TW202536184A - Respiratory syncytial virus vaccine compositions and their use - Google Patents

Abstract

The disclosure relates to respiratory virus ribonucleic acid (RNA) vaccines as well as methods of using the vaccines and compositions comprising the vaccines.

Description

Respiratory fusion virus vaccine composition and its uses

Human respiratory syncytial virus (RSV) is an infectious virus and a common cause of respiratory illnesses. Most infected adults experience only mild, flu-like symptoms. However, RSV infection can be life-threatening in infants, adults, and individuals with compromised immune systems through more severe symptoms such as bronchiolitis and pneumonia.

In fact, RSV is the leading cause of lower respiratory tract infections (LRTI, including bronchiolitis and pneumonia) in pediatrics worldwide. It is estimated that approximately 33 million children (<5 years old) worldwide suffer acute LRTI attacks each year due to RSV, resulting in approximately 3.2 million hospitalizations and about 120,000 deaths annually. About half of these deaths occur in infants under 6 months of age, and the vast majority of these cases occur in low- and middle-income countries.

For the elderly population, RSV is associated with actual morbidity and mortality, accounting for approximately 1.5 million LRTIs, and is particularly responsible for 4% to 10% mortality in those with heart or lung disease.

RSV is ubiquitous and relatively homogeneous in distribution worldwide. Almost all children are infected with the virus within the first two years of their lives. As host immunity to RSV weakens over time, reinfection is possible throughout life. The substantial medical burden associated with RSV infection has made RSV vaccine development a high priority in global public health and governmental organizations.

There is a need for RSV vaccines that are both highly effective and safe.

The invention described in this article provides, in particular, a highly effective and safe RSV mRNA vaccine to meet the current unmet medical needs of RSV vaccines.

This invention provides an RSV mRNA vaccine that encodes an optimized pre-fusion RSV F glycoprotein with a native cytoplasmic tail (CT) at the C-terminus and a T4 phage fibroin located between the extracellular and transmembrane domains. The efficacy of neutralizing antibodies generated against the antigen after administration of the antigen-modified mRNA vaccine encoded by this invention, and the host T cell response to the antigen, are also discussed. The T4 fibroin can be located between the EC and TM domains.

In contrast to the common practice of removing both the TM and CT regions from RSV vaccines (see GSK and Pfizer products) or only the CT region from RSV mRNA vaccines (such as those being developed by Moderna), the data revealed in this paper surprisingly demonstrate that the structure of an RSV vaccine design optimized by retaining both the TM and CT regions in the pre-fusion F protein antigen structure is optimal. Using three rounds of structure-based screening and optimization, this paper identifies and describes RSV mRNA vaccines that produce higher neutralizing antibody titers and better T-cell responses.

Therefore, in some phenotypes, this paper reveals modified polynucleotides (e.g., ribonucleotides or RNA) encoding the stable pre-fusion form of immunogenic RSV F glycoprotein, wherein the modified polynucleotides include: (a) a first polynucleotide encoding the transmembrane (TM) domain of RSV F glycoprotein; and (b) a second polynucleotide encoding the cytoplasmic tail (CT) region of RSV F glycoprotein.

This article also discloses compositions (e.g., pharmaceutical compositions) of mRNA vaccines, including the modified polynucleotides (e.g., mRNA) disclosed herein. In some embodiments, the composition comprises membrane-bound, pre-fusion stable form of mRNA encoding RSV F protein, wherein the p27-fusion protein domain is replaced by a short linker, has an extracellular trimeric domain, a complete transmembrane domain and a cytoplasmic tail region, and is complexed with ready-to-use (RTU) thermally stable lipid nanoparticles (LNPs).

The expression of the trimeric pre-F antigen in the present invention's mRNA vaccine was confirmed by in vivo transfection with human cells. Furthermore, following in vivo administration, the present invention's RSV vaccine induced potent RSV-neutralizing antibodies against the RSV F antigen and a robust T-cell response. Due to the robust T-cell response, the mRNA vaccine candidate is expected to promote complete RSV clearance.

The RSV mRNA vaccine described in this article can also induce higher levels of IgG antibodies against the pre-F antigen, exhibiting a balanced Th1/Th2 immune response profile rather than a Th2-biased one. The main antigenic form expressed by the present invention's mRNA vaccine in vivo is a trimeric pre-F protein (as opposed to the post-F protein), as confirmed in competitive ELISA using epitope-specific antibodies.

Compared to mRNA vaccines encoding the same ORF as mRNA-1345 (e.g., the first nearly commercially available RSV mRNA vaccine encoding the pre-F antigen), the RSV mRNA vaccine of this invention exhibits superior performance in neutralizing antibody titers. Furthermore, the target RSV mRNA vaccine described herein is thermally stable at room temperature for at least approximately one week, thereby greatly facilitating transportation, storage, and vaccination in most low- and middle-income countries.

