vaccine

Abstract

The present disclosure relates to an mRNA molecule encoding a polypeptide comprising an hMPV F protein, or an immunogenic fragment thereof, and a lumazine synthase (LuS) capable of multimerising to form a nanoparticle. Further aspects of the disclosure relate to compositions comprising the mRNA molecule and their use as a vaccine in the prevention of infectious diseases, including those caused by hMPV and RSV.

Description

[0001] hMPV- 100

[0002] VACCINE

[0003] FIELD

[0004] The present disclosure relates to an mRNA molecule encoding a polypeptide comprising an hMPV F protein, or an immunogenic fragment thereof, and a multimerization unit capable of multimerising to form a nanoparticle. Further aspects of the disclosure relate to compositions comprising the mRNA molecule and their use as a vaccine in the prevention of infectious diseases, including those caused by hMPV and RSV.

[0005] BACKGROUND

[0006] mRNA vaccines have several benefits compared to DNA-based vaccines, as well as to subunit, killed and live attenuated virus vaccines. One advantage is the safety profile of mRNA vaccines, with there being no risk of infection due to mRNA being non-infectious, unlike pathogen-containing vaccines. In addition, mRNA is a non-integrating platform, meaning that it does not interact with genomic DNA and, therefore, does not carry the risk of insertional mutagenesis. Another advantage of mRNA-based vaccines is their efficacy, as mRNA can be made more stable and highly translatable via various modifications. Furthermore, formulating mRNA into a carrier, or vehicle, enables efficient in vivo delivery through rapid uptake and expression in the cytoplasm. A third advantage of mRNA-based vaccines is their potential for inexpensive, rapid and scalable manufacturing.

[0007] Human metapneumovirus (hMPV) causes acute upper and lower respiratory tract infections in people of all ages, particularly in children, immunocompromised individuals and tire elderly. Symptoms of hMPV are similar to those of other viruses that cause upper and lower respiratory infections, and can include cough, fever, nasal congestion and shortness of breath. The hMPV infection can progress to more severe symptoms including bronchitis, bronchiolitis and pneumonia. hMPV is a negative-sense single-stranded RNA virus which is part of the Pneumoviridae family and comprises two genotypes, known as A and B. The hMPV fusion (F) glycoprotein is the primary target of both neutralising and protective antibodies against hMPV infection, and is therefore a suitable target for an hMPV vaccine. The hMPV F protein is a trimcric surface glycoprotein anchored to the viral envelope, and mediates viral fusion to facilitate viral entry into target cells. The protein is initially expressed as a single polypeptide precursor, designated Fo. The Fo polypeptide is cleaved once by a trypsin-like protease, generating two subunits referred to as F2 and Fi, which are covalently joined by disulphide bonds. F2 / F1 heterodimers then trimerise in a metastable, prefusion conformation, to form the mature F protein which is incorporated into virions. During membrane fusion, the conformation of the F protein changes into a highly stable post -fusion conformation (Hsieh et al., Nature Communications 13: 1299, 2022). hMPV- 100

[0008] Existing approaches for hMPV vaccines include that of WO2023 / 225562A1, which describes a multivalent vaccine comprising two or more virus-like particles (VLPs) in which one of the VLPs comprises a component comprising an hMPV F protein ectodomain or antigenic variant thereof. US2022 / 0378904A1 describes a vaccine comprising an mRNA encoding the full length wild-type hMPV F glycoprotein. However, it has also been suggested that modification of the F protein to stabilise it in its prefusion conformation would be advantageous, or even necessary, to produce an effective vaccine (Hsieh et al., supra). At present, there are no licensed vaccines available against hMPV infections. Therefore, there is a need to develop vaccines which are effective against hMPV infection.

[0009] SUMMARY

[0010] The present inventors have developed an mRNA vaccine against hMPV utilising an antigen derived from the hMPV F protein. The antigen may be an unmodified F protein, or alternatively may be stabilised in the prefusion conformation. The antigen is provided in the form of a fusion protein further comprising a multimerization unit. When expressed, the multimerization unit multimerises into a nanoparticle, with the hMPV F protein antigen arrayed on tire surface. The fusion protein thus constitutes a nanoparticle subunit (and the terms “fusion protein” and “nanoparticle subunit” are used interchangeably herein), as upon expression it is assembled into a nanoparticle.

[0011] According to a first aspect, the present disclosure provides a messenger RNA (mRNA) molecule encoding a polypeptide comprising a human metapneumovirus (hMPV) F protein, or an immunogenic fragment thereof, and a multimerization unit capable of multimerising to form a nanoparticle .

[0012] The polypeptide encoded by the mRNA molecule is thus the nanoparticle subunit, or fusion protein, discussed above. When expressed by a cell, the nanoparticle subunit multimerises via the multimerization unit to form a protein nanoparticle, of which the immunogen forms the exposed exterior.

[0013] In particular instances, the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO. 2, or a variant thereof, as described further below. Such an hMPV F protein may be a wild type hMPV F protein, or may be a modified hMPV F protein, including a modified F protein which is stabilised in its prefusion conformation. Particular prefusion -stabilised hMPV F proteins which may be used herein are provided in SEQ ID NOs: 4, 6 and 8.

[0014] In particular instances, the multimerization unit is lumazine synthase.

[0015] In particular instances, the protein nanoparticle comprises 60 self-assembled nanoparticle subunits (i.e. is a 60-mer). hMPV- 100

[0016] A second aspect of the disclosure provides a pharmaceutical composition comprising an mRNA molecule according to the first aspect of the disclosure.

[0017] In particular embodiments of the second aspect, the pharmaceutical composition comprises the mRNA molecule of the first aspect formulated in a lipid nanoparticle (LNP).

[0018] The pharmaceutical composition may further comprise a second mRNA molecule encoding a second polypeptide (specifically a second nanoparticle subunit) comprising a modified respiratory syncytial virus (RSV) F protein, or an immunogenic fragment thereof, and a lumazine synthase, wherein the modified RSV F protein is stabilised in the prefusion conformation

[0019] A third aspect of the disclosure is directed to an mRNA molecule of the first aspect or a pharmaceutical composition of the second aspect of the disclosure for use as a therapeutic or prophylactic agent. A fourth aspect of the disclosure is directed to an mRNA molecule of the first aspect or a pharmaceutical composition of the second aspect of the disclosure for use in raising an immune response in a subject. A fifth aspect of the disclosure is directed to an mRNA molecule of the first aspect or a pharmacal composition of the second aspect of the disclosure for use in a method of preventing and / or attenuating an infectious disease. The disease may be a disease caused by a respiratory virus, in particular a disease caused by hMPV or RSV.

[0020] A sixth aspect of the disclosure provides a method of inducing an immune response in a subject, comprising administering to the subject the mRNA molecule of the first aspect or the pharmaceutical composition of the second aspect of the disclosure.

[0021] A seventh aspect of the disclosure provides a method of preventing and / or attenuating an infectious disease in a subject, the method comprising administering an effective amount of the mRNA molecule of the first aspect or the pharmaceutical composition of the second aspect of the disclosure to the subject. The disease may be a disease caused by a respiratory virus, in particular a disease caused by hMPV or RSV.

[0022] An eighth aspect of the disclosure provides the use of the mRNA molecule of the first aspect or the pharmaceutical composition of the second aspect of the disclosure in the manufacture of a medicament for use in a method of inducing an immune response in a subject.

[0023] A ninth aspect of the disclosure provides the use of the mRNA molecule of the first aspect or the pharmaceutical composition of the second aspect of the disclosure in the manufacture of a medicament for use in preventing or attenuating a disease caused by hMPV or RSV infection.

[0024] A tenth aspect of the disclosure provides an expression vector comprising an expression cassette encoding an mRNA molecule of the first aspect of the disclosure. hMPV- 100

[0025] An eleventh aspect of the disclosure provides a cell comprising the expression vector of the tenth aspect of the disclosure.

[0026] A twelfth aspect of the disclosure provides a method of manufacturing an mRNA molecule of the first aspect of the disclosure, comprising expressing the mRNA from the expression vector of the tenth aspect of the disclosure. The mRNA may be expressed by in vitro transcription.

[0027] A thirteenth aspect of the disclosure provides a protein nanoparticle comprising a multimer of a polypeptide comprising a human metapneumovirus (hMPV) fusion (F) protein, or an immunogenic fragment thereof, and a lumazine synthase (i.e. a multimer of the nanoparticle subunit described above). The protein nanoparticle may be a 60-mer of the nanoparticle subunit.

[0028] DESCRIPTION OF THE FIGURES

[0029] Figure 1 is a graph showing in vitro expression of two different mRNA-hMPV prefusion-stabilised F protein vaccine candidates (hMPV v3B and hMPV DS-CavES2) displayed on a panel of protein nanoparticles. BA = beta-annulus; LuS = lumazine synthase. Expression was compared to a wild type F protein from hMPV strain TN / 92-4 (GenBank ABM67072.1) expressed as a soluble trimer.

[0030] Figure 2 (A and B) are graphs showing neutralising antibodies against hMPV elicited by a variety of hMPV F protein-scaffold mRNA vaccine constructs in naive mice. Figure 2 A shows neutralisation titers against hMPV A vims while Figure 2B shows neutralisation titers against hMPV B vims. In Fig. 2B, the response from all 6 animals in the DS-CavES2 PA group are at the lower limit of detection. BA = beta-annulus; IC50 = half-maximal inhibitory concentration; LuS = lumazine synthase.

[0031] Figure 3 is a graph showing the prefusion F-specific antibody titres (as determined by ELISA) elicited by a variety of hMPV F protein mRNA vaccine constmcts in naive mice. BA = beta-annulus; LuS = lumazine synthase. ** indicates significant difference between antibody titres relative to benchmark (i.e. transmembrane, wild type F), p < 0.01.

[0032] Figure 4 (A and B) are graphs showing neutralising antibody titres elicited by a variety of hMPV F protein mRNA vaccine constmcts in naive mice. Figure 4 A shows neutralisation titers against hMPV A vims while Figure 4B shows neutralisation titers against hMPV B vims. FL = full length; LuS = lumazine synthase; NT50 = half-maximal neutralisation titer; wt = wildtype.

[0033] Figure 5 (A, B, C and D) describes a vaccine efficacy study in hMPV-challenged cotton rats. Figure 5A depicts the two-dose immunization of cotton rats and subsequent virus challenge along with study endpoints to evaluate vaccine responses prior to (study day 42) and vaccine efficacy post vims challenge. Figure 5B is a graph showing the neutralising antibody titers measured on study day 42 as elicited by PBS control, 20 pg of mRNA vaccine encoding the full-length native hMPV A F protein, or hMPV- 100

[0034] (LuS) VLP scaffold. Figure 5C is a graph showing the levels of infectious virus recovered in the lungs of infected animals five days post virus challenge as determined by viral plaque assay. Figure 5D is a graph showing the levels of infectious virus recovered in the nasal tissues of infected animals five days post virus challenge as determined by viral plaque assay. LLD = lower limit of detection; IDso = half-maximal inhibitory dilution; LuS = lumazine synthase; PFU = plaque forming units; wt = wildtype.

[0035] Figure 6 (A and B) are graphs showing anti-hMPV neutralising antibody titres elicited by a single immunization of hMPV-experienced non-human primates with 120 pg of an RSV prefusion-stabilised F protein + adjuvant, 50 pg of an mRNA vaccine encoding a prefusion -stabilised RSV F protein, or a bivalent mRNA vaccine comprised of 15 pg of RNA encoding an hMPV F protein fused to a VLP (lumazine synthase) scaffold and 15 pg RNA encoding a prefusion-stabilised RSV F protein fused to a VLP (lumazine synthase) scaffold. The absolute neutralising antibody titers at baseline (day 0) and 14 and 92 days after immunization are shown along with the day 14 geometric mean titer (GMT) for each group . Figure 6A shows neutralisation titers against hMPV A virus while Figure 6B shows neutralisation titers against hMPV B virus. GMT = geometric mean titer; LLD = lower limit of detection; NT50 = half-maximal neutralisation titer; RSV = respiratory syncytial virus; VLP = virus-like particle.

[0036] DETAILED DESCRIPTION

[0037] All references referred to are incorporated herein by reference in their entireties.

[0038] Many modifications and other instances of the disclosures set forth herein will come to mind to one skilled in the art to which these disclosures pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific instances disclosed and that modifications and other instances are intended to be included within the scope of the appended claims.

[0039] Units, prefixes and symbols may be denoted in their ST accepted form. Unless otherwise indicated, nucleic acids arc written left to right in 5' to 3' orientation; amino acid sequences arc written left to right in amino to carboxy orientation, respectively. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

[0040] For the avoidance of doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. hMPV-100

[0041] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0042] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0043] Numeric ranges are inclusive of the numbers defining the range. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, values may be expressed as approximations. When values are expressed as approximations, by the use of the antecedent “about,” it will be understood that tire particular value fonns a specific instance of the disclosure . The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0044] Definitions

[0045] The term “nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e. a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides e.g. modified uridine. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary stmcture of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified nucleotides, or having non-standard linkages between nucleotides, as discussed further below.

[0046] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified, for example by disulphide bond formation, glycosylation, lipidation, acetylation or phosphorylation. “Percent identity” refers to the extent of identity between two sequences (e.g. nucleic acid sequences). Percent identity can be determined by aligning two sequences, introducing gaps to maximize identity between the sequences, as further discussed below. Percent identity should generally be calculated between the same types of nucleic acids, i.c. for DNA sequences or RNA sequences. Thus, it is understood, if a DNA sequence “corresponds to” an RNA sequence or if an RNA sequence “corresponds to” a DNA sequence, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence). hMPV-100

[0047] “5 ’ untranslated region (5’ UTR)” has the usual meaning recognised by a skilled person. It is the region of an mRNA molecule located 5’ of a coding sequence and which is not translated into protein. A 5’ UTR usually starts with the transcriptional start site and ends one nucleotide before the start codon of the coding sequence (or where multiple coding sequences are present, the first (5’) coding sequence). The 5’ UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites. The 5’ nucleotide of the mRNA molecule is located within the 5’ UTR, and as detailed below this is commonly capped.

[0048] “3 ’ untranslated region (3’ UTR)” has the usual meaning recognised by a skilled person. It is the region of an mRNA molecule located 3’ of a coding sequence and which is not translated into protein. A 3’ UTR is usually 3’ of a coding sequence. If an mRNA contains multiple coding sequences, the 3’ UTR is located 3’ to the final (i.e. 3’) coding sequence. If the molecule comprises a polyadenylation signal, the 3’ UTR is usually between the coding sequence and the polyadenylation signal. A 3’ UTR may commence immediately 3’ to the stop codon of tire CDS (or final CDS), or an intervening nucleotide or nucleotide sequence may be present.

[0049] “Coding sequence (CDS)” is a continuous stretch of DNA or RNA beginning with a start codon (e.g. methionine (ATG or AUG)) and ending with a stop codon (e.g. TAA, TAG or TGA, or UAA, UAG or UGA). A coding sequence typically encodes a polypeptide. The coding sequences disclosed herein arc operably linked to the 5’- and 3’- UTRs described herein.

[0050] “Messenger RNA (mRNA)” is any RNA that encodes a (at least one) protein (a naturall -occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U”, and vice-versa.

[0051] A "nucleoside" refers to a compound containing a sugar molecule (e.g. a pentose or ribose) or a derivative thereof in combination with an organic base (e g. a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides (a “nucleotide” refers to a nucleoside, including a phosphate group). hMPV- 100

[0052] “Expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g. by transcription); and (2) translation of an RNA into a polypeptide or protein.

[0053] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. The composition can be sterile.

[0054] As used herein, the terms “subject” and “patient” are used interchangeably. The subject can be an animal. In some aspects, the subject is a mammal. The mammal may be a non-human mammal (e.g. cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). Generally, the subject is a human. As used in the present disclosure and claims, the singular forms "a”, "an", and "the" include plural forms unless the context clearly dictates otherwise.

[0055] It is understood that wherever aspects are described herein with the language “comprising”, otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In this disclosure, “comprises”, “comprising”, “containing” and “having” and the like can mean “includes”, “including”, and the like; “consisting essentially of’ or “consists essentially of’ are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0056] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both “A and B”, “A or B”, “A” and “B”. Likewise, the term “and / or” as used in a phrase such as “A, B and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] Any molecules, vectors, compositions, uses, methods or other features provided herein can be combined with one or more of any of the other molecules, vectors, compositions, uses, methods or other features provided herein.

[0058] mRNA molecules

[0059] The present disclosure provides a nucleic acid molecule encoding a polypeptide comprising a human metapneumovirus (hMPV) F protein, or an immunogenic fragment thereof, and a lumazinc synthase (LuS). The polypeptide encoded by the nucleic acid molecule is thus a fusion protein comprising the hMPV F protein (or fragment thereof) joined to a lumazine synthase. As noted above, such a fusion hMPV-100

[0060] protein may be referred to herein as a nanoparticle subunit. In some embodiments the nucleic acid molecule is a messenger RNA (mRNA) molecule.

[0061] The mRNA molecule may encode multiple polypeptides or only a single polypeptide. Where an mRNA molecule encodes multiple polypeptides this may be in the context of a single open reading frame (ORF), e.g. utilising a 2A skipping sequence to divide them, or in the context of multiple open reading frames. Where an mRNA molecule comprises multiple open reading frames, translation can be initiated from an internal ribosome entry site (IRES). Generally, however, the mRNA molecules provided herein comprise a single open reading frame encoding a single polypeptide (the above-mentioned fusion protein).

[0062] The basic components of an mRNA molecule typically include at least one coding region (as discussed above), a 5' UTR, a 3' UTR, a 5' cap and a poly(A) tail. Generally, the mRNA molecules comprise a 5’ UTR, a 3’ UTR and a 5’ cap, wherein the 5’ UTR and the 3’ UTR are operably linked to the sequence encoding the nanoparticle subunit (the coding sequence). The mRNA molecules provided herein may have the structure of naturally-occurring mRNA, or may be distinguished from wild-type mRNA in their functional and / or structural design features.

[0063] In some instances, the mRNA molecule is a self-amplifying mRNA (saRNA) molecule. Unlike a traditional mRNA molecule, a saRNA molecule replicates itself after cellular entry. In addition to the basic mRNA components described above, an saRNA molecule comprises a second open reading frame (ORF) encoding an RNA-dependent RNA polymerase (along with its helper proteins), which is expressed within cells and replicates the mRNA molecule. This can allow smaller doses of mRNA to be used compared to vaccines using non-amplifying mRNA molecules, but substantially increases the size of the mRNA molecule to be delivered.

[0064] In some instances, the mRNA molecule is a circular RNA (circRNA) molecule. Synthesis of circRNA can be achieved by producing a traditional, linear mRNA molecule and then ligating the 5 ’ and 3 ’ ends to produce a circular structure (as described in e.g. Obi & Chen, Methods 196: 85-103, 2021). Due to the lack of termini and inaccessibility to exonucleases, circRNA can be more stable than linear mRNA, and so use of circRNA is another means by which the required mRNA dosage can be reduced.

[0065] mRNA Structure

[0066] The mRNA molecules provided herein may comprise one or more modified nucleotides or nucleosides. Such a modified nucleotide may be a modified adenosine (A), guanosine (G), uridine (U) or cytidine (C). By “modified” here is meant that the structure of the nucleotide is altered relative to the natural structure. For any given nucleotide, all instances of the nucleotide in the mRNA may be modified, all instances may be unmodified (i.e. have the native structure of the nucleotide), or a certain percentage of the nucleotide in the mRNA may be modified, e.g. about 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the hMPV-100

[0067] instances of the nucleotide in the mRNA may be modified. Where the mRNA contains a modified nucleotide, the percentage of the nucleotide in the mRNA which may be modified may be e.g. 5 to 20%, 5 to 25%, 5 to 50%, 5 to 60%, 5 to 70%, 5 to 80%, 5 to 90%, 5 to 95%, 10 to 20%, 10 to 25%, 10 to 50%, 10 to 60%, 10 to 70%, 10 to 80%, 10 to 90%, 10 to 95%, 10 to 100%, 20 to 25%, 20 to 50%, 20 to 60%, 20 to 70%, 20 to 80%, 20 to 90%, 20 to 95%, 20 to 100%, 50 to 60%, 50 to 70%, 50 to 80%, 50 to 90%, 50 to 95%, 50 to 100%, 70 to 80%, 70 to 90%, 70 to 95%, 70 to 100%, 80 to 90%, 80 to 95%, 80 to 100%, 90 to 95%, 90 to 100%, or 95 to 100%. A modified nucleotide may comprise a modified nucleobase (modified adenine, guanine, cytosine or uracil), a modified ribose sugar and / or a modified phosphate group.

[0068] Where a proportion of a particular nucleotide (e.g. adenosine) is modified, but not all instances of the nucleotide in the mRNA, the modified nucleotides may be evenly or randomly distributed across the mRNA, or may be localised in particular, chosen sequence regions. For instance, the modified nucleotide may be utilised in the coding region but not tire UTRs.

[0069] The mRNA molecule may contain modified versions of 1, 2, 3 or all 4 natural nucleosides, e.g. 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.

[0070] Where the mRNA molecule contains one or more modified nucleotides, the total proportion of nucleotides in the molecule which are modified may be anything up to 100%. For example, the mRNA may comprise about 10, 20, 30, 40, 50, 60, 70, 80 or 90% modified nucleotides. For instance, the mRNA may comprise 1 to 10%, 1 to 20%, 1 to 25%, 1 to 50%, 1 to 60%, 1 to 70%, 1 to 80%, 1 to 90%, 1 to 95%, 1 to 100%, 5 to 10%, 5 to 20%, 5 to 25%, 5 to 50%, 5 to 60%, 5 to 70%, 5 to 80%, 5 to 90%, 5 to 95%, 10 to 20%, 10 to 25%, 10 to 50%, 10 to 60%, 10 to 70%, 10 to 80%, 10 to 90%, 10 to 95%, 10 to 100%, 20 to 25%, 20 to 50%, 20 to 60%, 20 to 70%, 20 to 80%, 20 to 90%, 20 to 95%, 20 to 100%, 50 to 60%, 50 to 70%, 50 to 80%, 50 to 90%, 50 to 95%, 50 to 100%, 70 to 80%, 70 to 90%, 70 to 95%, 70 to 100%, 80 to 90%, 80 to 95%, 80 to 100%, 90 to 95%, 90 to 100%, or 95 to 100% modified nucleotides.

