanoparticle subunit comprises the amino acid sequence shown in SEQ ID NO: 6, or a variant thereof having at least 90% identity with SEQ ID NO:

6.

13. The RSV mRNA vaccine according to claim 12, wherein the nanoparticle subunit comprises the amino acid sequence shown in Sequence ID No.

6.

14. The RSV mRNA vaccine according to any one of claims 1 to 10, wherein the immunogen comprises the amino acid sequence shown in Sequence ID No.

2.

15. The RSV mRNA vaccine according to claim 14, wherein the nanoparticle subunit comprises the amino acid sequence shown in SEQ ID NO: 7, or a variant thereof having at least 90% identity with SEQ ID NO:

7.

16. The RSV mRNA vaccine according to claim 15, wherein the nanoparticle subunit comprises the amino acid sequence shown in Sequence ID No.

7.

17. The RSV mRNA vaccine according to any one of claims 1 to 16, wherein the amino acid numbering of the RSV F protein conforms to the reference sequence shown in SEQ ID NO:

19.

18. The aforementioned mRNA molecule (i) an immunoenzyme coding sequence comprising the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, or a sequence having at least 90% identity thereto, (ii) A scaffold coding sequence including the nucleotide sequence shown in Sequence ID No. 10, or a sequence having at least 90% identity thereto, and (iii) A linker code sequence including the nucleotide sequence shown in Sequence ID No. 11, or a sequence having at least 90% identity thereto. An RSV mRNA vaccine according to any one of claims 1 to 17, comprising:

19. The RSV mRNA vaccine according to claim 18, wherein the mRNA molecule comprises a nanoparticle subunit coding sequence including the nucleotide sequence shown in SEQ ID NO: 13 or SEQ ID NO:

14.

20. The RSV mRNA vaccine according to any one of claims 1 to 19, wherein the mRNA molecule comprises a 5'UTR having the nucleotide sequence shown in SEQ ID NO: 15, or a sequence having at least 90% identity thereto.

21. The RSV mRNA vaccine according to any one of claims 1 to 20, wherein the mRNA molecule comprises a 3'UTR having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 16, or a sequence having at least 90% identity therewith.

22. The RSV mRNA vaccine according to any one of claims 1 to 21, wherein the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18, or a sequence having at least 90% identity thereto.

23. The RSV mRNA vaccine according to claim 22, wherein the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

24. The RSV mRNA vaccine according to claim 23, wherein the mRNA molecule comprises the nucleotide sequence shown in Sequence ID No.

18.

25. The RSV mRNA vaccine according to any one of claims 1 to 24, wherein the mRNA molecule comprises a pseudouridine nucleotide or a modified pseudouridine nucleotide.

26. The RSV mRNA vaccine according to claim 24, wherein the modified pseudouridine is N1-methylpseudridine.

27. The RSV mRNA vaccine according to any one of claims 1 to 26, wherein the mRNA comprises a Cap1 or a 5' cap having a modified Cap1 structure.

28. The RSV mRNA vaccine according to any one of claims 1 to 27, wherein the mRNA molecule is formulated into lipid nanoparticles.

29. The RSV mRNA vaccine according to claim 28, wherein the lipid nanoparticles comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEG-lipids, and 30-50% cholesterol.

30. An RSV mRNA vaccine according to any one of claims 1 to 29, for use as a therapeutic or prophylactic agent.

31. An RSV mRNA vaccine according to any one of claims 1 to 29, for use in eliciting an immune response in a target.

32. An RSV mRNA vaccine according to any one of claims 1 to 29, for use in a method of preventing and / or attenuating RSV infection in a subject.

33. An RSV mRNA vaccine according to any one of claims 1 to 29, for use in the prevention of lower respiratory tract disease (LRTD) caused by respiratory syncytial virus (RSV) in a target population.

34. A method for inducing an immune response in a subject, comprising administering to the subject an RSV mRNA vaccine according to any one of claims 1 to 29.

35. A method for preventing lower respiratory tract disease (LRTD) caused by respiratory syncytial virus (RSV) in a subject, comprising administering the RSV mRNA vaccine described in any one of claims 1 to 29 to the subject.

36. Use of the RSV mRNA vaccine according to any one of claims 1 to 29 in the manufacture of a pharmaceutical product for use in a method of inducing an immune response in a subject.

37. Use of the RSV mRNA vaccine according to any one of claims 1 to 29 in the manufacture of a pharmaceutical product for use in preventing or reducing diseases caused by RSV infection.

38. An expression vector comprising an expression cassette encoding an mRNA molecule according to any one of claims 1 to 24.

39. A cell comprising the expression vector described in claim 38.

40. A method for producing an mRNA molecule according to any one of claims 1 to 26, comprising expressing mRNA from an expression vector according to claim 38, wherein the mRNA is optionally expressed by in vitro transcription.

41. Protein nanoparticles comprising a polymer of a nanoparticle subunit according to any one of claims 1 to 19, preferably a 60-mer of the nanoparticle subunit.

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