Some embodiments of the present invention provide compositions (e.g., immunogenic and/or vaccine compositions) comprising a modified polynucleotide (e.g., ribonucleotide or RNA) in a stable pre-fusion form encoding an immunogenic RSV F glycoprotein, wherein the modified polynucleotide comprises: (a) a first polynucleotide encoding a transmembrane (TM) domain of the RSV F glycoprotein; and (b) a second polynucleotide encoding a cytoplasmic tail (CT) region of the RSV F glycoprotein. In some embodiments, the RSV F glycoprotein is at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to SEQ ID NO:1 or a fragment thereof.

In some embodiments, any of the aforementioned modified polynucleotides further includes a third polynucleotide encoding an extracellular (EC) domain. In some embodiments, any of the aforementioned modified polynucleotides further includes a fourth polynucleotide encoding a trimeric domain of T4 fibroin.

In some embodiments, the RSV F glycoprotein encoded by the modified polynucleotide of any of the aforementioned states lacks the p27-fusion protein (p27-FP) domain. The p27-fusion protein domain of the RSV F glycoprotein can be replaced by a short linker (e.g., the GS linker). Therefore, in some embodiments, the p27-FP domain of the RSV F glycoprotein is replaced by a linker (e.g., the GS linker).

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes one or more amino acid substitutions selected from the group consisting of: S46X (e.g., S46G), E92X (e.g., E92D), P102X (e.g., P102A), S215X (e.g., S215P), I379X (e.g., I379V), L373X (e.g., L373R), M447X (e.g., M447V), and K465X (e.g., K465Q), wherein X is any amino acid other than the original amino acid.

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of A149C and Y458C.

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of S155C and S290C.

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S190X (e.g., S190F) and V207X (e.g., V207L).

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes amino acid substitution F572A.

In some embodiments, the RSV F glycoprotein includes a truncated CT (e.g., composed essentially of a peptide sequence of KAR/a truncated CT composed of it). In some embodiments, the truncated CT includes amino acid positions 4-24 of SEQ ID NO:16.

In some embodiments, the modified polynucleotide is an RNA with multiple (A) tails, the length of which, depending on the case, is between 50 and 150 nucleotides.

In some embodiments, the modified polynucleotides disclosed herein comprise nucleic acid sequences that are at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to fragments of SEQ ID NO:8, 9, 10, 11, 12, 13, 29, 32, 33, or 34 (e.g., SEQ ID NO:8, 11, 12, 13, 29, 32, 33, or 34). In some embodiments, the modified polynucleotides disclosed herein comprise the nucleic acid sequence of SEQ ID NO:45 or a fragment thereof. In some embodiments, the modified polynucleotides disclosed herein consist of the nucleic acid sequence of SEQ ID NO:45 or a fragment thereof.

In some embodiments, the RSV F glycoprotein is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to SEQ ID NO:17, 18, 19, 20, 21, or 22. In some embodiments, the RSV F glycoprotein includes the sequence of SEQ ID NO:17, 18, 19, 20, 21, or 22. In some embodiments, the RSV F glycoprotein includes the amino acid sequence of SEQ ID NO:46 or a fragment thereof. In some embodiments, the RSV F glycoprotein is composed of the amino acid sequence of SEQ ID NO:46 or a fragment thereof.

In some embodiments, the modified polynucleotide further includes a 5’ untranslated region (UTR) containing the nucleic acid sequence of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42 or 43. In some embodiments, the modified polynucleotide further includes a 3’ UTR containing the nucleic acid sequence of SEQ ID NO:5 or 44.

In some embodiments, the modified polynucleotide further includes a 5' cap (e.g., cap-0 or m7G(5')ppp(5')N1mpNp cap).

In some embodiments, the modified polynucleotide further includes chemical modification. In some embodiments, the chemical modification is the substitution of uridine in the polynucleotide with N1-methylpseuuridine; where applicable, all or substantially all of the uridine in the polynucleotide is substituted with N1-methylpseuuridine.

This article also discloses pharmaceutical compositions (e.g., vaccines) comprising any of the aforementioned states of modified polynucleotides. In some embodiments, the modified polynucleotides are formulated in lipid nanoparticles (LNPs).

In some embodiments, LNP includes a mixture of lipids containing the following lipids: (1) about 20%-60%, about 30%-50% or about 40% (moles percentage) of ionizable cationic lipids; (2) about 30%-70% (e.g., about 40%-60% or about 50%) (moles percentage) of sterol lipids; (3) about 5%-30% (e.g., about 5%-15% or about 10%) (moles percentage) of phospholipids; or (4) about 0%-5% (e.g., about 1%-3% or about 2%) (moles percentage) of occult lipids or PEG-modified lipids.

In some embodiments, the mixture of lipids includes ionizable cationic lipids, cholesterol, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), and 1,2-dimyristyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), wherein the ionizable cationic lipids are selected from the group consisting of lipids 2, 4, 5, and 8 having the following structures: Lipid 2 Lipid No. 4 Lipid No. 5 and lipid No. 8 .