[0071] In some instances, the mRNA does not contain any modified nucleotides, i.e. it contains only the native adenosine, cytidine, guanosine and uridine structures.

[0072] The mRNA molecule provided herein may be a modified mRNA molecule comprising modified intemucleoside linkages, e.g. it may comprise modifications to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone. hMPV-100

[0073] Modified mRNA molecules (i.e. mRNA molecules comprising one or more modified nucleotides and / or modified intemucleoside linkages) may be synthesised using modified nucleotides or may be modified post-synthesis.

[0074] The nucleobases found in the mRNA molecule provided herein are generally the standard RNA nucleobases, i.e. adenine, cytosine, guanine and uracil. Modified mRNA molecules may by synthesized by any suitable method, e.g. chemically, enzymatically, or recomb inantly, to include one or more modified or non-natural nucleotides or intemucleoside linkages.

[0075] Where a modified nucleotide comprises a modified ribose moiety, the 2' hydroxyl group (OH) can be modified or replaced with a number of 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 = an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar moiety); polyethyleneglycols (PEG); “locked” nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g. by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, wherein the amino group can be e g. an alkylamino, dialkylamino, heterocyclylamino, arylamino, diarylamino, heteroarylamino, diheteroaryl amino, ethylene diamine, polyamino or aminoalkoxy group).

[0076] “Deoxy” modifications include hydrogen, amino (e g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, hctcroaryl amino, dihctcroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N , and O.

[0077] The ribose moiety may alternatively or further comprise any additional suitable or desired modification at the 2’ OH group or at any other position on the sugar.

[0078] Where a modified nucleotide comprises a modified nucleobase, the nucleobase may be modified at any location by any suitable or desired modification, e.g. by addition of an amino group, a thiol group, an alkyl group, or a halo group.

[0079] Modified nucleosides which may be used in the mRNA molecules provided herein include: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2 -methylthio -N6-methyladeno sine ; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1 -methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine; 2 -methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-0-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methylthio-N6-isopentenyladenosine; 7 -de aza-adenosine; N1 -methyladenosine; N6-cis-hydroxy- hMPV-100

[0080] isopentenyladenosine; a-thio -adenosine; 2-aminoadenosine; 2-(aminopropyl)adenosine; 2-propyladcnosinc; 2'-amino-2'-dcoxyadcnosinc; 2,-azido-2'-dcoxyadcnosinc; 8-aminoadcnosinc; 8-hydroxyadenosine; 8-thioadenosine; 8-azidoadenosine; 8-azaadenosine; 7-deaza-8-aza-adenosine; 7-methyladenosine; 1 -deazaadenosine; 2'-fluoro-N6-benzoyl-deoxyadenosine; 2'-O-methyl-2 -aminoadenosine; 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-deazaadenosine; 2-thiocytidine; 3 -methylcytidine; 5 -hydroxymethyl cytidine; 5 -methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 5 -formyl -2'-O-mcthylcytidinc; lysidinc; N4,2'-O-dimcthylcytidinc; N4-acctyl-2'-O-mcthylcytidinc; N4-methylcytidine; N4,N4-dimethyl-2'-O-methylcytidine; 4 -methylcytidine; 5 -azacytidine; pseudoisocytidine; a-thio-cytidine; 2'-amino-2'-deoxycytidine; 2'-azido-2'-deoxycytidine; 3-deaza- 5 -azacytidine; 5-propynylcytidine; 5 -trifluoromethylcytidine; 5 -bromocytidine; 5 -iodocytidine; 6-azacytidinc; pscudoisocytidinc; 2'-O-mcthyl-5-mcthyl-cytidinc; 2-thio-5-mcthyl-cytidinc; 5-methylzebularine; zebularine; (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 -aminoallyl cytidine; cyanocytidine; 5 -etliynyl cytidine; 5 -methoxycytidine; N4 -aminocytidine; N4-benzoylcytidine; 7-m ethylguanosine; N2,2'-O-dimethylguanosine; N2 -methylguanosine; wyosine; l,2'-O-dimethylguanosine; 1 -methylguanosine; 2'-O-methylguanosine; 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archaeosine; methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6 -thioguano sine; 7-deazaguanosine; 8-oxoguanosine; a-thio-guanosine; 2'-amino-2'-deoxyguanosine; 2'-azido-2'-deoxyguanosine; 6-O-methylguanosine; 8-aminoguanosine; 8 -hydroxyguano sine; 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-mcthylinosinc; inosine; l,2'-O-dimcthylinosinc; 2'-O-mcthylinosinc; 7-mcthylinosinc; cpoxyqucuosinc; hMPV-100

[0081] galactosyl-queuosine; mannosyl-queuosine; queuosine; 2'-O-methyluridine; 2 -thiouridine; 3 -methyluridine; 5 -carboxymethyluridine; 5 -hydroxyuridine; 5 -methyluridine; 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'-0 -methyluridine ; 5 -carboxymethylamino methyl -2 -thiouridine ; 5 -carboxymethylaminomethyluridine ; 5-m ethoxycarbonylmethyl -2'-O-methyluridine; 5-methoxycarbonylmethyl -2-thiouridine; 5-methoxycarbonylmethyluridine; 5 -methoxyuridine; 5 -methyl -2 -thiouridine; 5 -m ethylaminomethyl -2-selenouridine ; 5 -methylaminomethyl -2 -thiouridine ; 5 -methylaminomethyluridine ; 5-methyldihydrouridine; uridine -5 -oxyacetic acid; methyluridine -5 -oxyacetic acid; 5-(isopentenylaminomethyl)uridine; 5-propynyluridine; a-thio -uridine; 2'-deoxyuridine; 2’-deoxy-2'-fluorouridinc; 2'-amino-2'-dcoxyuridinc; 2'-azido-2'-dcoxyuridinc; 4-thiopscudouridinc; 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-dcazauridinc; 2-thio-6-azauridinc and 2-thiopscudouridinc.

[0082] Where the mRNA molecule comprises a modified version of a particular nucleotide, it may contain one or more, e.g. 2, 3 or 4 different modified versions of that nucleotide.

[0083] In particular instances, the mRNA molecule comprises modified uridine nucleotides, in particular pseudouridine or modified pseudouridine nucleotides. Pseudouridine is an isomer of the natural uridine nucleoside in which uracil is attached to ribose via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Examples of modified pseudouridine nucleosides include those set out above. The mRNA molecule may comprise pseudouridine and / or one or more species of modified pseudouridine. The mRNA molecule may comprise exclusively pseudouridine or modified pseudouridine nucleosides (i.e. all uridine residues may be replaced with pseudouridine or modified pseudouridine residues), or only a proportion of uridine residues may be replaced with pseudouridine or modified pseudouridine residues.

[0084] Tn particular instances, the mRNA molecule comprises the modified pseudouridine N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine). The structure of N1 -methylpseudouridine is set out below in Formula I: hMPV-100

[0085]

[0086] In particular instances, the mRNA molecule contains exclusively N1 -methylpseudouridine. That is to say, in these instances all uridine residues are replaced with N1 -pseudouridine. In other instances the mRNA molecule comprises at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% N1 -methylpseudouridine (i.e. at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of uridine residues in the mRNA molecule may be replaced with N 1 -methylpseudouridine).

[0087] Where the intemucleoside linkages are modified, the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the nucleotides can be modified by the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups which may be used in the mRNA molecules provided herein include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) or carbon (bridged methylene-phosphonates).

[0088] mRNA molecules require a 5’ cap to avoid innate immune inactivation. The nucleic acid molecule of the disclosure may further comprise a 5’ cap, which is linked to the 5’ terminal nucleotide of the mRNA molecule. The cap may have any suitable structure. A 5' cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Generally, the 5' cap is linked to the 5' terminus via a 5 '-5 '-triphosphate linkage.

[0089] The cap may have a CapO structure, i.e. an N7-mcthylguanosinc connected to the 5' nucleotide of the mRNA molecule through a 5' to 5 ' triphosphate linkage. A CapO structure may be referred to as an m7G or m7GpppN cap (where N indicates any nucleotide).

[0090] Alternatively, the mRNA cap may have a Capl structure. The Capl structure differs to the CapO structure by the addition of a methyl group at the 2’0 position on the first residue of the mRNA. The Capl structure may be referred to as an m7GpppNm cap (where Nm refers to any 2’-O-methylated hMPV-100

[0091] nucleotide). This structure may alternatively be presented as m7Gppp(2’Om)N. Inclusion of a 5’ cap having a Cap 1 structure may be particularly suitable for the mRNA molecules provided herein. A Cap 1 structure is shown in Formula II below (showing guanosine as an exemplary first residue):

[0092]

[0093] The Capl structure may in particular have the structure m7GpppAm (i.e. wherein the first nucleotide in the mRNA molecule is adenosine). A Capl structure may be applied to mRNA molecules by any suitable process, e.g. chemically or enzymatically. For example, a Capl structure can be applied to mRNA using the CleanCap® Reagent AG (TriLink, CA, USA).

[0094] In other instances, the mRNA molecule provided herein comprises a 5’ cap having a modified Capl structure. A modified Capl structure is defined herein as a Capl structure (i.e. comprising the m7GpppNm structure) having an additional structural modification to the methylguanosine cap itself, the 5’ nucleotide of the mRNA molecule or the triphosphate linker between them. For example, the 5’ cap may have a Capl structure in which the N7-methylguanosine cap comprises an additional methyl group at the 3’0 position. Such a modified Capl may be referred to as an m7(3’Om)GpppNm cap. In particular instances, the mRNA molecule comprises a modified cap with the structure m7(3’Om)GpppAm (i.e. wherein the first nucleotide residue of the mRNA molecule is adenosine). Another example of a modified Capl 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 described above, further comprising an additional methyl group at position 6 of the adenosine residue at the 5’ terminus of the mRNA molecule. The modified Capl structures described above can be applied to mRNA using the CleanCap® Reagent AG (3’OMe) and the CleanCap® Reagent M6, respectively.

[0095] Further suitable cap structures and approaches for generating suitable cap structures are disclosed in WO2017 / 053297 and Tusup et al., Design of in vitro Transcribed mRNA Vectors for Research and Therapy, Chim Int J Chem. 2019;73(5):391-394, both of which are hereby incorporated by reference. '-capping of polynucleotides may be completed concomitantly during the in vztro -transcription reaction hMPV-100

[0096] using the following chemical RNA cap analogs to generate the 5 '-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5')G [the ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5' capping of modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the "CapO" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The Capl structure may be generated using both vaccinia virus capping enzyme and a 2'-O methyl-transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. The Cap2 structure maybe generated from the Capl structure followed by the 2'-O-methylation of the 5' antepenultimate nucleotide using a 2'-O methyl-transferase. The Cap3 structure may be generated from the Cap2 structure followed by the 2'-O-methylation of the 5' preantepenultimate nucleotide using a 2'-O methyl-transferase. Enzymes may be derived from a recombinant source. Further suitable means for generating suitable cap structures are disclosed in WO2016 / 193226, which is hereby incorporated by reference. Where the mRNA molecule is a circular RNA molecule, no cap is present.

[0097] UTRs and other Sequence Elements

[0098] As noted above, mRNA molecules naturally comprise a 5’ UTR and a 3’ UTR. The mRNA molecule provided herein may similarly comprise a 5 ’UTR and / or a 3’ UTR, generally both a 5’ UTR and a 3’ UTR. The coding sequence (encoding the nanoparticle subunit) is operably linked to the 5’ UTR and the 3’ UTR it is located between.

[0099] Any suitable 5 ’ and 3 ’ UTRs may be used in the mRNA molecules provided herein. The UTRs may be of any suitable length. For example, the 3’ UTR may be at least 20, 30, 40, 50 or 60 nucleotides long, e.g. 30 to 100, 40 to 90, 50 to 80 or 60 to 70 nucleotides long. The 5’ UTR may be at least 10, 15, 20, 25 or 30 nucleotides long, e.g. 10 to 50, 20 to 40 or 30 to 35 nucleotides long.

[0100] Some 5’ UTRs comprise a 5’ terminal oligopyrimidine tract (TOP). This is typically a stretch of pyrimidine nucleotides located in the 5' UTR. The sequence generally starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of e.g. about 3 to 30 pyrimidine nucleotides. For example, a TOP may comprise at least 3, 5, 10, 15, 20, 25 or 30 pyrimidine nucleotides. The pyrimidine stretch and thus the 5' TOP ends one nucleotide 5' to the first purine nucleotide located downstream of the TOP.

[0101] Any suitable combination of UTRs may be used in the mRNA molecule provided herein. The 5’ and 3’ UTRs may originate or be derived from a gene or may be synthetic. The 5’ and 3’ UTRs may have the same source or different sources, e.g. the 5 ’ UTR may be synthetic and the 3 ’ UTR may originate or be derived from a gene, or vice versa. A UTR originating from a gene refers to a UTR which is identical to a UTR found in nature, in the context of a gene. A UTR derived from a gene refers to a UTR which is based on a UTR found in nature, but which is modified relative to that native (parent) UTR. A UTR hMPV-100

[0102] derived from a gene may have e.g. at least 70, 75, 80, 85, 90 or 95% sequence identity to the parent UTR, and / or may comprise additional sequence elements 5’ and / or 3’ to the parent UTR.

[0103] Where a UTR originates or is derived from a gene, that gene is a eukaryotic gene, generally a mammalian gene, in particular a human gene.

[0104] Particular 5’ UTRs which may be used in the mRNA molecule provided herein include sequences originating from or derived from: the human chitinase-1 (CHIT1) 5’ UTR; the human protein kinase cAMP -activated catalytic subunit beta (PRKACB) 5’ UTR; and the human glutamic -oxaloacetic transaminase 1 (GOT1) 5’ UTR. Particular 3’ UTRs which may be used in the mRNA molecule provided herein include sequences originating from or derived from: the human citrate synthase (CS) 3’ UTR; and tire human CHIT1 3’ UTR.

[0105] The human CHIT1 5’ UTR may have the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the mRNA molecule comprises a 5’ UTR comprising or consisting of the nucleotide sequence of SEQ ID NO: 15, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 15.

[0106] The human PRKACB 5’ UTR may have the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the mRNA molecule comprises a 5’ UTR comprising or consisting of the nucleotide sequence of SEQ ID NO: 16, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 16.

[0107] The human GOT1 5’ UTR may have the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the mRNA molecule comprises a 5’ UTR comprising or consisting of the nucleotide sequence of SEQ ID NO: 17, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 17.

[0108] The human CS 3’ UTR may have the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the mRNA molecule comprises a 3’ UTR comprising or consisting of the nucleotide sequence of SEQ ID NO: 19, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 19.

[0109] The human CHIT1 3’ UTR may have the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the mRNA molecule comprises a 3’ UTR comprising or consisting of the nucleotide sequence of SEQ ID NO: 20, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 20.

[0110] In particular embodiments, the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 15. An extended UTR comprising SEQ ID NO: 15 with additional sequence elements at both ends is set forth hMPV-100

[0111] in SEQ ID NO: 18. In particular instances the 5’ UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 18, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto. In particular embodiments, the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 19. An extended UTR comprising SEQ ID NO: 19 with additional sequence elements at both ends is set forth in SEQ ID NO: 21. In particular instances the 3 ’ UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto. The 5’ and 3’ UTR sequences set forth above may be used in combination. Thus the mRNA molecule may comprise: (i) a 5’ UTR comprising or consisting of a nucleotide sequence as set forth in any one of SEQ ID NOs: 15-18, or a variant of any one of SEQ ID NOs: 15-18 having at least 80, 85, 90 or 95% sequence identity thereto; and (ii) a 3’ UTR comprising or consisting of a nucleotide sequence as set forth in any one of SEQ ID NOs: 19-21, or a variant of any one of SEQ ID NOs: 19-21 having at least 80, 85, 90 or 95% sequence identity thereto.

[0112] In particular instances, the mRNA molecule comprises a 5 ’ UTR and a 3 ’ UTR, wherein:

[0113] (i) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human CHIT1, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CS;

[0114] (ii) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human PRKACB, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CHIT1;

[0115] (iii) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human PRKACB, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CS;

[0116] (iv) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human CHIT1, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CHIT1;

[0117] (v) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human GOT1, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CS; or

[0118] (vi) the 5’ UTR comprises or consists of a sequence originating or derived from a 5’ UTR of human GOT1, and the 3’ UTR comprises or consists of a sequence originating or derived from a 3’ UTR of human CHIT1.

[0119] In particular instances, the mRNA molecule comprises a 5 ’ UTR and a 3 ’ UTR, wherein:

[0120] (i) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises hMPV-100

[0121] or consists of the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto;

[0122] (ii) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto;

[0123] (iii) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto;

[0124] (iv) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto;

[0125] (v) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto; or

[0126] (vi) the 5’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto, and the 3’ UTR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 80, 85, 90 or 95% sequence identity thereto.

[0127] It has previously been found that particularly high levels of protein expression are obtained from mRNA molecules comprising the 5’ UTR of SEQ ID NO: 15 (derived from the human CHIT1 5’ UTR) and a 3’ UTR comprising SEQ ID NO: 19 (derived from the human CS 3’ UTR) (WO 2024 / 153793). Thus in a particular instance, the mRNA molecule comprises: (i) a 5’ UTR comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 90 or 95% identity to SEQ ID NO: 15; and (ii) a 3’ UTR comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 90 or 95% identity to SEQ ID NO: 19.

[0128] Particularly high levels of protein expression have been found to be obtained when the 5’ UTR comprising SEQ ID NO: 15 is the 5’ UTR of SEQ ID NO: 18, and the 3’ UTR comprising SEQ ID NO: 19 is the 3’ UTR of SEQ ID NO: 21. Thus in a particular instance, the mRNA molecule comprises: (i) a 5’ UTR comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 90 or 95% identity to SEQ ID NO: 18; and (ii) a 3’ UTR comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 90 or 95% identity to SEQ ID NO: 21. hMPV-100

[0129] Where variants of the specified UTRs are used, the variants are generally at least functionally equivalent to the specified UTRs. By “functionally equivalent” in the context of UTR sequences is meant that the variant UTRs cause an equivalent (or similar) level of protein expression to the specified UTR sequences. A “similar” level of protein expression may mean that the variant UTR causes a protein expression level which is as high as, or at least 70, 75, 80, 85, 90 or 95% as high as, the specified, unmodified UTR sequence. That the variants are “at least functionally equivalent” to the specified sequences means that the variant sequences may be superior to the specified, unmodified UTR sequence, i.e. they may cause a higher level of protein expression than the specified, unmodified UTR sequence.

[0130] Tn some instances, the mRNA molecule provided herein comprises a polyadenylation signal (poly(A) tail) . The poly(A) tail is a long sequence of adenine residues which lies at the 3 ’ end of the molecule, 3 ’ to the 3’ UTR. The role of the poly(A) tail is two-fold: the poly(A) tail is essential for translation, with poly(A) binding proteins (PABP) recruiting translation factors to enhance translation levels. Furthermore, the poly(A) tail increases the stability of mRNA, as binding of PABP to the poly(A) tail of mRNA protects it against exonuclease digestion. The poly(A) tail is also known to play a key role in the transport of mRNA from the nucleus to the ribosomes (Shlake, T., et al., RNA Biol., (2012), 9(11), 1319-1330). In one instance, the mRNA molecule of the disclosure comprises a poly(A) tail of about 10 to about 500 adenosine nucleotides. For example, the poly(A) tail may contain about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine nucleotides. In some instances, the poly(A) tail contains 50 to 250 adenosine nucleotides. In some instances, the poly(A) tail contains 40 to 100 or 60 to 100 adenosine nucleotides, e.g. 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 or 75 to 80 adenosine nucleotides. In some instances, the poly(A) tail contains 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 adenosine nucleotides. In some instances, the poly(A) tail contains 77 adenosine nucleotides. In one instance of the disclosure, the nucleic acid molecule comprises a split poly(A) tail. A split poly(A) tail can comprise at least two adenosine -containing elements, optionally of between 30 and 60 adenosines each, separated by a spacer optionally of between 1 and 25 nucleotides.

[0131] Multimerization Unit

[0132] The nucleic acid molecule (particularly mRNA molecule) disclosed herein encodes a fusion polypeptide (or fusion protein), comprising an hMPV F glycoprotein (or immunogenic fragment thereof) linked to a multimerization unit. The multimerization unit is a protein which can self-assemble into a multimer, i.e. to form a protein complex comprising multiple copies of the multimerization unit. In some instances, the multimerization unit is a protein that can self-assemble into a protein nanoparticle. In these cases. hMPV- 100

[0133] the fusion protein encoded by the mRNA may be referred to as a “nanoparticle subunit”. In some instances, vims-like particles (VLPs) can be used to form stable nanoparticle structures.

[0134] The fusion protein encoded by the mRNA provided herein comprises the hMPV F protein joined to the multimerization unit (the multimerization unit may alternatively be referred to herein as a scaffold). The fusion protein may further comprise a linker joining the hMPV F protein to the scaffold. In some cases, the fusion protein comprises one or more additional elements, e.g. a signal peptide.

[0135] The components of the fusion protein may be arranged in either order. That is to say, the fusion protein may comprise hMPV F protein at its N-terminus and the scaffold at the C-terminus, or it may comprise hMPV F protein at its C-terminus and the scaffold at its N-terminus.

[0136] In some embodiments, tire multimerization unit is selected from tire group consisting of: foldon, ferritin, lumazine synthase or beta annulus.

[0137] In some embodiments, the multimerization unit is or comprises a lumazine synthase (LuS). The Examples below demonstrate that, unpredictably, hMPV F protein presented on a LuS scaffold induces a stronger immune response than the same antigen presented on different nanoparticle scaffolds. LuS is an enzyme responsible for the catalysis of the penultimate reaction in the riboflavin biosynthetic pathway. LuS is found in a range of organisms, including bacteria, archaea, fungi and plants. Some LuS enzymes form icosahedral capsids, which can be used in drug delivery or in vaccines to deliver antigens. LuS enzymes which form icosahedral capsids are generally derived from eubacteria and plants. Such capsids are generally referred to as protein cage nanoparticles. Herein, icosahedral LuS capsids are also referred to as vims-like particles (VLPs). Icosahedral LuS capsids are generally homomeric 60-mers, containing 60 identical lumazine synthase subunits (though capsids containing different numbers of protein subunits are also known). The 60-meric structures may be considered dodecamers of pentamers, with particularly strong interactions formed between the subunits within each pentamer, and weaker interactions formed between the pentamers which assemble to form the capsid.

[0138] In 60-meric lumazine synthase capsids, both the N- and C-terminus of each subunit is exposed on the outer surface. Lumazine synthase capsids can thus be used as nanoparticles for antigen display, by fusing a target antigen to either the N-terminus or C-terminus of the lumazine synthase enzyme. The presentation of antigens on the surface of the capsid results in a high concentration of antigens presented in an ordered array, which can elicit a strong immune response.

[0139] The lumazine synthase used as the scaffold in the present fusion proteins is thus a lumazine synthase which multimerises to form a protein nanoparticle, generally an icosahedral capsid. In particular, the lumazine synthase used in tire scaffold in tire fusion protein described herein may be a lumazine synthase which multimerises to form a 60-mer, i.e. a lumazine synthase which multimerises to form a 60-meric hMPV-100

[0140] icosahedral capsid, or a 60-meric nanoparticle. The lumazine synthase may originate or be derived from any species which produces such an enzyme, for instance a plant (e.g. spinach (Spinacia oleracea), tobacco (e.g. Nicotiana tabacum or Nicotiana rustled) or Arabidopsis thaliand) or a eubacterium. Bacteria which produce LuS enzymes which form 60-meric nanoparticles include those of the family Bacillaceae, e.g. Bacillus siibiihs. those of the family Enterobacteriaceae, e.g. Escherichia coll, and those of the family Aquificaceae, e.g. Aquifex aeolicus.

[0141] As set out above in respect of UTRs, a lumazine synthase originating from a particular species has the native sequence of the enzyme from that species. A lumazine synthase derived from a species is a modified version of the native enzyme. Where a modified version of a naturally occurring lumazine synthase is used, the modified version retains the ability to form multimeric complexes, and at least one of the N- and C-termini remain exposed on the outer surface of the capsid so that the immunogen can be fused to it and displayed on the capsid surface.

[0142] A modified version of a lumazine synthase which natively forms a 60-meric structure may retain the ability to form a 60-meric structure, or may form a different structure with more or fewer subunits than 60. Modified bacterial lumazine synthases are known which form 180-meric or 360-meric nanoparticles (Ladenstein & Morgunova, Biotechnology Reports 27: e00494, 2020), and such modified lumazine synthases may be used in the fusion proteins encoded by the mRNA molecules provided herein.

[0143] Alternatively, the lumazine synthase used herein may natively form smaller multimers, comprising e.g.

[0144] 5 or 10 subunits. Modified versions of such lumazine synthases may be used, which may form larger multimcrs than the native enzyme.

[0145] Where the lumazine synthase used is a variant of a native enzyme, the enzyme may be inactive. Since the enzyme is merely used as a scaffold, it is unimportant whether it retains its synthase activity.

[0146] In particular instances the lumazine synthase is the A. aeolicus lumazine synthase, or a derivative thereof. The A. aeolicus lumazine synthase has the amino acid sequence set out in SEQ ID NO: 10 (UniProt entry 066529). Thus the scaffold used in the fusion protein herein may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 10. In particular instances, the scaffold comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10.

[0147] Where a variant of SEQ ID NO: 10 is used, the variant retains the ability to form multimeric protein nanoparticles. In particular, the variant may retain the ability to form the 60-meric complexes discussed above. Assembly of A. aeolicus lumazine synthase pentamers into icosahedral capsids has been found to rely on 8 amino acid residues in each subunit, which form hydrophobic interactions (LI 21 and 1125), electrostatic interactions (E5, R21, D36, R40 and E145), and a hydrogen bond (H41), between neighbouring pentamers. Disruption of these interactions (particularly the hydrogen bond) by point hMPV- 100

[0148] mutation has been found to prevent dodecamerisation of the pentameric capsid building blocks, such that the enzyme forms only a pentameric structure (Hickman el al.. Proteins 83: 1733-1741, 2015). To avoid loss of the ability to form 60-meric or other larger structures, variants of the enzyme of SEQ ID NO: 10 may be unmodified at these locations, or contain conservative substitutions at these locations which do not disrupt the intersubunit interactions.

[0149] In some embodiments, the lumazine synthase protein is encoded in the mRNA molecule by the nucleotide sequence of SEQ ID NO: 9, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 9. In particular, the lumazine synthase protein may be encoded by a nucleotide sequence which has at least 80, 85, 90 or 95% identity to SEQ ID NO: 9, and which encodes an amino acid sequence having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 10. In some embodiments, the lumazine synthase is encoded by a nucleotide sequence which is degenerate with SEQ ID NO: 9 (i.e. a nucleotide sequence which encodes the same amino acid sequence as SEQ ID NO: 9, but which differs due to tire degeneracy of the genetic code).

[0150] Antigen

[0151] The antigen (or immunogen) is the hMPV F protein (or an immunogenic fragment thereof), against which it is desired to stimulate protective immunity. As set out above, the hMPV F protein is known to be a primary target for neutralising antibodies during hMPV infection. The hMPV F protein may originate or be derived from any hMPV strain, e.g. it may be from an hMPV A strain or an hMPV B strain.

[0152] While a full-length hMPV F protein may be used, generally only an immunogenic fragment of the F protein is used. By “immunogenic fragment” is meant a fragment of an hMPV F protein (i.e. an amino acid sequence corresponding to part of, but not all of, an hMPV F protein) which, when administered to or expressed in a subject (particularly a human), induces a neutralising antibody response against the hMPV F protein. An immunogenic fragment of an hMPV F protein may be truncated at the N- and / or C-terminus relative to the wild type F protein from which it is derived. The immunogenic fragment may be any length which is sufficient to induce a neutralising antibody response against the full-length protein, e.g. it may be at least 100, 150, 200, 250, 300, 350, 400 or 450 amino acids long. The immunogenic fragment may be less than 500, 450 or 400 amino acids long. In particular embodiments, the immunogenic fragment is between 450 and 500 amino acids long, e.g. 460-500, 470-500, 480-500, 460-490, 470-490 or 480-490 amino acids long.

[0153] In particular embodiments, the immunogenic fragment of the hMPV F protein is truncated at its C-terminus relative to the wild type protein, such that the transmembrane domain is deleted. Commonly, the ectodomain of the hMPV F protein is used in the constructs provided herein (i.e. the F protein domain which, in the context of an hMPV virion, is located on the virion surface). General references hMPV- 100

[0154] herein to an hMPV F protein encompass immunogenic fragments of the F protein, including hMPV F protein ectodomains, unless the context dictates otherwise .

[0155] In particular embodiments, the hMPV F protein included in the fusion protein encoded by the mRNA is, or is derived from, the hMPV F protein ectodomain set forth in SEQ ID NO: 2. Thus in some embodiments, the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO: 2, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 2. The F protein ectodomain of SEQ ID NO: 2 is aderivative of the F protein ofhMPV strain NL / 1 / 00, sublineage Al. In particular embodiments, the hMPV F protein used herein is a wild type hMPV F protein, in particular an ectodomain of a wild type hMPV F protein. By a “wild type” hMPV F protein is meant an F protein encoded by an hMPV isolate (i.e. an F protein with a sequence which has not been modified relative to the sequence encoded by the hMPV strain from which it originates).

[0156] As set out above, it had previously been thought that a wild type hMPV F protein would not be suitable for an hMPV vaccine, and that a modified F protein stabilized in its prefusion conformation would need to be used. Surprisingly, the present inventors have found that wild type hMPV F protein can be effective in an hMPV vaccine, at least using the vaccine design provided herein.

[0157] In some embodiments, the hMPV F protein used herein comprises or consists of the wild type hMPV F protein cctodomain set out in SEQ ID NO: 2, as exemplified below.

[0158] Alternatively, the F protein (or an immunogenic fragment thereof) from a different hMPV strain may be used. For example, the F protein (or an immunogenic fragment thereof) having the UniProt accession number A0A1B1VVH7, A0A1B1VVH8, A0A1B1VVH9, A0A1B1VV10, A0A1B1VV1I, A0A1B1VVI6, C5MRS7, C6F440, H6X1X4, H6X1Y3,Q6QQF7 or Q6WB98 may be used, or the F protein having the GenBank accession number AAQ67686.

[0159] In some embodiments, an cctodomain from the F protein of a different hMPV strain may be used, c.g. the ectodomain (which may be the sequence corresponding to SEQ ID NO: 2) from the hMPV F protein with the UniProt accession number A0A1B1VVH7, A0A1B1VVH8, A0A1B1VVH9, A0A1B1VVI0, A0A1B1VVI1, A0A1B1VVI6, C5MRS7, C6F440, H6X1X4, H6X1Y3,Q6QQF7 or Q6WB98, orthe F protein having the GenBank accession number AAQ67686.

[0160] Alternatively, a modified hMPV F protein, or immunogenic fragment thereof, may be used (for instance a modified hMPV F protein ectodomain). By a “modified” hMPV F protein is meant an hMPV F protein which is not found in nature, i.e. has been modified relative to the native sequence. Thus any hMPV F protein sequence which is not a wild type sequence is a modified F protein sequence.

[0161] In particular embodiments, a modified hMPV F protein is used which is stabilized in tire prefusion conformation (referred to herein as a “prefusion-stabilized F protein”). That is to say, the modified hMPV- 100

[0162] hMPV F protein may contain mutations relative to the wild type protein which cause the prefusion conformation of the protein to be stabilized relative to the wild type protein, such that the modified protein is less likely to spontaneously change into the post-fusion conformation than the wild type protein.

[0163] A prefusion -stabilized F protein may bind to prefusion F-specific antibody (or an antibody which binds prefusion F preferentially to post-fusion F) with a higher affinity than wild type F protein. High temperature promotes the change in F protein conformation from the prefusion to tire post-fusion conformation, and so a prefusion-stabilized F protein may in particular bind to prefusion F-specific antibody with a higher affinity than wild type F protein following incubation of the F protein at a high temperature, e g. at a temperature of about 30°C, 40°C or 50°C for about 10, 15, 20, 25 or 30 minutes. Prefusion F-specific antibodies which may be used in such an assessment are known in the art and include MPE8 (Corti et al., Nature 501: 439-443, 2013). Assays using MPE8 to assess stabilization of the prefusion conformation of modified hMPV F protein are described in Hsieh et al. (supra').

[0164] Mutations which stabilize the prefusion conformation of the F protein are known in the art. One such mutation is the A185P mutation (based on the position numbering of SEQ ID NO: 2). In some embodiments, the profusion-stabilized hMPV F protein comprises the A185P mutation. That is to say, in some embodiments, the profusion-stabilized hMPV F protein comprises a proline residue at the position corresponding to position 185 of SEQ ID NO: 2.

[0165] In some embodiments, the F protein used in the fusion protein comprises or consists of a variant of SEQ ID NO: 2 comprising a proline residue at the position corresponding to position 185 of SEQ ID NO: 2. In particular, the F protein may comprise or consist of a variant of SEQ ID NO: 2 having at least 90% identity to SEQ ID NO: 2 and comprising a proline residue at the position corresponding to position 185 of SEQ ID NO: 2.

[0166] In some embodiments, the profusion-stabilized F protein comprising the A185P mutation comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 4, wherein any such variant comprises a proline residue at tire position corresponding to position 185 of SEQ ID NO: 4. The protein of SEQ ID NO: 4 corresponds to the hMPV F protein of SEQ ID NO: 2 with the A185P mutation.

[0167] Other mutations which are known to stabilize the hMPV F protein in the prefusion conformation include: L219K, V231I, H368N and E453Q (amino acid position numbering in accordance with SEQ ID NO: 2). Other stabilizing mutations include N139G, E323D and replacement of amino acids 97-102 with a hexaglycine linker. Where a prefusion-stabilized hMPV F protein is used herein it may contain any one, or any combination, of these mutations relative to the wild type sequence. In particular embodiments, a prefusion-stabilised hMPV F protein comprises the A185P mutation plus one or more of the stabilizing mutations listed above. hMPV- 100

[0168] In some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0169] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2;

[0170] (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2;

[0171] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; and (iv) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2. In some embodiments, such a prefusion-stabilised hMPV F protein further comprises an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 2.

[0172] Thus in some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0173] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2;

[0174] (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2;

[0175] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; (iv) an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 2; and (v) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2. In some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0176] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2;

[0177] (ii) glycine residues at the positions corresponding to positions 97-102 and 139 of SEQ ID NO: 2; and

[0178] (iii) an aspartic acid residue at the position corresponding to position 323 of SEQ ID NO: 2. In addition to the stabilizing mutations described above, the hMPV F protein may be stabilized in its prefusion conformation by introducing pairs of cysteine residues which can form disulphide bonds which prevent conformational change into the post-fusion state. In some embodiments, the prefusion-stabilised hMPV F protein contains one or more of the following pairs of mutations introducing such cysteine residues into the protein: L110C / N322C, T365C / V463C, A140C / A147C, T127C / N153C and V84C / A249C (amino acid position numbering in accordance with SEQ ID NO: 2). The prefusion-stabilised hMPV F protein may comprise at least 1, 2, 3 or 4 such pairs of cysteine mutations.

[0179] In some embodiments, a prefiision-stabilised hMPV F protein comprises the A185P mutation plus one or more of the cysteine mutation pairs listed above.

[0180] In some embodiments, the prefusion-stabilized hMPV protein comprises cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2. In some embodiments, the prefusion-stabilized hMPV protein comprises cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2, and a proline residue at the position corresponding to position 1 5 of SEQ ID NO: 2.

[0181] In some embodiments, the prefiision-stabilized hMPV protein comprises cysteine residues at the positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 2. In some embodiments, the hMPV- 100

[0182] prefusion-stabilized hMPV protein comprises cysteine residues at the positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 2, and a proline residue at the position corresponding to position 185 of SEQ ID NO: 2.

[0183] The cysteine mutations may also be combined with the other stabilizing mutations described above. In some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0184] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2;

[0185] (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2;

[0186] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; (iv) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2; and (v) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2.

[0187] In some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0188] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2;

[0189] (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2;

[0190] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; (iv) an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 2; (v) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2; and

[0191] (vi) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2.

[0192] In some embodiments, such a prefusion-stabilised F protein comprises or consists of the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 6, and comprising:

[0193] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 6;

[0194] (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 6;

[0195] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 6; (iv) an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 6; (v) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 6; and

[0196] (vi) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 6.

[0197] The prefusion -stabilised F protein of SEQ ID NO: 6 is referred to in the Examples herein as DSCavES2. This is based on the DSCavES2 prefusion-stabilised F protein described in Hsieh et al (supra), which is incorporated herein by reference in its entirety. In some embodiments, the hMPV F protein used in the fusion protein encoded by the mRNA provided herein comprises or consists of the sequence of the hMPV- 100

[0198] DSCavES2 hMPV F protein described in Hsieh et al. supra), which has the amino acid sequence set forth in SEQ ID NO: 45.

[0199] In some embodiments, a prefusion-stabilised F protein comprises or consists of the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 45, and comprising:

[0200] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 45; (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 45;

[0201] (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 45; (iv) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 6; and (v) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 45.

[0202] In some embodiments, a prefusion-stabilised hMPV F protein used herein comprises:

[0203] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2; (ii) glycine residues at the positions corresponding to positions 97-102 and 139 of SEQ ID NO: 2;

[0204] (iii) an aspartic acid residue at the position corresponding to position 323 of SEQ ID NO: 2;

[0205] and

[0206] (iv) cysteine residues at the positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 2.

[0207] In some embodiments, such a profusion-stabilised F protein comprises or consists of the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 8, and comprising:

[0208] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 8; (ii) glycine residues at the positions corresponding to positions 97-102 and 139 of SEQ ID NO: 8;

[0209] (iii) an aspartic acid residue at tire position corresponding to position 323 of SEQ ID NO: 8;

[0210] and

[0211] (iv) cysteine residues at the positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 8

[0212] The profusion-stabilised F protein of SEQ ID NO: 8 is referred to in the Examples herein as v3B, which corresponds to the v3B prefusion-stabilised F protein described in Stewart-Jones et al., PNAS 118(39): e2106196118, 2021 , which is incorporated herein by reference in its entirety.

[0213] As set out above, the prefusion-stabilised hMPV F protein sequences contain a number of mutations relative to the wild type sequence. These mutations are at positions defined as “corresponding to” a particular position in a reference sequence. Corresponding amino acid positions can be identified by hMPV- 100

[0214] sequence alignment to the reference sequence. A corresponding amino acid position is the position which corresponds to (or aligns with) the specified position in the reference sequence. Thus for example, the position in an hMPV F protein sequence of interest corresponding to position 185 of SEQ ID NO: 2 is the position which corresponds to (or aligns to) position 185 of SEQ ID NO: 2 when the F protein sequence of interest is aligned to the F protein sequence of SEQ ID NO: 2.

[0215] In some embodiments the hMPV F protein is encoded in the mRNA molecule by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 1. In particular, the hMPV F protein may be encoded by a nucleotide sequence which has at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 1, and which encodes an amino acid sequence having at least 80, 85, 90 or 95% identity to SEQ ID NO: 2.

[0216] In some embodiments, the hMPV F protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence which is degenerate with SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 encodes the wild type F protein ectodomain of SEQ ID NO: 2.

[0217] In some embodiments, the hMPV F protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence which is degenerate with SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 encodes the prefusion-stabilised F protein ectodomain of SEQ ID NO: 4, which corresponds to SEQ ID NO: 2 with the A185P mutation. In some embodiments, the hMPV protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence which is degenerate with SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 5 encodes the prefusion-stabilised F protein ectodomain of SEQ ID NO: 6, which is described above and is referred to in the Examples below as the DSCavES2 hMPV F protein.

[0218] In some embodiments, the hMPV protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence which is degenerate with SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 7 encodes the prefusion-stabilised F protein ectodomain of SEQ ID NO: 8, which is described above and is referred to in the Examples below as the v3B hMPV F protein .

[0219] Linkers

[0220] In some embodiments of the present disclosure, the fusion protein encoded by the mRNA molecule comprises a linker between the F protein and the multimerization unit. That is to say, the F protein and the multimerization unit may be joined by a linker. hMPV-100

[0221] Inclusion of a linker may be advantageous to provide physical separation (or distance) between the multimerization unit multimer and the immunogens arrayed on its surface, to improve accessibility of the F protein. The linker may be of any suitable length to achieve this separation, e.g. at least 3, 4, 5, 6, 7 or 8 amino acids long. The linker may have a maximum length of e.g. 20, 18, 16, 14, 12, 10 or 8 amino acids. For example, the linker may be 4 to 12 or 4 to 8 amino acids long.

[0222] The sequence of the linker is not particularly limited, so long as its length is suitable for the above -described purpose. Various linker sequences for joining fusion protein constituents are known in the art and can be used in the present fusion protein, including glycine-serine linkers (i.e. linkers consisting of glycine and serine residues). Examples of glycine -serine linkers include [G]n, [S]n, [A]n, [GS]n, [GG]n, [SA]n, [GGS]n, [GGG]n, [GSG]n, [GGGS]n, [GGGGS]n, [GGSG]n, [GSGG]n, [SGGG]n, [SSGG]n, [SSSG]n, [SAGS]n or [GGGSG]n, wherein ‘n’ is between 1 and 20 (a suitable value of ‘n’ is dependent on the length of the repeating unit - a suitable value for n' can be selected to yield a linker of a length falling within the ranges set out above).

[0223] In some embodiments, the linker is a glycine-serine linker with the sequence [GSG]n. In particular embodiments, the linker is a glycine-serine linker with the amino acid sequence |GSG ]?, i.e. GSGGSG (SEQ ID NO: 12). In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or a variant of SEQ ID NO: 12 comprising up to 3, up to 2, or 1 amino acid substitution, deletion and / or insertion relative to SEQ ID NO: 12. Amino acid substitutions relative to SEQ ID NO: 12 may be conservative amino acid substitutions. The linker of SEQ ID NO: 12 may be encoded by the nucleotide sequence of SEQ ID NO: 11.

[0224] In other embodiments, the linker is a glycine-serine linker with sequence SGGSSGSSGGS (SEQ ID NO: 43). In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, or a variant of SEQ ID NO: 43 comprising up to 3, up to 2, or 1 amino acid substitution, deletion and / or insertion relative to SEQ ID NO: 43. Amino acid substitutions relative to SEQ ID NO: 43 may be conservative amino acid substitutions. The linker of SEQ ID NO: 43 may be encoded by the nucleotide sequence of SEQ ID NO: 44.

[0225] The skilled person will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acid molecules provided herein). The skilled person will likewise appreciate that other polycistronic constructs (nucleic acid molecules encoding more than one antigen / polypeptide separately within the same molecule) may be suitable for use as provided herein.

[0226] Fusion Protein

[0227] In some embodiments, tire polypeptide (i.e. fusion protein or nanoparticle subunit) encoded by the mRNA molecule provided herein comprises or consists of the amino acid sequence set forth in SEQ ID hMPV- 100

[0228] NO: 14, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 14. The fusion protein of SEQ ID NO: 14 comprises the wild type hMPV F protein of SEQ ID NO: 2 at the N-terminus and the lumazine synthase of SEQ ID NO: 10 at the C-terminus, joined by the linker of SEQ ID NO: 12.

[0229] In some embodiments, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 14, and comprising:

[0230] (a) an hMPV F protein having at least 80, 85, 90 or 95% identity to the amino acid sequence ofSEQ ID NO: 2; and

[0231] (b) a lumazine synthase having at least 80, 85, 90 or 95% identity to the amino acid sequence ofSEQ ID NO: 10;

[0232] optionally joined by a linker, optionally wherein the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or a variant of SEQ ID NO: 12 comprising up to 3, up to 2, or 1 amino acid substitution relative to SEQ ID NO: 12.

[0233] In some embodiments, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 (the fusion protein comprising the A185P F protein joined to lumazine synthase). In some embodiments, the fusion protein comprises or consists of a variant of SEQ ID NO: 23 having at least 80, 85, 90 or 95% identity to SEQ ID NO: 23, and comprising:

[0234] (a) an hMPV F protein (or immunogenic fragment thereof) having at least 80, 85, 90 or 95% identity to the amino acid sequence of SEQ ID NO: 2, and comprising a proline residue at the position corresponding to position 185 of SEQ ID NO: 2; and

[0235] (b) a lumazine synthase having at least 80, 85, 90 or 95% identity to the amino acid sequence ofSEQ ID NO: 10;

[0236] optionally joined by a linker, optionally wherein the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or a variant of SEQ ID NO: 12 comprising up to 3, up to 2, or 1 amino acid substitution relative to SEQ ID NO: 12.

[0237] In some embodiments, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 (the fusion protein comprising the DSCavES2 F protein joined to lumazine synthase). In some embodiments, the fusion protein comprises or consists of a variant of SEQ ID NO: 25 having at least 80, 85, 90 or 95% identity to SEQ ID NO: 25, and comprising:

[0238] (a) an hMPV F protein having at least 80, 85, 90 or 95% identity to the amino acid sequence of SEQ ID NO: 2, and comprising:

[0239] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2; (ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2; (iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; hMPV-100

[0240] (iv) an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 2;

[0241] (v) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2; and

[0242] (vi) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2; and

[0243] (b) a lumazine synthase having at least 80, 85, 90 or 95% identity to the amino acid sequence ofSEQ ID NO: 10;

[0244] optionally joined by a linker, optionally wherein the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12 or 43, or a variant of SEQ ID NO: 12 or 43 comprising up to 3, up to 2, or 1 amino acid substitution relative to SEQ ID NO: 12 or 43.

[0245] In some embodiments, the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 (tire fusion protein comprising tire v3B F protein joined to lumazine synthase), hi some embodiments, the fusion protein comprises or consists of a variant of SEQ ID NO: 27 having at least 80, 85, 90 or 95% identity to SEQ ID NO: 27, and comprising:

[0246] (a) an hMPV F protein having at least 80, 85, 90 or 95% identity to the amino acid sequence of SEQ ID NO: 2, and comprising:

[0247] (i) a proline residue at the position corresponding to position 185 of SEQ ID NO: 2; (ii) glycine residues at the positions corresponding to positions 97-102 and 139 of SEQ ID NO: 2;

[0248] (iii) an aspartic acid residue at the position corresponding to position 323 of SEQ ID NO: 2; and

[0249] (iv) cysteine residues at the positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 2; and

[0250] (b) a lumazine synthase having at least 80, 85, 90 or 95% identity to the amino acid sequence ofSEQ ID NO: 10;

[0251] optionally joined by a linker, optionally wherein the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or a variant of SEQ ID NO: 12 comprising up to 3, up to 2, or 1 amino acid substitution relative to SEQ ID NO: 12.

[0252] In some embodiments, the fusion protein encoded by the mRNA molecule provided herein is encoded by the nucleotide sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 13 (SEQ ID NO: 13 encodes the fusion protein of SEQ ID NO: 14). In particular, the fusion protein may be encoded by the nucleotide sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 13 and which encodes a fusion protein having at least 80, 85, 90 or 95% identity to SEQ ID NO: 14, in particular wherein it encodes a fusion protein which is a variant of SEQ ID NO: 14 as described above. hMPV-100

[0253] In some embodiments, the fusion protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence which is degenerate with SEQ ID NO: 13.

[0254] In some embodiments, the fusion protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 22, or a nucleotide sequence which is degenerate with SEQ ID NO: 22. The nucleotide sequence of SEQ ID NO: 22 encodes the fusion protein of SEQ ID NO: 23 (comprising tire A185P F protein).

[0255] In some embodiments, the fusion protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence which is degenerate with SEQ ID NO: 24. Tire nucleotide sequence of SEQ ID NO: 24 encodes the fusion protein of SEQ ID NO: 25 (comprising the DSCavES2 F protein).

[0256] In some embodiments, the fusion protein is encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence which is degenerate with SEQ ID NO: 26. The nucleotide sequence of SEQ ID NO: 26 encodes the fusion protein of SEQ ID NO: 27 (comprising the v3B F protein).

[0257] Complete mRNAs

[0258] In some embodiments, the mRNA molecule provided herein comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 28 to 31, or a variant thereof having at least 80, 85, 90 or 95% identity to any one of SEQ ID NOs: 28 to 31.

[0259] SEQ ID NO: 28 is a full-length mRNA comprising the fusion protein coding sequence of SEQ ID NO: 13, the 5’ UTRof SEQ ID NO: 18, the 3 ’UTR of SEQ ID NO: 21 and a 77 nucleotide poly(A) sequence. Where the mRNA is a variant of SEQ ID NO: 28, the various elements (fusion protein coding sequence, UTR sequences and poly(A) sequence) are generally as described above.

[0260] SEQ ID NO: 29 corresponds to SEQ ID NO: 28, except that it encodes the A 185P F protein ectodomain, rather than the wild type ectodomain. SEQ ID NO: 30 corresponds to SEQ ID NO: 28 except that it encodes the DSCavES2 F protein ectodomain, rather than the wild type ectodomain. SEQ ID NO: 31 corresponds to SEQ ID NO: 28, except that it encodes the v3B protein ectodomain, rather than the wild type ectodomain. As for SEQ ID NO: 28, where the mRNA is a variant of any one of SEQ ID NOs: 29-31, the various elements (fusion protein coding sequence, UTR sequences and poly(A) sequence) are generally as described above. hMPV-100

[0261] Pharmaceutical Compositions

[0262] Tine present disclosure provides a composition comprising an mRNA molecule as defined anywhere herein. The pharmaceutical composition may be provided within, or take the form of, a vaccine. Thus also provided herein is a vaccine comprising an mRNA molecule as described herein or a pharmaceutical composition as described herein.

[0263] In the pharmaceutical composition, the mRNA molecule may be formulated in a suitable delivery vehicle for administration to a subject, in particular a lipid-based delivery system such as a lipid nanoparticle, a liposome or a lipoplex. Lipid nanoparticles may be particularly suitable .

[0264] Lipid Nanoparticles

[0265] In the pharmaceutical compositions provided herein, the mRNA molecule is generally formulated in a lipid nanoparticle. A lipid nanoparticle is an essentially spherical particle with a surface comprising a lipid layer surrounding a core. The lipid layer may be a monolayer or a bilayer. The core may be lipophilic, such as a solid lipid matrix, and may be stabilised by surfactants.

[0266] Lipid nanoparticle (LNP) characteristics and behaviour in vivo can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to the surface. Furthermore, LNPs can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, or carbohydrates) to their surface or to the terminal end of the attached PEG chains (Front Pharmacol. 2015 Dec 1;6:286). The mRNA molecule formulated in a lipid nanoparticle is generally encompassed by the lipid nanoparticle, i.e. the mRNA molecule is located in the core of the nanoparticle, entirely surrounded by lipid and is inaccessible to external milieu, hi the core, cationic and / or ionisable lipids can organise into inverted micelles around the encapsulated, negatively charged mRNA molecules.

[0267] Lipid nanoparticles (LNPs) as used herein may comprise a cationic lipid and an aggregation reducing agent (such as a PEGylated lipid, also referred to herein as a polyethylene glycol (PEG) -modified lipid or a PEG-lipid), and optionally a non-cationic lipid (such as a neutral lipid) and / or a sterol. Generally, the LNP comprises a cationic lipid, anon-cationic lipid, a PEG-lipid and a sterol.

[0268] LNPs may include any cationic lipid suitable for forming a lipid nanoparticle. The cationic lipid may carry a net positive charge at physiological pH. The cationic lipid may comprise a fatty acid chain of any suitable length, e.g. CIO, C12 or C14 to C22 or C24. The fatty acid chain may be saturated, monounsaturated or polyunsaturated (used herein to mean comprising two or more carbon-carbon double bonds).

[0269] The cationic lipid may be an amino lipid. As used herein, the term "amino lipid" is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid at physiological pH. hMPV-100

[0270] Suitable amino lipids include those having alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-). In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0271] In some instances, amino or cationic lipids have at least one protonatable or deprotonatable group (which may also be referred to as ionisablc lipids), such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the invention.

[0272] In some instances, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g. a pKa of about 5 to about 7.

[0273] LNPs can include two or more cationic lipids. Tire cationic lipids can be selected to contribute different advantageous properties. For example, cationic lipids that differ in 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 the LNP. In particular, the cationic lipids can be chosen so that the properties of the mixed LNP arc more desirable than the properties of a single LNP of individual lipids. In some instances, the ratio of cationic lipid to nucleic acid in the LNP is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.

[0274] The non-cationic lipid can be a neutral lipid, an anionic lipid, or an amphipathic lipid. Neutral lipids, when present, can be any of a number of lipid species which exist either 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 consideration of, e.g., LNP size and stability of the LNP in the bloodstream.

[0275] Neutral lipids may comprise a fatty acid chain of any suitable length, e.g. CIO, C12 or C14 to C22 or C24. The fatty acid chain may be saturated, monounsaturated or polyunsaturated. In some instances, the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of CIO to C20. In other instances, neutral lipids with monounsaturated or diunsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used. hMPV-100

[0276] Amphipathic lipids are those in which the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients towards the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, sphingolipids, glycosphingolipids, diacylglycerols and beta-acyloxyacids.

[0277] Tire sterol is generally cholesterol.

[0278] The aggregation reducing agent can be a lipid capable of reducing aggregation. Examples of such lipids include, but are not limited to, polyethylene glycol (PEG)-modified lipids, mono sialoganglioside Gml, and polyamide oligomers (PAO). Other compounds with uncharged, hydrophilic, steric -barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATTA, can also be coupled to lipids.

[0279] The composition of LNPs maybe influenced by, inter alia, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, the ratio of all components and biophysical parameters such as its size.

[0280] In some instances, the LNPs comprise a cationic lipid, a non-cationic lipid, a PEG-lipid and cholesterol. In some instances, LNPs may comprise about 35 to 45%, 40 to 50%, 40 to 60%, 50 to 60% or 55 to 65% cationic lipid on a molar basis (i.e. mol%). In some instances, the cationic lipid is present in a ratio of from about 50 mol% to about 60, 65, 70, 75, 80, 85, 90 or 95 mol% of the total lipid present in the LNP, e.g. from about 60 mol% to about 70, 75, 80, 85, 90 or 95 mol% of the total lipid present in the LNP, or from about 70 mol% to about 80, 85, 90 or 95 mol% of the total lipid present in the LNP. The cationic lipid may be present in a ratio of about 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 mol% of the total lipid present in tire LNP.

[0281] In some instances, LNPs may comprise about 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10%, 3 to 25%, 3 to 20%, 3 to 15%. 3 to 10%, 5 to 25%, 5 to 20%, 5 to 15% or 5 to 10% non-cationic lipid on a molar basis (i.e. mol%). In some instances, the non-cationic lipid is present in a ratio of from about 5 mol% to about 90 mol%, 50 mol%, 40 mol%, 30 mol%, 20 mol%, 15 mol% or about 10 mol% of the total lipid present in the LNP, e.g. from about 10 mol% to about 90 mol%, 50 mol%, 40 mol%, 30 mol% or 20 mol% of the total lipid present in the LNP. The non-cationic lipid may be present in a ratio of about 5, 10, 15, 20, 25, 30, 35 or 40 mol% of the total lipid present in the LNP.

[0282] In some instances, the molar ratio or weight ratio of lipid to mRNA in the nanoparticle ranges from about 5 : 1 to about 20:1, from about 10:1 to about 25:1, from about 15:1 to about 30 : 1 , or at least 30:1. In some instances, the LNPs may comprise about 0.1 to 5 % aggregation -reducing agent (e.g. PEG-lipid) on a molar basis (i.e. mol%), e g. 0.1 to 4 mol%, 0.1 to 3 mol%, 0.1 to 2 mol%, 0.1 to 1 mol%, 0.5 to 5 mol%, 0.5 to 4 mol%, 0.5 to 3 mol%. 0.5 to 2 mol%, 0.5 to 1 mol%, 1 to 5 mol%, 1 to 4 mol%, 1 to 3 mol% or 1 to 2 mol% aggregation-reducing agent. hMPV-100

[0283] The average molecular weight of the PEG moiety in PEG-modified lipids can range from about 1000 to about 8000 Daltons (e.g., from about 1000 to about 4000 Daltons). In some instances, the average molecular weight of the PEG moiety is about 2000 Daltons.

[0284] In some instances, the LNPs may comprise about 10 to 70 mol% sterol (e.g. cholesterol), e.g. about 20 to 60 mol% or 30 to 50 mol% sterol.

[0285] In some instances, the LNPs may comprise about 40 to 60% cationic lipid, 5 to 15% non-cationic lipid, 1 to 2% PEG-lipid, and 30-50% cholesterol by weight.

[0286] In some instances, LNPs have a median diameter size of about 50 to 300 nm, such as about 50 to 250 nm, 50 to 200 nm, 50 to 150 nm, 100 to 300 nm, 100 to 250 nm, 100 to 200 nm or 150 to 300 nm. Alternatively, nucleic acids may be delivered using smaller LNPs which may comprise a median diameter from about 1 to 100 nm, e.g. 1 to 10 nm, 1 to 20 nm, 1 to 30 nm, 1 to 40 nm, 1 to 50 nm, 1 to 60 nm, 1 to 70 nm, 1 to 80 nm, 1 to 90 nm, 5 to 100 nm, 5 to 10 nm, 5 to 20 nm, 5 to 30 nm, 5 to 40 nm, 5 to 50 nm, 5 to 60 nm, 5 to 70 nm, 5 to 80 nm, 5 to 90 nm, 10 to 50 nm, 20 to 50 nm, 30 to 50 nm, 40 to 50 nm, 20 to 60 nm, 30 to 60 nm, 40 to 60 nm, 20 to 70 nm, 30 to 70 nm, 40 to 70 nm, 50 to 70 nm, 60 to 70 nm, 20 to 80 nm, 30 to 80 nm, 40 to 80 nm, 50 to 80 nm, 60 to 80 nm, 20 to 90 nm, 30 to 90 nm, 40 to 90 nm, 50 to 90 nm, 60 to 90 nm and / or 70 to 90 nm.

[0287] Alternatively, nucleic acids may be delivered using larger LNPs having a median diameter greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0288] In other instances, LNPs have a single mode particle size distribution (i.e. they are not bi-or poly-modal).

[0289] LNPs may further comprise one or more lipids and / or other components in addition to those mentioned above. Other lipids may be included in the LNP compositions for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the LNP surface. Any of a number of lipids may be present in LNPs, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination. Additional components that may be present in a L P include bilaycr stabilizing components such as polyamide oligomers (see, e.g., U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety), peptides, proteins, and detergents.

[0290] Liposomes

[0291] The mRNA molecule may be formulated in liposomes in the vaccine. Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids (e.g. mRNA) via electrostatic interactions, hMPV-100

[0292] resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the nucleic acid is then released from the endosome / carrier into the cytoplasm.

[0293] Liposomes typically consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, which encloses 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 single-layered, referred to as unilamellar, or multi-layered, referred to as multilamcllar.

[0294] Liposome characteristics and behaviour in vivo can be modified by addition of a hydrophilic polymer coating, e.g. 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 its surface or to the terminal end of the attached PEG chains.

[0295] Liposomes can be of different types and sizes, ranging from a small unicellular vesicle (SUV) which may be smaller than 100 nm in diameter, to a giant unicellular vesicle (GUV) which may be over a micrometre in diameter. Large unilamellar vesicles (LUVs) which are 100-1000 nm in diameter may also be used. Other suitable species of liposome include multilamellar vesicle (MLVs) which may be hundreds of nanometres or over a micron in diameter. MLVs contain a series of concentric bilayers separated by narrow aqueous compartments; and multi vesicular liposomes (MVLs), in which multiple smaller, non-concentric vesicles are encapsulated within a single large vesicle, which may have a diameter of hundreds of nanometres or over a micron. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to a particular tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0296] Lipoplexes

[0297] Alternatively, the mRNA molecule may be formulated as lipoplexes, i.e. cationic lipid bilayers sandwiched between nucleic acid layers. Cationic lipids, such as described above, can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction.

[0298] Composition Components and Administration

[0299] The pharmaceutical composition provided herein may be provided in a liquid form, or in a lyophilised form for reconstitution in liquid. The active agents (e.g. mRNA molecule, optionally formulated in a lipid nanoparticle or suchlike as set out above) may be constituted (or reconstituted) in a suitable buffer, particularly a buffer suitable for injection, e.g. an aqueous solution such as Ringer’s lactate solution, hMPV-100

[0300] Ringer’s solution or a phosphate buffer solution. The pharmaceutical composition provided herein may comprise one or more pharmaceutically -acceptable carriers, diluents or excipients.

[0301] A buffer suitable for injection may be used as a carrier in the composition or simply for resuspending the composition or composition components. Such a buffer suitable for injection may contain salts selected from, for example, sodium chloride (NaCl), calcium chloride (CaCl) or potassium chloride (KC1), wherein further anions may be present additional to the chlorides. The injection buffer may be hypertonic, isotonic or hypotonic with reference to relevant bodily fluids such as blood, lymph or cytosolic liquids. Suitable concentrations may be selected so as not to lead to cell damage due to osmosis or other concentration effects.

[0302] Additionally, liquid phamraceutical compositions may include one or more of the following: polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as dextrose. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions are preferably sterile.

[0303] The choice of a pharmaceutically acceptable carrier or diluent for the composition is determined, in principle, by the manner, in which it is to be administered. The composition can be administered, systemically or locally, generally locally. Routes for systemic administration include, for example, oral and parenteral routes, including intravenous, intraarterial, and intraperitoneal injections and / or intranasal administration routes. Routes for local administration include, for example, topical, intradermal, transdermal, subcutaneous and intramuscular injections. In a particular instance, the pharmaceutical composition (or vaccine) is administered intramuscularly. Intramuscular administration is generally by injection, which may be via a needle or may be needle-free.

[0304] The pharmaceutical composition may comprise an adjuvant. An adjuvant may be used in order to enhance the immunostimulatory properties of the pharmaceutical composition / vaccine provided herein. In this context, an adjuvant may be understood as any compound which initiates or increases an immune response by the innate immune system, i.e. a non-specific immune response, when administered to a subject. In other words, when administered, the pharmaceutical composition / vaccine provided herein initiates an adaptive immune response against the immunogen (i.e. the hMPV F protein) encoded by the mRNA molecule. Additionally, the pharmaceutical composition / vaccine may generate a (supportive) innate immune response due to addition of an adjuvant as defined herein to the vaccine. In other instances, the pharmaceutical composition / vaccine does not comprise an adjuvant, or does not contain a dedicated adjuvant component, e.g. the pharmaceutical composition / vaccine may comprise an mRNA molecule carrier which also functions as an adjuvant. For example, lipid nanoparticles may have an immunostimulatory or adjuvant effect, and so a pharmaceutical composition / vaccine hMPV- 100

[0305] comprising an mRNA molecule formulated in an LNP may utilise the intrinsic adjuvant activity of the LNP, and not comprise an additional adjuvant component.

[0306] Combination Compositions

[0307] In some embodiments, the composition further comprises an mRNA molecule (i.e. a second mRNA molecule) encoding a second immunogenic polypeptide (i.e. an immunogenic polypeptide which is different to the hMPV F protein -LuS fusion protein described above). Tire second immunogenic polypeptide generally comprises a different immunogen to the hMPV F protein immunogen used in the fusion proteins described above. Generally, the second immunogenic polypeptide comprises an immunogen from a different infectious agent to the first polypeptide, i.e. generally the second immunogenic polypeptide comprises an immunogen from an infectious agent (e.g. virus) which is not hMPV.

[0308] Tn some embodiments, the second immunogenic polypeptide comprises an immunogen from respiratory syncytial virus (RSV). In particular, the second immunogenic polypeptide may comprise an RSV fusion (F) protein. References to “an RSV F protein” herein encompass immunogenic fragments of the RSV F protein. Generally, the RSV F protein used is a prefusion-stabilised RSV F protein. Prefusion-stabilised RSV F proteins arc well known in the art (sec c.g. McLellan et al., Science 342: 592-598, 2013).

[0309] In particular embodiments, the second immunogenic polypeptide is a second fusion protein or nanoparticlc subunit, comprising a prefusion -stabilised RSV F protein joined to a lumazinc synthase. In particular, the RSV F protein may be joined to the same lumazine synthase (i.e. a lumazine synthase having the same sequence) as the lumazine synthase to which the hMPV F protein is joined in the first polypeptide . Without being bound by theory, provision of two such nanoparticle subunits together, in the same composition, may result in co-production of the two nanoparticle subunits, resulting in the assembly of lumazine synthase nanoparticles with both the hMPV and RSV antigens presented on their exterior, which can thus induce immune responses specific to both viruses.

[0310] The second mRNA molecule, encoding the second immunogenic polypeptide (e.g. RSV F proteinlumazine synthase fusion protein) may also be provided in a delivery vehicle, as described above, particularly an LNP. In some embodiments, both the first and second mRNA molecules are formulated in the same LNPs. That is to say, the pharmaceutical composition may comprise LNPs in which both the first mRNA molecule and the second mRNA molecule are formulated / encapsulated. Alternatively, the pharmaceutical composition may comprise two separate populations of LNP: a first LNP population in which the first polypeptide is formulated, and a second LNP population in which the second polypeptide is formulated. hMPV- 100

[0311] Said compositions may comprise an effective amount of the mRNA molecule or nucleic acid vector as defined herein. An effective amount of the mRNA molecule or nucleic acid vector to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. In one instance, the effective amount of the mRNA molecule or nucleic acid vector as defined anywhere herein within the pharmaceutical composition is effective to treat or prevent an infectious disease, optionally a disease cause by a respiratory virus, optionally a disease caused by hMPV or RSV.

[0312] Uses

[0313] Tire present disclosure provides a vaccine comprising an mRNA molecule or pharmaceutical composition as described anywhere herein.

[0314] The mRNA molecule, pharmaceutical composition or vaccine provided herein may be used in therapy, in particular in a method of vaccination. Thus provided herein is an mRNA molecule, pharmaceutical composition or vaccine as described above for use as a therapeutic agent. In particular, the mRNA molecule, pharmaceutical composition or vaccine is provided for use as a prophylactic agent. A “therapeutic agent” as referred to herein is an agent used for treatment or prevention of an illness or condition; a “prophylactic agent” as referred to herein is an agent used in medicine for prophylaxis of an illness or condition. Specifically, the mRNA molecule, pharmaceutical composition or vaccine provided herein can be used for prophylaxis of disease caused by hMPV and / or RSV infection.

[0315] Prophylaxis of disease caused by hMPV or RSV infection is broadly defined herein as reducing the likelihood of an individual developing a disease caused by hMPV or RSV infection or reducing the severity of (i.e. attenuating) that disease in the event that the individual develops a disease caused by hMPV or RSV infection. A disease caused by hMPV or RSV infection may have reduced severity if e.g. the individual has a reduced likelihood of death, hospitalisation, admission to intensive care or requiring mechanical ventilation; the individual has symptoms which are milder and have a reduced impact on the individual’s daily life, e.g. reducing the likelihood of the individual being bedridden and increasing their ability to care for themselves or others, work, etc; or the individual recovers more quickly, e.g. requires a shorter stay in hospital, intensive care, period of mechanical ventilation, etc., compared to if they were not vaccinated, and / or compared to an equivalent unvaccinated individual. An equivalent unvaccinated individual is an individual of similar size and age, of the same sex and in a similar condition of general health.

[0316] A prophylactic agent, as used herein, may be administered to an individual who is at particular risk of developing a disease caused by hMPV or RSV infection or, more commonly, suffering severely from a disease caused by hMPV or RSV infection. For example, the individual may be at high risk of exposure to hMPV or RSV, or may be at risk of developing severe illness if infected by hMPV or RSV due to e.g. hMPV- 100

[0317] age and / or other health conditions. For instance, the prophylactic agent may be administered as part of a routine or regular schedule of immunisation for individuals of a particular age, for example to older adults over the age of e.g. 50, 60 or 70. The prophylactic agent may be administered repeatedly, e.g. annually, to boost protection against hMPV and RSV. For example, a vaccine may be administered to an at-risk individual, such as an elderly person, annually or every 2, 3, 4 or 5 years or more, as needed. Such a reduction in risk of developing a disease caused by hMPV or RSV infection, or suffering severely from a disease caused by hMPV or RSV infection, may be a reduction of e.g. at least 50, 60, 70, 80 or 90% compared to an unvaccinated individual.

[0318] The present disclosure also provides an mRNA molecule, pharmaceutical composition or vaccine as described above for use in a method of preventing and / or attenuating an infectious disease. Tn some embodiments the disease is caused by a respiratory virus. In one embodiment, the disease is caused by hMPV. In one embodiment, the disease is caused by RSV.

[0319] The mRNA molecule, pharmaceutical composition or vaccine for use according to this aspect may prevent hMPV or RSV infection. Alternatively, the mRNA molecule, pharmaceutical composition or vaccine may not prevent hMPV / RSV infection, but may prevent symptomatic disease in the case of infection by hMPV or RSV, or when symptomatic disease occurs, may reduce the severity of the disease. That is to say, the mRNA molecule, pharmaceutical composition or vaccine may reduce the likelihood of the subject becoming infected with hMPV or RSV, e.g. in a particular context when there is a risk of exposure to the virus, or in day-to-day life, compared to an unvaccinated subject. Similarly, the mRNA molecule, pharmaceutical composition or vaccine may reduce the risk of the subject developing symptomatic disease caused by hMPV or RSV compared to an unvaccinated subject, or may reduce the risk of the subject developing a severe disease caused by hMPV or RSV (e.g. a disease requiring hospitalisation) compared to an unvaccinated subject. Such reductions in risk may be e.g. of at least 50, 60, 70, 80 or 90% compared to an unvaccinated subject.

[0320] In particular embodiments, the infectious disease caused by hMPV and / or RSV which is prevented or attenuated using tire mRNA molecule, pharmaceutical composition or vaccine provided herein is a lower respiratory tract disease (LRTD) caused by hMPV or RSV. As standard in the art, the lower respiratory tract may be defined as the respiratory tract from the vocal cords downwards, comprising the trachea, lungs, bronchi and bronchioles. Examples of LRTDs caused by hMPV or RSV include bronchiolitis, bronchitis and pneumonia.

[0321] The subject in which an infectious disease caused by hMPV or RSV, e.g. an LRTD, is prevented or attenuated may be the subject to whom the mRNA molecule, pharmaceutical composition or vaccine is administered. The subject is generally a human patient, e.g. a person over the age of 1, 2, 5, 12, 16, 18 or 21. In particular, the subject may be an adult, generally an older person over the age of e.g. 50, 55, 60, 65 or 70. Alternatively or additionally, the subject may suffer from a health condition putting them hMPV- 100

[0322] at greater risk of serious disease from hMPV or RSV infection, e.g. cancer, an immunodeficiency or a respiratory condition such as COPD or suchlike. In adults and older children (e.g. over the age of 5, or over the age of 2), the LRTD prevented or attenuated may in a particular instance be pneumonia. In other instances, the subject may be a small child, e.g. an infant, baby or toddler, e.g. a child in the age range from birth to about 6 months, 12 months, 18 months or 24 months. In small children, the LRTD prevented or attenuated may in a particular instance be bronchiolitis.

[0323] Alternatively, the subject in which an infectious disease caused by hMPV or RSV, e.g. an LRTD, is prevented or attenuated may not be the same subject to whom the mRNA molecule, pharmaceutical composition or vaccine is administered. In particular, the subject protected by the vaccine (i.e. in which an infectious disease caused by hMPV or RSV is prevented or attenuated) may be an infant child of a woman administered the mRNA molecule, pharmaceutical composition or vaccine prior to giving birth to the infant. In particular, the subject to whom the mRNA molecule or vaccine is administered may be a woman e g. 20 to 38 weeks pregnant, e g. 22-36 weeks, 24-36 weeks, 24-34 weeks, 24-32 weeks, 26-36 weeks, 26-34 weeks or 26-32 weeks pregnant. Vaccinating pregnant women in this way may lead to anti -HMPV and / or anti -RSV antibodies being passed to unborn children through the mother’s placenta, providing passive protection against hMPV and / or RSV infection and disease in the early months of a child’s life. Such protection may be effective for at least e.g. 1, 2, 3, 4, 5 or 6 months after the birth of the child.

[0324] The above-discussed aspect may alternatively be seen as providing a method for preventing or attenuating an infectious disease in a subject, particularly an infectious disease caused by hMPV or RSV. The disease prevented or attenuated may be an LRTD as described above. As detailed above, such a method may comprise administering the mRNA molecule, pharmaceutical composition or vaccine provided herein to the subject. Alternative, if the subject is an infant, the method may comprise administering the mRNA molecule or vaccine to the mother of the infant, while she is pregnant with the infant, as detailed above. Similarly, the above -discussed aspect may alternatively be seen as providing the use of an mRNA molecule, pharmaceutical composition or vaccine as described above in the manufacture of a medicament for use in preventing or attenuating a disease caused by an hMPV infection or an RSV infection in a subject, particularly an LRTD. The subject may be as described above.

[0325] More broadly, provided herein is a method of inducing an immune response in a subject, comprising administering to the subject an mRNA molecule, pharmaceutical composition or vaccine as described above. This aspect may alternatively be seen as providing an mRNA molecule, pharmaceutical composition or vaccine as described above for use in a method of inducing an immune response in a subject, or as providing the use of an mRNA molecule, pharmaceutical composition or vaccine as described above in the manufacture of a medicament for inducing an immune response in a subject. In hMPV- 100

[0326] this aspect, the immune response is an immune response against hMPV, specifically against the hMPV F protein. In an embodiment, the immune response is also an immune response against RSV, particularly the RSV F protein. Thus the method may be for inducing an immune response against hMPV, and optionally also against RSV.

[0327] Such a specific immune response may entail a cellular (i.e. T cell) response and / or a humoral (i.e. antibody) response. Generally the immune response comprises at least an antibody response against the hMPV F protein, i.e. tire subject is induced to produce antibodies against tire hMPV F protein. Tire antibodies produced by the subject may include neutralising antibodies against hMPV F protein. The immune response may also include an antibody response against the RSV F protein, including neutralising antibodies against the RSV F protein (particularly against the profusion conformation of the RSV F protein.

[0328] An antibody response induced by the mRNA molecule, pharmaceutical composition or vaccine may be identified and / or measured by ELISA (enzyme-linked immunosorbent assay). The size of an antibody response may be measured based on the antibody titre, e.g. by ELISA. An antibody titre is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g. the hMPV F protein) or epitope of an antigen. Antibody titre is typically expressed as the inverse of the greatest dilution that provides a positive result.

[0329] In some instances, induction of an immune response results in an antibody titre in the subject which is increased by at least 1 order of magnitude relative to the titre prior to vaccination (i.e. relative to the titre in the same individual prior to vaccination). For example, the titre may be increased by at least 1.5, 2, 2.5 or 3 orders of magnitude relative to the titre prior to vaccination. In other instances, induction of an immune response results in an antibody titre in the subject which is increased at least 2-fold relative to the titre prior to vaccination. For example, the titre may be increased at least 3, 4, 5, 6, 7, 8 or 9-fold relative to prior to vaccination.

[0330] An antibody titre may measure the total amount of all antibodies against a particular antigen in a subject, or may measure just a particular type of antibody, e.g. neutralising antibody. In some instances, the increase in titre may be an increase in the titre of neutralising antibodies against hMPV. In some instances, the increase in titre may also be an increase in the titre of neutralising antibodies against RSV. Tine titre of neutralising antibodies against a virus, such as hMPV, may be determined by a neutralisation assay. For instance, the titre of neutralising antibodies against hMPV may be determined in an hMPV neutralisation assay. A reliable neutralisation assay is the plaque reduction neutralisation test (PRNT), in which heat-inactivated serum of interest is applied with live virus to human cells. Viral infection of cells causes the formation of plaques in the cell culture dish, and the neutralising antibody titre can be calculated by the reduction in plaques. Both hMPV and RSV neutralisation assays are described in detail in Caban el al., Nature Communications 14: 798, 2023, incorporated herein by reference. hMPV- 100

[0331] The induced immune response may be long-lasting, e.g. the increased antibody titre or neutralising antibody titre may remain for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 18 months or more after vaccination.

[0332] The induced immune response may include a memory response, i.e. a memory B cell response against hMPV, and optionally RSV, may be generated. Memory B cells may be identified by e.g. staining PBMCs with a fluorescent-labelled antigen (e.g. hMPV F protein) and then identifying the antigenspecific memory B cells by flow cytometry based on binding of tire fluorescent -labelled antigen and expression of memory B cell markers (e.g. CD27).

[0333] In the aspects described above, administration of the mRNA molecule provided herein (encoding a fusion protein comprising the hMPV F protein joined to lumazine synthase), or a pharmacal composition or vaccine comprising the mRNA molecule, may be particularly effective in inducing an immune response against hMPV, and preventing and / or attenuating infectious disease caused by hMPV infection. Administration of the mRNA molecule provided herein together with the second mRNA molecule described above, encoding an RSV antigen, for instance in the context of a composition comprising both mRNA molecules, may be particularly effective in inducing an immune response against both hMPV and RSV, and preventing and / or attenuating both an infectious disease caused by hMPV infection and an infectious disease caused by RSV infection.

[0334] Thus in some embodiments the mRNA molecule provided herein encoding a fusion protein comprising the hMPV F protein joined to lumazine synthase, or a pharmaceutical composition or vaccine comprising the mRNA molecule, is administered to the subject in combination with a second mRNA molecule as described above (i.e. encoding an RSV immunogen, particularly an RSV F protein, particularly a prefiision-stabilised RSV F protein, most particularly a fusion protein comprising a prefusion-stabilised RSV F protein joined to lumazine synthase), or a second pharmaceutical composition comprising the second mRNA molecule, or a second vaccine comprising the second mRNA molecule or second pharmaceutical composition.

[0335] The mRNA molecule provided herein, or pharmaceutical composition or vaccine comprising the mRNA molecule, may be administered in combination with the second mRNA molecule described above, or second pharmaceutical composition or vaccine described above, in order to induce an immune response against both hMPV and RSV. Tire mRNA molecule provided herein, or pharmacal composition or vaccine comprising the mRNA molecule, may be administered in combination with the second mRNA molecule described above, or second pharmaceutical composition or vaccine described above, in order to prevent and / or attenuate an infectious disease caused by hMPV and / or RSV.

[0336] Where the mRNA molecule provided herein is administered in combination with a second mRNA molecule, as described above, the two mRNA molecules may be administered separately or together. As used herein, administration “together” means that the two mRNA molecules are administered to the hMPV-100

[0337] subject in a single composition. Compositions comprising both the first and second mRNA molecules are described above. As used herein, “separate” administration means that the first and second mRNA molecules are administered to the subject separately, i.e. not in a single composition. For example the two mRNA molecules (or compositions or vaccines comprising them) may be administered by separate injections. Generally the two mRNA molecules (or compositions or vaccines comprising them) are administered by the same route. When administered separately, the two mRNA molecules may be administered at the same time, one after the other.

[0338] Vectors And Cells

[0339] Also provided herein is a vector encoding the mRNA molecule provided herein. Such a vector is a DNA vector, and may be e.g. a cloning vector or an expression vector.

[0340] An “expression vector” as used herein is a DNA molecule used for expression of foreign genetic material in a cell or in an in vitro system. Any suitable vectors known in the art may be used. Suitable vectors include DNA plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). An expression vector may be circular or linear. An expression vector comprises an expression cassette, from which the mRNA molecule provided herein may be expressed.

[0341] An “expression cassette” as used herein is a polynucleotide sequence that is capable of effecting transcription of an expression product. Typically, the expression cassette comprises a promoter operably linked to the sequence encoding the mRNA molecule. The term “operably linked” in this context means that the mRNA molecule sequence and promoter arc covalently linked in such a way as to place the expression of the mRNA molecule under the influence or control of the promoter. Thus, a promoter is operably linked to the mRNA sequence if the promoter is capable of effecting transcription of the mRNA sequence in order to yield an mRNA molecule as described above.

[0342] Any suitable promoter known in the art may be used in the expression cassette providing it functions in the cell type being used. For example, where the cell is a mammalian cell, the promoter may be a cytomegalovirus (CMV) promoter. Where tire expression cassette is to be expressed in vitro, tire promoter may be a phage promoter, e.g. the SP6, T7 or T3 promoter. The promoter used in an expression cassette as provided herein may be a constitutive promoter or an inducible promoter.

[0343] An expression cassette may comprise additional elements useful for control of transcription, e g. one or more enhancer sequences and one or more transcription termination (terminator) sequences.

[0344] The vector may additionally comprise other standard sequence elements such as a selectable marker, e g. encoding an antibiotic resistance gene or suchlike. hMPV- 100

[0345] Also provided herein is a cell comprising the vector provided herein. Such a cell may be e.g. a cloning host. Suitable cells include prokaryotic cells, e.g. bacterial cells, and eukaryotic cells, e.g. mammalian cells. Suitable bacterial cells include e.g. E. colt cells or B. subtilis cells.

[0346] mRNA Manufacture

[0347] The mRNA molecule provided herein may be manufactured in any suitable manner. The mRNA may be expressed in and purified from cells, e.g. mammalian cells, particularly human cells. Generally, the mRNA molecule is manufactured in vitro, in particular by in vitro transcription. The mRNA molecule may be transcribed from the expression vector described above.

[0348] Thus provided herein is a method of manufacturing the mRNA molecule provided herein, the method comprising expressing the mRNA molecule from the expression vector provided herein. Generally, the mRNA is expressed by in vitro transcription.

[0349] The expression vector used for in vitro transcription is generally linear, e g. a linearised plasmid. Transcription may be performed using a phage RNA polymerase, e.g. the T7 RNA polymerase, SP6 RNA polymerase or T3 RNA polymerase. The expression vector contains a promoter recognised by the chosen polymerase. In vitro transcription can be performed using a commercially available kit in accordance with the manufacturer’s instructions, e.g. a HiScribc® kit from New England Biolabs (NEB, USA) or a RiboMAX™ kit from Promega (USA).

[0350] Protein Nanoparticles

[0351] Also provided herein is a protein nanoparticle comprising the nanoparticle subunit described above. The protein nanoparticle may be as described above in the context of the nanoparticle subunit encoded by the mRNA molecule.

[0352] The protein nanoparticle comprises subunits comprising an hMPV F protein (as described above) joined to a lumazine synthase. The protein nanoparticle may be a homomultimer, comprising only the hMPV F protein-lumazine synthase subunits. In other embodiments, the protein nanoparticle is a heteromultimer comprising both hMPV F protein-lumazine synthase subunits as provided herein, and subunits comprising a second antigen joined to lumazine synthase. In such cases the second antigen is not the hMPV F protein. The second antigen is generally from a different pathogen (i.e. a pathogen other than hMPV). The second antigen may be derived from a vims other than hMPV, or a non-viral pathogen such as a bacterium or a fungus. The second antigen may be derived from RSV, in particular it may be an RSV F protein, particularly a prefusion-stabilised RSV F protein, as described above. The nanoparticle subunit may be expressed from an expression vector, as described above. Such an expression vector may be introduced into a host production cell, e.g. a mammalian cell, such as a human hMPV-100

[0353] cell, and the protein expressed from it. Where the nanoparticle is a heteromultimer, both or each different subunit(s) may be expressed from either the same or different expression vectors. Where different expression vectors are used, the multiple expression vectors encoding the two or more nanoparticle subunits are introduced into the same production cell and the proteins expressed from them. The nanoparticle subunit may be expressed with a signal peptide so that it is exported from the cell and the multimer forms extracellularly, in which case the protein nanoparticle may be isolated from the culture supernatant. Otherwise, the multimer may form intracellularly, in which case the cells may be lysed to enable isolation of the protein nanoparticle. Cell lysis may be performed by any method known in the art, e.g. mechanical lysis using a French press, sonication or chemical lysis using a lysis agent.

[0354] Protein nanoparticle isolation / purification may be performed using standard methods in the art, e.g. the fusion protein may be expressed with an affinity tag (such as a His-tag) to enable affinity purification. A protein nanoparticle as provided herein could be administered to a subject as described above as a protein vaccine .

[0355] Accordingly, in some instances, the disclosure provides a recombinant protein nanoparticle comprising 60 of the nanoparticle subunits described herein self-assembled into a nanoparticle. In some instances, when self-assembled, the nanoparticlc subunits do not comprise a signal peptide.

[0356] Sequence Identity And Variants

[0357] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990)), FASTA (which uses the method of Pearson and Lipman (1988)), or the Smith-Waterman algorithm (Smith and Waterman (1981)), tire TBLASTN program, of Altschul et al. (1990) supra, or Emboss Needle (Madeira et al, Nucleic Acids Research 5O(W1): W276-W279), generally employing default parameters. A sequence alignment, however performed, is performed across the entirety of the reference sequence.

[0358] Where the disclosure makes reference to a particular amino acid or nucleotide sequence having at least 90% sequence identity to a reference amino acid or nucleotide sequence, this includes the amino acid or nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence identity to the reference amino acid or nucleotide sequence (including as rounded to the nearest integer percentage). hMPV-100

[0359] Where an amino acid or nucleotide sequence has less than 100% identity to a reference sequence (e.g. about 90 or 95% identity to a reference sequence), that amino acid or nucleotide sequence comprises one or more sequence alterations relative to the reference sequence. The term “sequence alterations” as used herein is includes the substitution, deletion and / or insertion of an amino acid residue or nucleotide. Thus, a protein containing one or more amino acid sequence alterations compared to a reference sequence contains one or more substitutions, one or more deletions and / or one or more insertions of an amino acid residue as compared to tire reference sequence, hr relation to peptide or polypeptide sequences, the term “amino acid mutation” is herein used interchangeably with “sequence alteration”, unless the context clearly identifies otherwise.

[0360] In some instances in which one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example according to the following table. In some instances, amino acids in the same block in the middle column are substituted, i.e. a non-polar amino acid is substituted for another non-polar amino acid for example. In some instances, amino acids in the same line in the rightmost column are substituted, i.e. G is substituted for A or P for example.

[0361]

[0362] In some instances, substitution(s) may be functionally conservative. That is, in some instances the substitution may not affect (or may not substantially affect) one or more functional properties of the protein containing the substitution as compared to the equivalent unsubstituted protein.

[0363] Similarly, a nucleic acid molecule (e.g. RNA molecule) containing one or more nucleotide sequence alterations compared to a reference sequence contains one or more substitutions, one or more deletions and / or one or more insertions of a nucleotide compared to the reference sequence. In relation to nucleic acid sequences, the term “nucleotide mutation” is herein used interchangeably with “sequence alteration”, unless the context clearly identifies otherwise.

[0364] Where a nucleic acid (e.g. RNA) sequence includes one or more sequence alterations, the alterations may similarly not affect (or not substantially affect) the functional properties of the nucleic acid. Where the nucleic acid sequence is a protein coding sequence, whether the functional properties of the sequence are affected by an alteration depends on whether the function or expression of the encoded hMPV- 100

[0365] protein is affected. Where the nucleic acid sequence is a non-coding nucleotide sequence, the effect of sequence alterations on the function of the non-coding sequence is assessed.

[0366] Nucleotide sequences may also be defined by degeneracy to a reference sequence. As a result of the degeneracy of the genetic code, there are many nucleotide sequences that may encode any given amino acid sequence. By degenerate nucleotide sequences is meant two (or more) nucleotide sequences which encode the same peptide or polypeptide (or amino acid sequence), specifically in the open reading frame of the reference nucleotide sequence which begins at position 1 (i.e. in which codon 1 of the encoding sequence corresponds to positions 1 -3 of the reference nucleotide sequence). Thus for example, a nucleotide sequence degenerate with SEQ ID NO: 1 is a nucleotide sequence which is different to SEQ ID NO: 1 but which, due to the degeneracy of the genetic code, encodes the same protein sequence as SEQ ID NO: 1 (i.e. the hMPV F protein of SEQ ID NO: 2).

[0367] EXAMPLES

[0368] The examples below describe the development of an mRNA vaccine against hMPV by targeting the F protein. Various prefusion-stabilized and wild type F protein constructs displayed on self-assembling protein nanoparticles (“nanoparticles”) were generated. Multiple nanoparticle scaffolds were evaluated, such as: foldon, a trimcrization domain; ferritin, a 24-valcnt scaffold derived from Helicobacter pylori,' lumazine synthase (LuS), a 60-valent scaffold derived from Aquifex aeolicus'. and beta-annulus (PA), a 180-valent scaffold derived from Tomato bushy stunt virus.

[0369] Example 1: Generation of Plasmid DNA Expressing hMPV Prefusion F Proteins Displayed on Protein Nanoparticles and Expression from HEK293 Cells

[0370] 1.1 METHODS

[0371] 1.1.1. Prefusion F Cloning

[0372] Two prefusion-stabilised F protein sequences (SEQ ID NOs: 6 and 8, see Table 1) were cloned through standard molecular biology techniques into a DNA expression vector wherein tire codon-optimized, prefusion-stabilized F protein sequences were genetically linked at the C-terminus to a panel of selfassembling scaffolds (SEQ ID NOs in Table 2). The prefusion-stabilised F protein sequences were joined to the self-assembling scaffolds via a linker of SEQ ID NO: 12, except for the fusion of the DSCavES2 F protein (SEQ ID NO: 6) to the lumazine synthase scaffold (SEQ ID NO: 10), in which the two components of the fusion protein were joined by the linker of SEQ ID NO: 43. The individual F protein antigen-scaffold fusion protein sequences are listed in Table 3. hMPV- fOO

[0373] Table 1: hMPV F protein antigens used

[0374]

[0375] Table 2: self-assembling scaffolds used

[0376]

[0377] Table 3: antigen-scaffold fusions used

[0378]

[0379] 1.1.2. Transfection ofHEK293 Cells

[0380] HEK293 cells were grown in T175 flasks in complete medium (DMEM supplemented with 10% FBS) at 37°C, 5% CO2. Cells were harvested using trypsin for 5 minutes at 37°C and plated at 300,000 cells per well in 24-well plates on day -1. On day 0, cells were transfected with plasmid DNA (500 ng / well) encoding the hMPV antigen-scaffold fusions using Lipofectamine 2000 according to the manufacturer’s hMPV- 100

[0381] protocol (Thermo Fisher Scientific). On day 3, cell culture supernatant was harvested and evaluated by ELISA for production of hMPV nanoparticles.

[0382] 1.1.3. Sandwich ELISA

[0383] Cell culture supernatants from transfected cells were analysed by a sandwich ELISA. Briefly, 384-well ELISA plates were coated overnight at 4°C with an anti-hMPV F protein-specific mAb (MPE33, Stewart-Jones etal., supra) at 3 pg / ml. Plates were washed and incubated with diluted cell supernatant for 1 hr at room temperature. Plates were then washed and hMPV nanoparticles detected with a cocktail of anti-hMPV F protein-specific mouse antibodies. Plates were washed and incubated with an antimouse IgG-HRP antibody (Sigma-Aldrich). Plates were washed and incubated with TMB substrate for 5 minutes and colour development stopped with 2 N H2SO4. Plates were then read with an Envision at 450 nm wavelength.

[0384] 1.2 RESULTS

[0385] To detect F protein nanoparticle production, HEK293 cells were transfected and cell culture supernatant assessed by sandwich ELISA using an hMPV F protein-specific capture antibody. Cells expressing the hMPV_DS-CavES2 nanoparticle constructs produced nanoparticles that were captured in greater abundance than those constructs presenting the hMPV_v3B profusion-stabilised F protein antigen (Figure 1). With regards to the scaffold presenting the hMPV DS-CavES2 prefusion-stabilised F protein antigen, there was a greater signal observed for the foldon and beta-annulus scaffolds in comparison to ferritin and lumazine synthase. These results suggest that the nanoparticles are produced to varying extents and present the F protein trimer.

[0386] Example 2: hMPV F Protein Antigens Presented on Scaffolds Elicit High-Titre Virus-Neutralising Antibodies in Naive Mice

[0387] Mice were immunised with a panel of mRNA vaccines encoding the prefusion -stabilised constructs linked to the different scaffolds and the immunogenicity evaluated for F protein-specific binding antibody titers (ELISA) as well as virus-neutralizing antibody titers.

[0388] 2.1 METHODS

[0389] 2.1.2. mRNA Synthesis using in vitro Transcription (IVT)

[0390] Sequences encoding a control (full-length wildtype hMPV F protein as a transmembrane-anchored protein, SEQ ID NO: 46) or the different prefiision-stabilized F protein-scaffold designs were cloned into an mRNA production vector comprised of a T7 promoter element, 5 ’ and 3 ’ UTRs, and a poly(A) tail. The template plasmid was linearised by digesting the plasmid DNA with BspQI (New England Biolabs) for 4 hr at 50°C. mRNAs were synthesized by in vilro transcription (New England Biolabs) hMPV- 100

[0391] incorporating N 1 -methylpseudouridine and CleanCap-AG (TriLink BioTechnologies). IVT reactions were incubated for 5 hr at 37°C, followed by digestion with RNase -free DNase. The mRNAwas purified using LiCl precipitation, formulated in lipid nanoparticles (LNP), and stored at -80°C.

[0392] 2.1.2. Mouse Immunizations

[0393] Mouse studies were approved by the Institutional Animal Care and Use Committee at AstraZeneca. Groups of 6 female BALB / c mice aged 5-7 weeks were immunised with the mRNA vaccines. mRNA / LNP formulations were diluted in PBS to achieve the target dose. Mice were immunised intramuscularly with a 50 pl volume of mRNA / LNP vaccine on days 0 and 21. Mice were bled on days 14 and 35 to evaluate the immune response.

[0394] 2.1.3. Antibody Binding ELISA

[0395] ELISA assays were performed to evaluate prefusion F protein-specific antibody binding titres in immunised mice. 384-well plates were coated with 3 pg / ml of purified recombinant hMPV prefiision-stabilised F protein (DSCavES2, SEQ ID NO: 6) overnight at 4°C. Plates were then washed and blocked in PBS supplemented with 5% milk and 3% BSA for 1 hr at room temperature. Mouse sera was then diluted in blocking buffer and incubated with the coated plates for 1 hr at room temperature. Plates were washed, incubated with an HRP -conjugated anti -mouse IgG secondary antibody (Sigma-Aldrich) for 1 hr, washed, and then incubated with TMB substrate for 5 minutes. Colour development was stopped with 2 N H2SO4 and plates read with an Envision at 450 nm wavelength.

[0396] 2.1.4. hMPV Neutralisation Assay

[0397] Sera from immunised mice were analysed for virus neutralisation activity at the indicated time points. Sera were heat-inactivated and serially diluted in a 96-well plate . Diluted sera were then incubated with the indicated virus (hMPV A or hMPV B) at an MOI of 0.04 for 1 hr at 37°C. 20,000 Hep2 cells were then added to the virus / sera mixtures and the plates were incubated for 5 days at 37°C. To visualise infection, cells were fixed in acetone, washed, and stained with a biotinylated anti -hMPV F protein -specific antibody. Cells were then washed and incubated with a streptavidin-HRP antibody (Sigma-Aldrich). Plates were washed and incubated with TMB substrate for 7 minutes and colour development was stopped with 2 N H2SO4. Plates were read with an Envision at 450 nm wavelength.

[0398] 2.2 RESULTS

[0399] 2.2.1. hMPV DS-CavES2 F Protein on LuS Scaffold Elicits Potent Neutralisation Activity Against hMPV A Virus

[0400] The stabilising substitutions found in hMPV_DS-CavES2 and hMPV_v3B have previously demonstrated that immunogenicity could be improved as compared to immunisation with wild-type F protein. We next wanted to determine if grafting these prefusion-stabilized F proteins on different antigen scaffolds - as an mRNA / LNP vaccine - resulted in enhanced virus neutralizing antibody hMPV- 100

[0401] responses. To test this, mice were immunised twice intramuscularly on days 0 and day 21 with mRNA encoding either native, wildtype hMPV F protein or our different prefusion -stabilized F protein-scaffold constructs. On day 35 (14 days after the second immunization), sera were collected and assessed for virus-neutralizing antibody titers against hMPV_A (Figure 2A) and hMPV_B (Figure 2B) viruses. Mice immunized with hMPV_DS-CavES2 presented on the LuS scaffold elicited the highest virus neutralizing titer against hMPV A virus (Figure 2A) . Mice immunized with the construct encoding the hMPV_v3B F protein presented on either foldon, ferritin, or LuS resulted in comparable neutralizing antibody titers against hMPV B virus (Figure 2B). Moreover, the mice immunized with hMPV DS-CavES2 presented on the LuS scaffold elicited the highest levels of prefusion F protein-specific antibody binding titers (Figure 3).

[0402] 2.2.2. Wildtype hMPV F Protein Grafted on LuS Scaffold Induced Potent Neutralisation Activity Against hMPV Virus

[0403] Although stabilising substitutions engineered into the hMPV F protein have previously demonstrated that immunogenicity could be improved in comparison to wild-type F protein, we sought to determine if simply grafting less engineered or even wildtype F protein onto the LuS scaffold - as an mRNA / LNP vaccine - would result in sufficient virus neutralizing antibody responses compared to a full profusion -stabilized hMPV F protein presented on the LuS scaffold. To test this, mice were immunised twice intramuscularly on days 0 and 21 with mRNA encoding either native, wildtype hMPV F protein or hMPV_DS-CavES2 F protein on LuS. Additionally, four additional mRNA vaccine constructs were tested: an hMPV F protein harboring a single stabilizing modification (A185P) on either foldon scaffold or LuS scaffold; abd a wildtype hMPV F protein on either foldon or LuS scaffold. On day 35 (14 days after the second immunization), sera were collected and assessed for virus neutralizing antibody titers against hMPV_A (Figure 4A) and hMPV_B (Figure 4B) viruses.

[0404] Mice immunized with any of the constructs expressing either of the three hMPV F proteins (DS-CavES2, A185P, or wildtype) presented on the LuS scaffold elicited the highest virus -neutralizing titers against hMPV A (Figure 4A) and hMPV B (Figure 4B) virus. Constructs employing tire foldon domain did not give rise to substantial virus-neutralizing antibody titers (Figure 4). Taken together, these data suggest that, unexpectedly, the prefusion stabilizing modifications engineered into hMPV_ DS-CavES2 or even the single A185P substitution are dispensable when the wildtype F protein cctodomain is fused onto and presented by the LuS scaffold as an mRNA vaccine.

[0405] Example 3: hMPV mRNA- VLP Vaccine Protects Infected Cotton Rats from Virus Replication in the Lungs and Nasal Tissue

[0406] In order to demonstrate vaccine efficacy, groups of naive cotton rats were immunised with control PBS or two different mRNA / LNP vaccines expressing either the wildtype hMPV F protein as a hMPV- 100

[0407] transmembrane-anchored protein or the wildtype F protein presented on the LuS scaffold. Vaccine responses were evaluated prior to live hMPV virus challenge. Five days after intranasal administration of virus, animals were euthanized to measure virus replication in the lungs and nasal tissue.

[0408] 3.1 METHODS

[0409] 3.1.1. mRNA Synthesis Using in vitro Transcription (IVT)

[0410] Sequences encoding a wildtype hMPV F protein as a transmembrane -anchored protein or tire wildtype hMPV F protein-LuS scaffold design were cloned into an mRNA production vector comprised of a T7 promoter element, 5’ and 3’ UTRs, and a polyA tail. The template plasmid was linearised by digesting the plasmid DNA with BspQT (New England Biolabs) for 4 hr at 50°C. mRNAs were synthesized by in vitro transcription (New England Biolabs) incorporating N 1 -methylpseudouridine and CleanCap-AG (TriLink BioTechnologies). IVT reactions were incubated for 5 hr at 37°C, followed by digestion with RNase-free DNase. Tire mRNA was purified using LiCl precipitation, formulated in lipid nanoparticles (LNP), and stored at -80°C.

[0411] 3.1.2. Cotton Rat Immunization and Virus Challenge

[0412] The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at AstraZeneca and conducted at Sigmovir Biosystems, Inc. (Rockville, MD). Three groups (n = 8 per group) of cotton rats received two intramuscular (IM) injections of either PBS control or mRNA vaccine (20 pg) on study days 1 and 21. Sera samples were obtained 21 days after second immunization (study day 42) just prior to hMPV vims challenge . On study day 42, all animals were administered live hMPV vims intranasally and subsequently euthanized five days later (study day 47) at which point lungs and nasal tissues were harvested to assess vims replication by viral plaque assay.

[0413] 3.1.3. hMPV Neutralisation Assay

[0414] Sera from immunised animals were analysed for vims neutralisation activity at the indicated time points. Sera were heat-inactivated and serially diluted in a 96-well plate. Diluted sera were then incubated with hMPV A vims at an MOI of 0.04 for 1 hr at 37°C. 20,000 Hep2 cells were then added to the vims / sera mixtures and the plates were incubated for 5 days at 37°C. To visualise infection, cells were fixed in acetone, washed, and stained with a biotinylated anti -hMPV F protein-specific antibody. Cells were then washed and incubated with a strepavidin-HRP antibody. Plates were washed and incubated with TMB substrate for 7 minutes and colour development was stopped with 2 N H2SO4. Plates were read with an Envision at 450 nm wavelength.

[0415] 3.1.4. Tissue Collection and Processing

[0416] Animals were euthanized five days after vims challenge on study day 47. Lungs were collected immediately after thoracotomy and nasal tissue isolated. The right lung lobe was weighed prior to sectioning. Two pieces of right lung - one for viral plaque assay and one for RT-qPCR-based assay of hMPV- 100

[0417] virus replication - were collected and snap frozen by immediately placing tubes on dry ice after collection then storing at < -70°C until further analysis. Nasal tissues were weighed, processed, and stored at < -70°C until further analysis.

[0418] 3.2 RESULTS

[0419] The hMPV mRNA-VLP expressing the wildtype F protein on the lumazine synthase scaffold elicited a potent virus -neutralizing antibody response and provided complete protection from vims challenge (Figure 4). We next wanted to compare the vaccine efficacy of our hMPV mRNA-VLP vaccine in the gold standard cotton rat model of hMPV vims challenge. To test this, we immunized groups of cotton rats on study days 1 and 21 with either control PBS, 20 pg of an mRNA vaccine expressing wildtype, full-length, membrane-anchored hMPV F protein, or 20 pg of our mRNA-VLP vaccine expressing the wildtype hMPV F protein ectodomain fused to the lumazine synthase (LuS) scaffold (Figure 5 A). Prior to live hMPV vims challenge on study day 42, all groups were profiled for vims-neutralizing antibody titers (Figure 5B). Both mRNA vaccines elicited high neutralizing antibody titres with a greater level measured in animals having received the hMPV mRNA-VLP (Figure 5B). Five days after intranasal vims challenge, all groups were euthanized with the lungs and nasal tissues harvested to assess vims replication (Figure 5C - D). The levels of vims replication in the lungs - a measure of severe disease -among all vaccinated groups was substantially reduced compared to the PBS control which measured approximately 105infectious units per gram of lung tissue (Figure 5C). A substantial level of vims replication was measured in the nasal cavities of the PBS control animals (Figure 5D). No infectious vims was recovered in the mRNA-VLP group whereas one of the eight animals in the native F mRNA vaccine group possessed extensive vims replication suggesting a ‘breakthrough’ infection occurred. In contrast, there were no ‘breakthrough’ infections among the mRNA-VLP vaccine group suggesting sterilizing immunity. Given that both mRNA vaccines were administered at the same dose, these results taken together further strengthen the value of the VLP antigen design in maximizing the immune response and providing complete protection from vims challenge in the cotton rat model.

[0420] Example 4: hMPV mRNA-VLP Vaccine Elicits High-Titre Neutralising Antibodies in RSV-Experienced Non-Human Primates

[0421] Groups of RSV-experienced non-human primates (NHPs) were immunised with either a benchmark RSV prefusion-stabilised F protein vaccine (120 pg of DS-CAV1 + adjuvant), an mRNA vaccine expressing the prefusion-stabilised F protein DS-CAV1 (50 pg), or a combination mRNA vaccine expressing both the RSV prefusion-stabilised F protein as a VLP (LuS scaffold) and hMPV F protein as a VLP (LuS scaffold) administered as 15 pg of each mRNA. The immunogenicity was evaluated by vims neutralisation assays against hMPV A and hMPV B vimses. hMPV- 100

[0422] 4.1 METHODS

[0423] 4.1.1. RNA Synthesis Using in vitro Transcription (IVT)

[0424] Sequences encoding the RSV F DS-CAV1 protein, an RSV prefusion-stabilised F-LuS antigen, and the hMPV F-LuS antigen designs were cloned into an mRNA production vector comprised of a T7 promoter element, 5 ’ and 3 ’ UTRs, and a polyA tail. The template plasmid was linearised by digesting the plasmid DNA with BspQI (New England Biolabs) for 4 hr at 50°C. mRNAs were synthesized by in vitro transcription (New England Biolabs) incorporating N1 -methylpseudouridine and CleanCap-AG (TriLink BioTechnologies). IVT reactions were incubated for 5 hr at 37°C, followed by digestion with RNase-free DNase. The mRNA was purified using LiCl precipitation, formulated in lipid nanoparticles (LNP), and stored at -80°C.

[0425] 4.1.2. NHP Immunizations

[0426] The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at AstraZeneca and conducted at BIOQUAL, Inc. (Rockville, MD). 24 cynomolgus macaques (Macaca fascicularis) were divided into three groups. Individual animals received 1 mL intramuscular (IM) injections of either protein or mRNA vaccine on study day 1. Serum samples were obtained 1 day prior to and 14 and 92 days after immunization (study days 0, 14, and 92, respectively) to evaluate baseline and post-immunization antibody levels, respectively.

[0427] 4.1.3. hMPV Neutralisation Assay

[0428] Sera from immunised animals were analysed for neutralisation activity at the indicated time points. Sera were heat-inactivated and serially diluted in a 96 -well plate. Diluted sera were then incubated with the indicated virus (hMPV A or hMPV B) at an MOI of 0.04 for 1 hr at 37°C. 20,000 Hep2 cells were then added to the virus / sera mixtures and the plates incubated for 5 days at 37°C. To visualise infection, cells were fixed in acetone, washed, and then stained with biotinylated mAB 133-1H. Cells were washed and incubated with a strep-HRP antibody (Sigma-Aldrich). Plates were washed and incubated with TMB substrate for 7 minutes and colour development was stopped with 2 N H2SO4. Plates were read with an Envision at 450 nm wavelength.

[0429] 4.2 RESULTS

[0430] The immunogenicity of the hMPV mRNA-VLP vaccine was tested in combination with a related RSV mRNA-VLP vaccine among hMPV- and RSV-experienced non-human primates (NHPs) as a combination vaccine (15 pg of each mRNA) and compared against an adjuvanted RSV protein vaccine (120 pg) and an mRNA vaccine expressing a profusion-stabilised RSV F protein antigen (50 pg). Prior to study start (day 0) all animals were profiled for hMPV virus neutralizing antibody titres against both hMPV A (Figure 6A) and hMPV B (Figure 6B) viruses and deemed seronegative. 14 and 92 days after hMPV- 100

[0431] immunization, only the mRNA-VLP vaccine group elicited substantial hMPV A or hMPV B virus neutralizing titers (Figure 6A and 6B, respectively) compared to the other two RSV vaccine groups. hMPV- 100

[0432] SEQUENCE LISTING

[0433] Upper ease sequences arc protein sequences. Lower ease sequences arc RNA sequences. The sequences set out the basic amino acid / nucleotide sequences of the elements discussed above. As set out above, the standard nucleotides of the RNA sequences may be replaced with modified versions of the same.

[0434] SEQ ID NO: 1 - WT hMPV F protein augagcuggaagguggugauaauauucagccuuuugauuacgccacagcacggucugaaagagagcuaccuugaagaga gcugcucuacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaaugucuuuacucucgaggucgg cgaugucgaaaaucuuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaagucugcccuucgc gagcuucggacugucuccgcugaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuuguguugg gugcuaucgcgcuugggguagcuaccgccgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaauc agaagugacggcaauaaagaaugcucuuaaaaaaacgaaugaagcugucaguacgcuggguaacgggguacgaguccuu gccacugcagucagggaacugaaagauuucgugucuaaaaaucugacgagggcgauuaauaagaacaagugugauaucg cggaucucaagauggcggucaguuucagccaauuuaauagacgcuucuugaacgugguuagacaguuuucugauaacgc cgggauuacgcccgcgauuagccuugaccucaugaccgacgccgaacucgcgagggcagucucuaacaugccgacaucag cuggccagaucaaguugauguuggaaaaccgggcgaugguaaggagaaaagguuuugguauacucaucggcguauacgg uucaucagucauauauauggugcagcugccaauauucggcgugauugacacuccguguuggauagugaaagcagccccg uccuguucagaaaagaaggguaauuacgccugcuuguugagagaagaucagggaugguacugucaaaacgccgggucua cgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucugcgacaccgcggcgggaauaaacgu agcagagcagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaagcaccgggagacauccca uuuccaugguagcauugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguauuggauccaaca gaguggguauuauuaagcaauugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauagauaac acaguuuaucaauugucuaagguugaaggugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccgg ucaaguuucccgaagaucaauucaacguggcguuggaccaaguauucgagucuaucgaaaauagucaagcauugguuga ucaaagcaaccggauacugucaagcgcugaaaaaggcaacaca

[0435] SEQ ID NO: 2 - WT hMPV F protein MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNT hMPV-100

[0436] SEQ ID NO: 3 - hMPV F protein A185P augagcuggaagguggugauaauauucagccuuuugauuacgccacagcacggucugaaagagagcuaccuugaagagagcugcuc uacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaaugucuuuacucucgaggucggcgaugucgaaaauc uuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaagucugcccuucgcgagcuucggacugucuccgcu gaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuuguguugggugcuaucgcgcuugggguagcuaccgc cgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaaucagaagugacggcaauaaagaaugcucuuaaaaaaac gaaugaagcugucaguacgcuggguaacgggguacgaguccuugccacugcagucagggaacugaaagauuucgugucuaaaaauc ugacgagggcgauuaauaagaacaagugugauaucccggaucucaagauggcggucaguuucagccaauuuaauagacgcuucuug aacgugguuagacaguuuucugauaacgccgggauuacgcccgcgauuagccuugaccucaugaccgacgccgaacucgcgagggca gucucuaacaugccgacaucagcuggccagaucaaguugauguuggaaaaccgggcgaugguaaggagaaaagguuuugguauacu caucggcguauacgguucaucagucauauauauggugcagcugccaauauucggcgugauugacacuccguguuggauagugaaag cagccccguccuguucagaaaagaaggguaauuacgccugcuuguugagagaagaucagggaugguacugucaaaacgccgggucu acgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucugcgacaccgcggcgggaauaaacguagcagag cagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaagcaccgggagacaucccauuuccaugguagca uugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguauuggauccaacagaguggguauuauuaagcaau ugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauagauaacacaguuuaucaauugucuaagguugaag gugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccggucaaguuucccgaagaucaauucaacguggcguug gaccaaguauucgagucuaucgaaaauagucaagcauugguugaucaaagcaaccggauacugucaagcgcugaaaaaggcaacaca

[0437] SEQ ID NO: 4 - hMPV F protein A185P MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFG1L1GVYGSSV1YMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESTENSQALVDQSNRTLSSAEKGNT

[0438] SEQ ID NO: 5 - hMPV F protein DSCavES2 auguccuggaagguugucaucauauuuagucucuugaucacgccucaacaugggcugaaagagagcuacuuggaggaaagcuguag cacuauuacggaaggcuaucuuaguguguugcgaaccggaugguacaccaacguguucacauuggaggugggugacguggaaaauc ugaccugcgccgacgguccgucuuugauaaaaaccgaauuggaucuuaccaagucagcucuucgggaacucagaacugucuccgcug aucagcuggcgcgagaagagcaaauagaaaauccgagacgccgcagguuuguacugggugccauagcgugcgggguagcaacugcg gccgcggucacugccggggucgcaaucgcaaaauguauccgguuggagucugaaguaaccgccaucaaaaacugccugaagaaaacc aacgagugcguuaguacucugggcugcggcgugagagucuuggcuacagcuguaagggagcugaaggauuuugucucuaaaaaccu gacccgagcuauaaauaagaacaaaugcgacauaccagaccucaaaauggcgguuucuuuuagucaguucaaucggagauuccugaa cguggugcgacaauuuagugacaaugcugguauaacgccagcuauuucuaaagauuugaugacggaugcggaguuggcucgagcaa uuuccaauaugcccaccagugcuggucaaaucaaauugauguuggaaaaucgcgccaugguaaggcgaaagggguucgggauucuc auuggaguuuaugggagcagcgugauauauaugguccaguugccgauauucggaguuauagauacuccgugcuggauugugaagg cagcucccucuugcagugagaagaaagggaacuacgcaugucugcugcgcgaagaucaaggcugguauugucaaaaugcugguuca acaguauauuauccaugcgaaaaagacugugagacccgcggcgaccacguuuucugcgacacugcugcugguauuaacgucgcagag caaaguaaagaauguaacauaaacaucaguacaacuaauuauccauguaaagucagcugcggacggaaccccauuaguauggucgccc ucucuccccuuggcgcacugguugcaugcuacaaaggcgucucuuguaguaucgguaguaacagaguaggaauuauaaagcaacuc aacaaaggcugcaguuauauuacaaaucaggacgcugauacggucacgauugacaacacugucuaucaacugucuaaaguggaaggg gagcagcacgucauuaagggacgaccugucuccagcucuuucgauccggucaaguucccacaggaucaauucaacguagcacuugac caaugcuuugaaucaauugaaaauagccaagcucuuguagaccaaucaaacagaauccuuagcagcgcugagaagggaaacacg hMPV- 100

[0439] SEQ ID NO: 6 - hMPV F protein DSCavES2 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAK CTRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKM AVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMV RRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNT

[0440] SEQ ID NO: 7 - hMPV F protein v3B auguccuggaaggugaugauuauuauaaguuuguugaucaccccgcagcacggccugaaagaauccuaccuggaagaaaguuguuc aacaauaaccgagggauauuugucuguccugcgcacugggugguauacgaaugucuucacucuggaggugggugacguagaaaacc uuacuugcacagaugguccaaguuugauuaaaacugaguuggaucugacaaagagugcgcucagggaguugaaaacguguagugcg gaccaacucgcacgggaagaacaaauugaagggggugguggaggaggguuuguccucggagccauugcucuuggcguagcaacagc ggcagcggucacugccggaauagcuauagcaaaaaccauucgacucgagagugaggugaaugcgaucaaaggcuguuugaaaacaac uaaugaaugcgucaguacgcugggaaacgggguacgcgucuuggcgacagccguucgcgaguugaaggaguuuguuaguaagaacc ucaccucugcgaucaauaagaauaaaugugacauacccgauuugaaaauggccguuucauuuucacaguucaaucggagauuccuga auguagugcgccaauucagcgacaaugcugguaucacgccagcuaucagucuugaucuuaugacagacgcugaguuggcacgagcu gugaguuacaugccgacaagugcuggacagauaaaguugaugcuugaaaaucgaugcaugguacgacggaaaggauuuggaauccu uaucggagucuacgguucaucugucauauacaugguucaguugccuauauucggcguaauagacacgccauguuggauuaucaagg cggcgcccagcuguucugagaaggauggaaauuaugccugucuucuucgagaggaucagggcugguacugcaagaaugcuggauca acuguguacuauccuaacgacaaagacugugaaacaaggggcgaccacgucuucugugauacggcggcggguauaaacguggcagaa caaagucgagaaugcaauaucaacauaaguaccacuaauuauccuugcaaaguaucaaccggcagacacccuaucaguaugguggcuc uuuccccucucggcgcucuuguugcauguuacaaaggugucucauguuccauagguucuaaucgagugggaauuauuaaacaacuc ccuaaagguugcagcuacauuacuaaccaagacgcagauaccgugacaaucgacaauacuguuuaccagcuuucaaaaguagagggc gaacaacaugugaucaagggcaggccgguaagcagcagcuuugacccaauaaaguuucccgaggaccaguuuaacguagcuuuggac cagguguucgagucaauugagaauucucaggcgcuuguagaccagucaaacaagauccuuaacagugcagag

[0441] SEQ ID NO: 8 - hMPV F protein v3B MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGP SLIKTELDLTKSALRELKTCSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTI RLESEVNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIPDLKMA VSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVR RKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAG STVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPL GALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRP VSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAE

[0442] SEQ ID NO: 9 - Lumazine Synthase augcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgc cuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagauauaacucugguuagaguuccugguaguuggg agauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgccaucggcguauugauucgcggggcuaccc cgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuuggaguc aucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauu gaaauggcgaaccuguuu aaauc acugagg

[0443] SEQ ID NO: 10 — Lumazine Synthase hMPV-100

[0444] MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAA GELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLR SEQ ID NO: 11 - Linker

[0445] ggguccgguggcucuggg

[0446] SEQ ID NO: 12 - Linker

[0447] GSGGSG SEQ ID NO: 13 - WT F protein-LuS augagcuggaagguggugauaauauucagccuuuugauuacgccacagcacggucugaaagagagcuaccuugaagagagcugcuc uacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaaugucuuuacucucgaggucggcgaugucgaaaauc uuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaagucugcccuucgcgagcuucggacugucuccgcu gaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuuguguugggugcuaucgcgcuugggguagcuaccgc cgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaaucagaagugacggcaauaaagaaugcucuuaaaaaaac gaaugaagcugucaguacgcuggguaacgggguacgaguccuugccacugcagucagggaacugaaagauuucgugucuaaaaauc ugacgagggcgauuaauaagaacaagugugauaucgcggaucucaagauggcggucaguuucagccaauuuaauagacgcuucuug aacgugguuagacaguuuucugauaacgccgggauuacgcccgcgauuagccuugaccucaugaccgacgccgaacucgcgagggca gucucuaacaugccgacaucagcuggccagaucaaguugauguuggaaaaccgggcgaugguaaggagaaaagguuuugguauacu caucggcguauacgguucaucagucauauauauggugcagcugccaauauucggcgugauugacacuccguguuggauagugaaag cagccccguccuguucagaaaagaaggguaauuacgccugcuuguugagagaagaucagggaugguacugucaaaacgccgggucu acgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucugcgacaccgcggcgggaauaaacguagcagag cagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaagcaccgggagacaucccauuuccaugguagca uugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguauuggauccaacagaguggguauuauuaagcaau ugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauagauaacacaguuuaucaauugucuaagguugaag gugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccggucaaguuucccgaagaucaauucaacguggcguug gaccaaguauucgagucuaucgaaaauagucaagcauugguugaucaaagcaaccggauacugucaagcgcugaaaaaggcaacaca ggguccgguggcucugggaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaa ccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagauauaacucugguua gaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgccaucggcgua uugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauuggaguugcgaaa gccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcuggga agcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugagg

[0448] SEQ ID NO: 14 - WT F protein-LuS MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGSGGSGMQIYE GKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELAR KEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHG NKGWEAALSAIEMANLFKSLR hMPV-100

[0449] SEQ ID NO: 15 - CHIT1 5’ UTR

[0450] auugugcugcauc

[0451] SEQ ID NO: 16 - PRKACB 5’ UTR

[0452] auucugcuguuugcuccuugccagguucaac

[0453] SEQ ID NO: 17 - GOT1 5’ UTR

[0454] aaaaucucuugauuccuagucucucgau

[0455] SEQ ID NO: 18 - Extended CHIT1 5’ UTR

[0456] aggauugugcugcaucaagcuugccgccacc

[0457] SEQ ID NO: 19 - CS 3’ UTR

[0458] aacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaa

[0459] SEQ ID NO: 20 - CHIT1 3’ UTR gucgcuaaagccccuccagucccagcuuugaggcugggcccaggaucacucuacagccugccuccuggguuuucccugggggccgc aaucuggcuccugcaggccuuucuguggucuuccuuuauccaggcuuucugcucucagccuugccuuccuuuuuucugcgacuccu ggg cugccccuuucacuugcaaaauaaa

[0460] SEQ ID NO: 21 - Extended CS 3’ UTR ugaugauaauaggacuaguggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccuagcguac guaaaaaauggaaagaaccuagcguacg

[0461] SEQ ID NO: 22 - A185P F Protein-LuS Fusion augagcuggaagguggugauaauauucagccuuuugauuacgccacagcacggucugaaagagagcuaccuugaagagagcugcuc uacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaaugucuuuacucucgaggucggcgaugucgaaaauc uuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaagucugcccuucgcgagcuucggacugucuccgcu gaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuuguguugggugcuaucgcgcuugggguagcuaccgc cgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaaucagaagugacggcaauaaagaaugcucuuaaaaaaac gaaugaagcugucaguacgcuggguaacgggguacgaguccuugccacugcagucagggaacugaaagauuucgugucuaaaaauc ugacgagggcgauuaauaagaacaagugugauaucccggaucucaagauggcggucaguuucagccaauuuaauagacgcuucuug aacgugguuagacaguuuucugauaacgccgggauuacgcccgcgauuagccuugaccucaugaccgacgccgaacucgcgagggca gucucuaacaugccgacaucagcuggccagaucaaguugauguuggaaaaccgggcgaugguaaggagaaaagguuuugguauacu caucggcguauacgguucaucagucauauauauggugcagcugccaauauucggcgugauugacacuccguguuggauagugaaag cagccccguccuguucagaaaagaaggguaauuacgccugcuuguugagagaagaucagggaugguacugucaaaacgccgggucu acgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucugcgacaccgcggcgggaauaaacguagcagag cagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaagcaccgggagacaucccauuuccaugguagca uugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguauuggauccaacagaguggguauuauuaagcaau ugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauagauaacacaguuuaucaauugucuaagguugaag gugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccggucaaguuucccgaagaucaauucaacguggcguug gaccaaguauucgagucuaucgaaaauagucaagcauugguugaucaaagcaaccggauacugucaagcgcugaaaaaggcaacaca ggguccgguggcucugggaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaa ccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagauauaacucugguua gaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgccaucggcgua uugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauuggaguugcgaaa gccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcuggga agcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugagg hMPV-100

[0462] SEQ ID NO: 23 - A185P F Protein-LuS Fusion MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTV YYPNEKDCETRGDHVFCDTAAG1NVAEQSKECN1N1STTN YPCKVSTGRHP1SMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGSGGSGMQIYE GKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELAR KEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHG NKGWEAALSAIEMANLFKSLR SEQ ID NO: 24 - DSCavES2-LuS Fusion auguccuggaagguugucaucauauuuagucucuugaucacgccucaacaugggcugaaagagagcuacuuggaggaaagcuguag cacuauuacggaaggcuaucuuaguguguugcgaaccggaugguacaccaacguguucacauuggaggugggugacguggaaaauc ugaccugcgccgacgguccgucuuugauaaaaaccgaauuggaucuuaccaagucagcucuucgggaacucagaacugucuccgcug aucagcuggcgcgagaagagcaaauagaaaauccgagacgccgcagguuuguacugggugccauagcgugcgggguagcaacugcg gccgcggucacugccggggucgcaaucgcaaaauguauccgguuggagucugaaguaaccgccaucaaaaacugccugaagaaaacc aacgagugcguuaguacucugggcugcggcgugagagucuuggcuacagcuguaagggagcugaaggauuuugucucuaaaaaccu gacccgagcuauaaauaagaacaaaugcgacauaccagaccucaaaauggcgguuucuuuuagucaguucaaucggagauuccugaa cguggugcgacaauuuagugacaaugcugguauaacgccagcuauuucuaaagauuugaugacggaugcggaguuggcucgagcaa uuuccaauaugcccaccagugcuggucaaaucaaauugauguuggaaaaucgcgccaugguaaggcgaaagggguucgggauucuc auuggaguuuaugggagcagcgugauauauaugguccaguugccgauauucggaguuauagauacuccgugcuggauugugaagg cagcucccucuugcagugagaagaaagggaacuacgcaugucugcugcgcgaagaucaaggcugguauugucaaaaugcugguuca acaguauauuauccaugcgaaaaagacugugagacccgcggcgaccacguuuucugcgacacugcugcugguauuaacgucgcagag caaaguaaagaauguaacauaaacaucaguacaacuaauuauccauguaaagucagcugcggacggaaccccauuaguauggucgccc ucucuccccuuggcgcacugguugcaugcuacaaaggcgucucuuguaguaucgguaguaacagaguaggaauuauaaagcaacuc aacaaaggcugcaguuauauuacaaaucaggacgcugauacggucacgauugacaacacugucuaucaacugucuaaaguggaaggg gagcagcacgucauuaagggacgaccugucuccagcucuuucgauccggucaaguucccacaggaucaauucaacguagcacuugac caaugcuuugaaucaauugaaaauagccaagcucuuguagaccaaucaaacagaauccuuagcagcgcugagaagggaaacacgucc ggcggaaguucuggcucuagugggggaagcaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagc cucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagaua uaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaauc gccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauu ggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaa uaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugagg

[0463] SEQ ID NO: 25 - DSCavES2-LuS Fusion MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAK CIRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKM AVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMV RRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNTSGGSSGSSGGSMQI YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGEL ARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTK HGNKGWEAALSAIEMANLFKSLR hMPV-100

[0464] SEQ ID NO: 26 - v3B-LuS Fusion auguccuggaaggugaugauuauuauaaguuuguugaucaccccgcagcacggccugaaagaauccuaccuggaagaaaguuguuc aacaauaaccgagggauauuugucuguccugcgcacugggugguauacgaaugucuucacucuggaggugggugacguagaaaacc uuacuugcacagaugguccaaguuugauuaaaacugaguuggaucugacaaagagugcgcucagggaguugaaaacguguagugcg gaccaacucgcacgggaagaacaaauugaagggggugguggaggaggguuuguccucggagccauugcucuuggcguagcaacagc ggcagcggucacugccggaauagcuauagcaaaaaccauucgacucgagagugaggugaaugcgaucaaaggcuguuugaaaacaac uaaugaaugcgucaguacgcugggaaacgggguacgcgucuuggcgacagccguucgcgaguugaaggaguuuguuaguaagaacc ucaccucugcgaucaauaagaauaaaugugacauacccgauuugaaaauggccguuucauuuucacaguucaaucggagauuccuga auguagugcgccaauucagcgacaaugcugguaucacgccagcuaucagucuugaucuuaugacagacgcugaguuggcacgagcu gugaguuacaugccgacaagugcuggacagauaaaguugaugcuugaaaaucgaugcaugguacgacggaaaggauuuggaauccu uaucggagucuacgguucaucugucauauacaugguucaguugccuauauucggcguaauagacacgccauguuggauuaucaagg cggcgcccagcuguucugagaaggauggaaauuaugccugucuucuucgagaggaucagggcugguacugcaagaaugcuggauca acuguguacuauccuaacgacaaagacugugaaacaaggggcgaccacgucuucugugauacggcggcggguauaaacguggcagaa caaagucgagaaugcaauaucaacauaaguaccacuaauuauccuugcaaaguaucaaccggcagacacccuaucaguaugguggcuc uuuccccucucggcgcucuuguugcauguuacaaaggugucucauguuccauagguucuaaucgagugggaauuauuaaacaacuc ccuaaagguugcagcuacauuacuaaccaagacgcagauaccgugacaaucgacaauacuguuuaccagcuuucaaaaguagagggc gaacaacaugugaucaagggcaggccgguaagcagcagcuuugacccaauaaaguuucccgaggaccaguuuaacguagcuuuggac cagguguucgagucaauugagaauucucaggcgcuuguagaccagucaaacaagauccuuaacagugcagagggguccgguggcuc ugggaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggcauuguagccucucgcuucaaccacgcacucguug accgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgaggaagauauaacucugguuagaguuccugguag uugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgcaguaaucgccaucggcguauugauucgcgggg cuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuu ggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagcaugguaauaagggcugggaagcggcgcucagu gccauugaaauggcgaaccuguuuaaaucacugagg

[0465] SEQ ID NO: 27 - v3B-LuS Fusion MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGP SLIKTELDLTKSALRELKTCSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTI RLESEVNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIPDLKMA VSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVR RKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAG STVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPL GALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRP VSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEGSGGSGMQIYEGKLTAEGLR FGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAI GVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL SAIEMANLFKSLR hMPV-100

[0466] SEQ ID NO: 28 - Full-length mRNA - WT antigen aggauugugcugcaucaagcuugccgccaccaugagcuggaagguggugauaauauucagccuuuugauuacgccacagcacgguc ugaaagagagcuaccuugaagagagcugcucuacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaauguc uuuacucucgaggucggcgaugucgaaaaucuuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaaguc ugcccuucgcgagcuucggacugucuccgcugaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuugugu ugggugcuaucgcgcuugggguagcuaccgccgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaaucagaa gugacggcaauaaagaaugcucuuaaaaaaacgaaugaagcugucaguacgcuggguaacgggguacgaguccuugccacugcaguc agggaacugaaagauuucgugucuaaaaaucugacgagggcgauuaauaagaacaagugugauaucgcggaucucaagauggcggu caguuucagccaauuuaauagacgcuucuugaacgugguuagacaguuuucugauaacgccgggauuacgcccgcgauuagccuug accucaugaccgacgccgaacucgcgagggcagucucuaacaugccgacaucagcuggccagaucaaguugauguuggaaaaccggg cgaugguaaggagaaaagguuuugguauacucaucggcguauacgguucaucagucauauauauggugcagcugccaauauucggc gugauugacacuccguguuggauagugaaagcagccccguccuguucagaaaagaaggguaauuacgccugcuuguugagagaaga ucagggaugguacugucaaaacgccgggucuacgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucu gcgacaccgcggcgggaauaaacguagcagagcagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaa gcaccgggagacaucccauuuccaugguagcauugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguau uggauccaacagaguggguauuauuaagcaauugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauaga uaacacaguuuaucaauugucuaagguugaaggugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccgguca aguuucccgaagaucaauucaacguggcguuggaccaaguauucgagucuaucgaaaauagucaagcauugguugaucaaagcaacc ggauacugucaagcgcugaaaaaggcaacacaggguccgguggcucugggaugcagauuuaugagggcaagcucacagcagaaggc uugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggca ugggggucgcgaggaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaa gaagacaucgacgcaguaaucgccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaa gggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacg ggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugaggugau gauaauaggacuaguggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccuagcguacguaa aaaauggaaagaaccuagcguacgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaa hMPV-100

[0467] SEQ ID NO: 29 - Full-length mRNA - A185P antigen aggauugugcugcaucaagcuugccgccaccaugagcuggaagguggugauaauauucagccuuuugauuacgccacagcacgguc ugaaagagagcuaccuugaagagagcugcucuacuauuaccgagggcuaccugucaguucuuagaacggguugguacacaaauguc uuuacucucgaggucggcgaugucgaaaaucuuaccugugcggauggcccuucuuugaucaagacugaguuggaucuuaccaaguc ugcccuucgcgagcuucggacugucuccgcugaucaauuggcacgggaagaacagaucgagaauccucgacgcaggagauuugugu ugggugcuaucgcgcuugggguagcuaccgccgcugccguaaccgccgggguugcaaucgcgaaaaccauacgacuugaaucagaa gugacggcaauaaagaaugcucuuaaaaaaacgaaugaagcugucaguacgcuggguaacgggguacgaguccuugccacugcaguc agggaacugaaagauuucgugucuaaaaaucugacgagggcgauuaauaagaacaagugugauaucccggaucucaagauggcggu caguuucagccaauuuaauagacgcuucuugaacgugguuagacaguuuucugauaacgccgggauuacgcccgcgauuagccuug accucaugaccgacgccgaacucgcgagggcagucucuaacaugccgacaucagcuggccagaucaaguugauguuggaaaaccggg cgaugguaaggagaaaagguuuugguauacucaucggcguauacgguucaucagucauauauauggugcagcugccaauauucggc gugauugacacuccguguuggauagugaaagcagccccguccuguucagaaaagaaggguaauuacgccugcuuguugagagaaga ucagggaugguacugucaaaacgccgggucuacgguuuauuaucccaaugagaaagauugcgaaacacggggggaccacguauucu gcgacaccgcggcgggaauaaacguagcagagcagucuaaagaguguaauaucaauauaagcacaaccaauuauccuugcaaaguaa gcaccgggagacaucccauuuccaugguagcauugaguccgcuuggugccuugguagcuuguuacaagggcguaucuuguaguau uggauccaacagaguggguauuauuaagcaauugaacaaggggugcuccuacaucacuaaccaagacgcggacacagugaccauaga uaacacaguuuaucaauugucuaagguugaaggugagcagcauguaaucaaagggaggcccguaaguuccaguuuugauccgguca aguuucccgaagaucaauucaacguggcguuggaccaaguauucgagucuaucgaaaauagucaagcauugguugaucaaagcaacc ggauacugucaagcgcugaaaaaggcaacacaggguccgguggcucugggaugcagauuuaugagggcaagcucacagcagaaggc uugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggca ugggggucgcgaggaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaa gaagacaucgacgcaguaaucgccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaa gggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacg ggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugaggugau gauaauaggacuaguggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccuagcguacguaa aaaauggaaagaaccuagcguacgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaa hMPV-100

[0468] SEQ ID NO: 30— Full-length mRNA— DSCavES2 antigen aggauugugcugcaucaagcuugccgccaccauguccuggaagguugucaucauauuuagucucuugaucacgccucaacaugggc ugaaagagagcuacuuggaggaaagcuguagcacuauuacggaaggcuaucuuaguguguugcgaaccggaugguacaccaacgug uucacauuggaggugggugacguggaaaaucugaccugcgccgacgguccgucuuugauaaaaaccgaauuggaucuuaccaaguc agcucuucgggaacucagaacugucuccgcugaucagcuggcgcgagaagagcaaauagaaaauccgagacgccgcagguuuguacu gggugccauagcgugcgggguagcaacugcggccgcggucacugccggggucgcaaucgcaaaauguauccgguuggagucugaag uaaccgccaucaaaaacugccugaagaaaaccaacgagugcguuaguacucugggcugcggcgugagagucuuggcuacagcuguaa gggagcugaaggauuuugucucuaaaaaccugacccgagcuauaaauaagaacaaaugcgacauaccagaccucaaaauggcgguuu cuuuuagucaguucaaucggagauuccugaacguggugcgacaauuuagugacaaugcugguauaacgccagcuauuucuaaagau uugaugacggaugcggaguuggcucgagcaauuuccaauaugcccaccagugcuggucaaaucaaauugauguuggaaaaucgcgc caugguaaggcgaaagggguucgggauucucauuggaguuuaugggagcagcgugauauauaugguccaguugccgauauucgga guuauagauacuccgugcuggauugugaaggcagcucccucuugcagugagaagaaagggaacuacgcaugucugcugcgcgaaga ucaaggcugguauugucaaaaugcugguucaacaguauauuauccaugcgaaaaagacugugagacccgcggcgaccacguuuucu gcgacacugcugcugguauuaacgucgcagagcaaaguaaagaauguaacauaaacaucaguacaacuaauuauccauguaaaguca gcugcggacggaaccccauuaguauggucgcccucucuccccuuggcgcacugguugcaugcuacaaaggcgucucuuguaguauc gguaguaacagaguaggaauuauaaagcaacucaacaaaggcugcaguuauauuacaaaucaggacgcugauacggucacgauugac aacacugucuaucaacugucuaaaguggaaggggagcagcacgucauuaagggacgaccugucuccagcucuuucgauccggucaag uucccacaggaucaauucaacguagcacuugaccaaugcuuugaaucaauugaaaauagccaagcucuuguagaccaaucaaacagaa uccuuagcagcgcugagaagggaaacacguccggcggaaguucuggcucuagugggggaagcaugcagauuuaugagggcaagcuc acagcagaaggcuugagauucggcauuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacug uaucguucggcaugggggucgcgaggaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagag cuggcuagaaaagaagacaucgacgcaguaaucgccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucu gaaguaaguaagggacuggcaaaucucucauuggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaa gccauugaacgggcggguaccaagcaugguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaauc acugaggugaugauaauaggacuaguggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccu agcguacguaaaaaauggaaagaaccuagcguacgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaa hMPV-100

[0469] SEQ ID NO: 31 — Full-length mRNA— v3B antigen aggauugugcugcaucaagcuugccgccaccauguccuggaaggugaugauuauuauaaguuuguugaucaccccgcagcacggcc ugaaagaauccuaccuggaagaaaguuguucaacaauaaccgagggauauuugucuguccugcgcacugggugguauacgaauguc uucacucuggaggugggugacguagaaaaccuuacuugcacagaugguccaaguuugauuaaaacugaguuggaucugacaaagag ugcgcucagggaguugaaaacguguagugcggaccaacucgcacgggaagaacaaauugaagggggugguggaggaggguuugucc ucggagccauugcucuuggcguagcaacagcggcagcggucacugccggaauagcuauagcaaaaaccauucgacucgagagugagg ugaaugcgaucaaaggcuguuugaaaacaacuaaugaaugcgucaguacgcugggaaacgggguacgcgucuuggcgacagccguu cgcgaguugaaggaguuuguuaguaagaaccucaccucugcgaucaauaagaauaaaugugacauacccgauuugaaaauggccgu uucauuuucacaguucaaucggagauuccugaauguagugcgccaauucagcgacaaugcugguaucacgccagcuaucagucuug aucuuaugacagacgcugaguuggcacgagcugugaguuacaugccgacaagugcuggacagauaaaguugaugcuugaaaaucga ugcaugguacgacggaaaggauuuggaauccuuaucggagucuacgguucaucugucauauacaugguucaguugccuauauucgg cguaauagacacgccauguuggauuaucaaggcggcgcccagcuguucugagaaggauggaaauuaugccugucuucuucgagagg aucagggcugguacugcaagaaugcuggaucaacuguguacuauccuaacgacaaagacugugaaacaaggggcgaccacgucuucu gugauacggcggcggguauaaacguggcagaacaaagucgagaaugcaauaucaacauaaguaccacuaauuauccuugcaaaguau caaccggcagacacccuaucaguaugguggcucuuuccccucucggcgcucuuguugcauguuacaaaggugucucauguuccaua gguucuaaucgagugggaauuauuaaacaacucccuaaagguugcagcuacauuacuaaccaagacgcagauaccgugacaaucgac aauacuguuuaccagcuuucaaaaguagagggcgaacaacaugugaucaagggcaggccgguaagcagcagcuuugacccaauaaag uuucccgaggaccaguuuaacguagcuuuggaccagguguucgagucaauugagaauucucaggcgcuuguagaccagucaaacaa gauccuuaacagugcagagggguccgguggcucugggaugcagauuuaugagggcaagcucacagcagaaggcuugagauucggca uuguagccucucgcuucaaccacgcacucguugaccgccuugucgaaggugcuauugacuguaucguucggcaugggggucgcgag gaagauauaacucugguuagaguuccugguaguugggagauccccguugcggcaggagagcuggcuagaaaagaagacaucgacgc aguaaucgccaucggcguauugauucgcggggcuaccccgcauuucgauuacauagcgucugaaguaaguaagggacuggcaaauc ucucauuggaguugcgaaagccgauuacuuuuggagucaucaccgccgacacccuggaacaagccauugaacgggcggguaccaagc augguaauaagggcugggaagcggcgcucagugccauugaaauggcgaaccuguuuaaaucacugaggugaugauaauaggacuag uggauccaacuggagacugggugaaagugacuaccagaaagugaggaagccuaaauaaaccuagcguacguaaaaaauggaaagaac cuagcguacgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0470] SEQ ID NO: 32 - Foldon

[0471] YIPEAPRDGQAYVRKDGEWVLLSTFL

[0472] SEQ ID NO: 33 - Ferritin DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS SEQ ID NO: 34 - Beta-annulus

[0473] INHVGGTGGAIMAPVAVTRQLVGS SEQ ID NO: 35 - hMPV_v3B-Foldon MSWKVM111SLL1TPQHGLKESYLEESCST1TEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGP SLIKTELDLTKSALRELKTCSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTI RLESEVNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIPDLKMA VSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVR RKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAG STVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPL GALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRP VSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEGSGGSGYIPEAPRDGQAYVR KDGEWVLLSTFL hMPV-100

[0474] SEQ ID NO: 36 - hMPV_v3B-Ferritin MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGP SLIKTELDLTKSALRELKTCSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTI RLESEVNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDTPDLKMA VSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVR RKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAG STVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPL GALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRP VSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEGSGGSGDIEKLLNEQVNKEM QSSN L YMSMSS WC YTHSLDGAGLFLFDHA AEE YEHAKKL11FLN EN N VP V QLTS1S APEHKFE GLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIG NENHGLYLADQYVKGIAKSRKS SEQ ID NO: 37 - hMPV_v3B-P-annulus MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGP SLIKTELDLTKSALRELKTCSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTI RLESEVNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIPDLKMA VSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVR RKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAG STVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPL GALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRP VSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESGGSSGSSGGSINHVGGTGG AIMAPVAVTRQLVGS SEQ ID NO: 38 - hMPV_DS-CavES2-Foldon MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAK CIRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKM AVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMV RRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNTGSGGSGYIPEAPRD GQAYVRKDGEWVLLSTFL SEQ ID NO: 39 - hMPV_DS-CavES2-Ferritin MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAK CIRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKM AVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMV RRKGFG1L1GV YGSSV1YMVQLP1FGV1DTPCW1VKAAPSCSEKKGN YACLLREDQGW YCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNTGSGGSGDIEKLLNE QVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS APEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDIL DKIELIGNENHGLYLADQYVKGIAKSRKS hMPV- 100

[0475] SEQ ID NO: 40 - hMPV_DS-CavES2-P-annulus MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAK CTRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRATNKNKCDTPDLKM AVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMV RRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNTSGGSSGSSGGSINH VGGTGGA1MAPVAVTRQLVGS

[0476] SEQ ID NO: 41 - hMPV_wt-Foldon MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTYIPEAPRDGQA YVRKDGEWVLLSTFL SEQ ID NO: 42 - hMPV_A185P-Foldon MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSL1KTELDLTKSALRELRTVSADQLAREEQ1ENPRRRRFVLGA1ALGVATAAAVTAGVA1AK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLK MAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVA LSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVI KGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNT SEQ ID NO: 43 - Linker

[0477] SGGSSGSSGGS SEQ ID NO: 44 - Linker

[0478] uccggcggaaguucuggcucuagugggggaagc

[0479] SEQ ID NO: 45 - Alternative hMPV F protein DSCavES2 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADG PSL1KTELDLTKSALRELRTVSADQLAREEQ1ENPRRRRFVLGA1ACGVATAAAVTAGVA1AK CIRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKM AVSFSQFNRRFLNVVRQFSDNAGTTPATSKDLMTDAELARATSNMPTSAGQTKLMLENRAMV RRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRHPISMVALS PLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKG RPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNT SEQ ID NO: 46 - Full-Length hMPV wt F protein (GenBank ABM67072.1) hMPV-100

[0480] MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGP SLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVAAAAAVTAGVAIAKTI RLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDTDDLKMAV SFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRR KGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAG1NVAEQSKECN1N1STTNYPCKVSTGRHP1SMVALSPLG ALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPV SSSFDPTKFPEDQFNVALDQVFENTENSQALVDQSNRTLSSAEKGNTGFTTVTTLTAVLGSSMTLVST FIIIKKTKKPTGAPPELSGVTNNGFIPHN

Claims

hMPV- 100CLAIMS1. A messenger RNA (mRNA) molecule encoding a polypeptide comprising a human metapneumovirus (hMPV) fusion (F) protein, or an immunogenic fragment thereof, and a multimerization unit capable of multimerising to form a nanoparticle.

2. The mRNA molecule of claim 1, wherein the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO. 2, or a variant thereof having at least 90% identity to SEQ ID NO: 2.

3. The mRNA molecule of any one of claims 1 or 2, wherein the hMPV F protein is a wild type hMPV F protein.

4. The mRNA molecule of claim 3, wherein the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO: 2.

5. The mRNA molecule of any one of claims 1 or 2, wherein the hMPV F protein is a modified F protein which is stabilised in its prefusion conformation.

6. The mRNA molecule of claim 5, wherein the modified F protein comprises a proline residue at the position corresponding to position 185 of SEQ ID NO: 2.

7. The mRNA molecule of claim 6, wherein the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 90% identity to SEQ ID NO: 4.

8. The mRNA molecule of claim 6, wherein the hMPV F protein further comprises:(i) cysteine residues at the positions corresponding to positions 110, 127, 140, 147, 153, 322, 365 and 463 of SEQ ID NO: 2;(ii) a lysine residue at the position corresponding to position 219 of SEQ ID NO: 2;(iii) an isoleucine residue at the position corresponding to position 231 of SEQ ID NO: 2; (iv) an asparagine residue at the position corresponding to position 368 of SEQ ID NO: 2; and (v) a glutamine residue at the position corresponding to position 453 of SEQ ID NO: 2.hMPV- 1009. The mRNA molecule of claim 8, wherein the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 90% identity to SEQ ID NO: 6.

10. The mRNA molecule of claim 6, wherein the hMPV F protein further comprises:(i) cysteine residues at positions corresponding to positions 84, 140, 147 and 249 of SEQ ID NO: 2;(ii) glycine residues at the positions corresponding to positions 97-102 and 139 of SEQ ID NO: 2; and(iii) an aspartic acid residue at the position corresponding to position 323 of SEQ ID NO: 2.

11. The mRNA molecule of claim 10, wherein the hMPV F protein comprises or consists of the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 90% identity to SEQ ID NO: 8.

12. The mRNA molecule of any one of claims 1 to 2, wherein the hMPV F protein is encoded by (i) the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 70% identity to SEQ ID NO: 1; (ii) the nucleotide sequence of SEQ ID NO: 3; (iii) the nucleotide sequence of SEQ ID NO: 5; or (iv) the nucleotide sequence of SEQ ID NO: 7.

13. The mRNA molecule of any one of claims 1 to 12, wherein the multimerization domain is selected from the group consisting of: foldon, ferritin, lumazine synthase, and beta-annulus.

14. The mRNA molecule of any ne of claims 1 to 13, wherein the multimerization unit is lumazine synthase.

15. The mRNA molecule of claim 14, wherein the lumazine synthase multimerises to form a 60-meric nanoparticle.

16. The mRNA molecule of any one of claims 14 or 15, wherein the lumazine synthase comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least 90% identity to SEQ ID NO: 10.

17. The mRNA molecule of claim 16, wherein the lumazine synthase is encoded by the nucleotide sequence of SEQ ID NO: 9, or a variant thereof having at least 90% identity to SEQ ID NO: 9.hMPV- 10018. The mRNA molecule of any one of claims 1 to 17, wherein the hMPV F protein is located at the N-terminus of the polypeptide and the multimerization unit is located at the C-terminus of the polypeptide.

19. The mRNA molecule of any one of claims 1 to 18, wherein the hMPV F protein or fragment thereof and the multimerization unit are joined by a linker.

20. The mRNA molecule of claim 19, wherein the linker is 4 to 12 amino acids long.

21. The mRNA molecule of claim 20, wherein the linker comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence comprising up to 3 amino acid substitutions relative thereto.

22. The mRNAmolecule of any one of claims 1 or 2, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 14 or a variant thereof having at least 90% identity to SEQ ID NO: 14.

23. The mRNA molecule of claim 22, wherein the polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 13, or a variant thereof having at least 70% identity to SEQ ID NO: 13.

24. The mRNAmolecule of any one of claims 1 to 23, further comprising:(i) a 5 ’ untranslated region (5 ’ UTR),(ii) a 3 ’ untranslated region (3’ UTR), and(iii) a 5 ’ cap structurewherein the 5’ UTR and the 3’ UTR are operably linked to the sequence encoding the polypeptide.

25. The mRNA molecule of claim 24, wherein the 5’ UTR sequence originates or is derived from:(a) the 5’ UTR of human chitinase-1 (CHIT1);(b) the 5’ UTR of human protein kinase cAMP-activated catalytic subunit beta (PRKACB); or (c) the 5’ UTR of human glutamic-oxaloacetic transaminase 1 (GOT1).

26. The mRNAmolecule of any one of claims 24 or 25, wherein the 3’ UTR sequence originates or is derived from:(a) the 3’ UTR of human citrate synthase (CS); or(b) the 3’ UTR of human chitinase- 1 (CHIT1).hMPV-10027. The mRNA molecule of any one of claims 24 to 26, wherein:(i) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human CHIT1 and the 3’ UTR comprises a sequence originating or derived from the 3’ UTR of human CS; (ii) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human PRKACB and the 3 ’ UTR comprises a sequence originating or derived from the 3 ’ UTR of human CHIT1;(iii) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human PRKACB and the 3 ’ UTR comprises a sequence originating or derived from the 3 ’ UTR of human CS;(iv) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human CHIT1 and the 3’ UTR comprises a sequence originating or derived from the 3’ UTR of human CHIT1; (v) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human GOTl and the 3’ UTR comprises a sequence originating or derived from the 3’ UTR of human CS; or (vi) the 5’ UTR comprises a sequence originating or derived from the 5’ UTR of human GOTl and the 3 ’ UTR comprises a sequence originating or derived from the 3 ’ UTR of human CHIT1.

28. The mRNA molecule of any one of claims 24 to 27, wherein:(i) the 5’ UTR originating or derived from the 5’ UTR of human CHIT1 comprises the nucleotide sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 80% identity to SEQ ID NO: 15;(ii) the 5 ’ UTR originating or derived from the 5 ’ UTR of human PRKACB comprises the nucleotide sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 80% identity to SEQ ID NO: 16;(iii) the 5’ UTR originating or derived from the 5’ UTR of human GOTl comprises the nucleotide sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 80% identity to SEQ ID NO: 17;(v) the 3’ UTR originating or derived from the 3’ UTR of human CS comprises the nucleotide sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 80% identity to SEQ ID NO: 19; and / or(vi) the 3’ UTR originating or derived from the 3’ UTR of human CHIT1 comprises the nucleotide sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 80% identity to SEQ ID NO: 20.hMPV-10029. The mRNA molecule of any one of claims 24 to 28, wherein:(i) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence having at least 80%, 85%, 90% or 95% identical thereto;(ii) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence having at least 80%, 85%, 90% or 95% identity thereto;(iii) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence having at least 80%, 85%, 90% or 95% identity thereto;(iv) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence having at least 80%, 85%, 90% or 95% identity thereto;(v) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence having at least 80%, 85%, 90% or 95% identity thereto; or(vi) the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence having at least 80%, 85%, 90% or 95% identity thereto, and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence having at least 80%, 85%, 90% or 95% identity thereto.

30. The mRNA molecule of any of claims 24 to 29, wherein the 5’ UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 18, or a variant thereof having at least 90% identity to SEQ ID NO: 18; and the 3’ UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having at least 90% identity to SEQ ID NO: 21.

31. The mRNA molecule of any one of claims 1 to 30, further comprising a 3’ polyadenylation sequence comprising 60 to 100 adenine nucleotides.

32. The mRNA molecule of any one of claim 1 or 2, wherein the mRNA molecule comprises or consists of the nucleotide sequence of any one SEQ ID NOs: 28-31, or a variant thereof having at least 90% identity to any one of SEQ ID NOs: 28-31.hMPV-10033. The mRNA molecule of any one of claims 1 to 32, wherein the mRNA molecule comprises pseudouridine or modified pseudouridine nucleotides.

34. The mRNA molecule of claim 33, wherein the modified pseudouridine is N1 methylpseudouridine.

35. The mRNA molecule of any one of claims 1 to 34, wherein the mRNA comprises a 5’ cap having a Capl or modified Capl structure.

36. A pharmaceutical composition comprising the mRNA molecule of any one of claims 1 to 35.

37. The pharmaceutical composition according to claim 36, wherein the mRNA molecule is formulated in a lipid nanoparticle (LNP).

38. The pharmaceutical composition of claim 36 or 37, further comprising a second mRNA molecule encoding a second polypeptide comprising a modified respiratory syncytial virus (RSV) F protein, or an immunogenic fragment thereof, and a lumazine synthase, wherein the modified RSV F protein is stabilised in the prefusion conformation.

39. The pharmaceutical composition of any one of claims 36 to 38, further comprising a pharmaceutically -acceptable carrier, diluent or excipient.

40. The mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 39, for use as a therapeutic or prophylactic agent.

41. The mRNA molecule of any one of claims 1 to 35, the pharmaceutical composition of any one of claims 36 to39, for use in raising an immune response in a subject.

42. The mRNA molecule of any one of claims 1 to 35, the pharmaceutical composition of any one of claims 36 to 39, for use in a method of preventing and / or attenuating an infectious disease, optionally a disease caused by a respiratory virus.

43. The mRNA molecule or pharmaceutical composition for use according to claim 42, wherein the disease is caused by hMPV or RSV.

44. The mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 37, for use in a method of prophylaxis or therapy, wherein the method compriseshMPV- 100administering the mRNA molecule or pharmaceutical composition to a subject in combination with a second mRNA molecule as defined in claim 38, or a second pharmaceutical composition comprising the second mRNA molecule.

45. Amethod of inducing an immune response in a subject, comprising administering to the subject the mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 39.

46. A method of preventing and / or attenuating an infectious disease in a subject, the method comprising administering an effective amount of the mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 39 to the subject, optionally wherein the disease is caused by a respiratory virus.

47. The method of claim 46, wherein the disease is a lower respiratory tract disease.

48. The method of claim 46 or 47, wherein the disease is caused by hMPV or RSV.

49. Use of the mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 39 in the manufacture of a medicament for use in a method of inducing an immune response in a subject.

50. Use of the mRNA molecule of any one of claims 1 to 35 or the pharmaceutical composition of any one of claims 36 to 39 in the manufacture of a medicament for use in preventing or attenuating a disease caused by hMPV or RSV infection.

51. An expression vector comprising an expression cassette encoding an mRNA molecule as defined in any one of claims 1 to 35.

52. A cell comprising the expression vector of claim 51.

53. A method of manufacturing an mRNA molecule as defined in any one of claims 1 to 35, comprising expressing the mRNA from the expression vector of claim 51, optionally wherein the mRNA is expressed by in vitro transcription.

54. A protein nanoparticle comprising a multimer of a polypeptide as defined in any one of claim 1 to 35.

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