In some embodiments, the molar ratio of ionizable cationic lipids, cholesterol, DSPC and DMG-PEG2000 is approximately 40:48:10:2.

In some embodiments, the modified polynucleotide (e.g., mRNA) is formulated into lipid nanoparticles (LNPs) by mixing the modified polynucleotide (e.g., mRNA) with the LNP in a container to achieve the following final mRNA concentrations: approximately 10 μg/mL to approximately 100 μg/mL, approximately 20 μg/mL to approximately 100 μg/mL, approximately 40 μg/mL to approximately 100 μg/mL, approximately 50 μg/mL to approximately 100 μg/mL, approximately 60 μg/mL to approximately 100 μg/mL, approximately 20 μg/mL to approximately 80 μg/mL, approximately 40 μg/mL to approximately 80 μg/mL, approximately 40 μg/mL to approximately 60 μg/mL, approximately 50 μg/mL to approximately 150 μg/mL, approximately 100 to approximately 200 μg/mL, and approximately 150 μg/mL to approximately 250 μg/mL. The concentrations are approximately 200 μg/mL to approximately 300 μg/mL, approximately 30 μg/mL, approximately 40 μg/mL, approximately 50 μg/mL, approximately 60 μg/mL, approximately 70 μg/mL, approximately 80 μg/mL, approximately 90 μg/mL, approximately 100 μg/mL, approximately 120 μg/mL, approximately 150 μg/mL, approximately 170 μg/mL, approximately 200 μg/mL, approximately 220 μg/mL, approximately 250 μg/mL, approximately 270 μg/mL, or approximately 300 μg/mL, wherein the mixing comprises inverting the container to mix thoroughly for approximately 30 seconds. In some embodiments, the final mRNA concentration is approximately 50 μg/mL to approximately 100 μg/mL. In some embodiments, the final mRNA concentration is approximately 50 μg/mL.

In some embodiments, after (e.g.) storage in solution or suspension at about 1 µg/µL or in freeze-dried powder form at -60°C to -80°C, the modified polynucleotide (e.g., mRNA) is equilibrated to room temperature for about 30 minutes before being mixed with LNP; wherein the LNP is stored at 4°C and protected from light. In some embodiments, the mRNA is stored in freeze-dried powder form before use.

In some embodiments, the mixture of modified polynucleotides (e.g., mRNA) and LNPs is incubated at room temperature for about 10 minutes before being used for immunization.

In some embodiments, the average diameter of the LNP is less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, or less than 60 nm; (e.g., about 50-70 nm or about 60 nm).

In some embodiments, the polydispersity index (PDI) of LNP is about 0.01 to about 0.15 or about 0.05 to about 0.10.

In some embodiments, the zeta potential of the LNP is about 1.00 mV to about 5.00 mV or about 1.50 mV to about 4.00 mV.

This article also discloses a method for treating RSV infection in individuals, which includes administering to an individual a therapeutically effective amount of any of the aforementioned modified polynucleotides or any of the aforementioned pharmaceutical compositions.

In some implementations, the individual's immune system is compromised. In some implementations, the individual has lung disease.

In some embodiments, the individual is 5 years of age or younger. In other embodiments, the individual is 60 years of age or older.

In some embodiments, the method includes administering at least one dose of the combination to an individual. In some embodiments, the method includes administering at least two doses of the combination to an individual.

In some implementations, administration of a pharmaceutical compound induces an individual’s Th1/Th2 balance.

It should be understood that, unless such combination is expressly denied or incorrect, any embodiment of the invention described herein (including embodiments described only in the scope of the patent application or embodiments described only in one aspect of the invention) may be combined with any one or more additional embodiments of the invention.

This article also discloses a modified polynucleotide including a 5' untranslated region (UTR), which comprises a nucleic acid sequence having a secondary structure substantially identical to any of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 and their fragments, and, where applicable, is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to any of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 and their fragments. In some embodiments, the 5'UTR comprises a nucleic acid sequence that is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to SEQ ID NO:4. Secondary structures may include one or more (e.g., all) of the following characteristics: 1) having a length of approximately 50 nucleotides; 2) having binding sites for translation initiation factors and ribosomes; 3) having a GC-rich hairpin region for stable secondary structures; 4) having a short AT-rich region for rapid ribosome passage; 5) having a GC-rich sequence adjacent to a Kozak sequence for efficient binding of ribosomes to the AUG start codon; and/or 6) lacking a translational repression domain (e.g., lacking a non-canonical start codon).

In some embodiments, the modified polynucleotide further includes a 3’ UTR. In some embodiments, the 3’ UTR includes the nucleic acid sequence of SEQ ID NO:5 or 44.

In some embodiments, the modified polynucleotide further includes a nucleic acid sequence encoding a polypeptide. In some embodiments, the antigen is a viral antigen (e.g., hRSV antigen). In some embodiments, the antigen induces a higher antibody titer when expressed in a host mammal (e.g., human) compared to the same antigen encoded by a control polynucleotide lacking the 5'UTR.

References to related applications: This application claims priority and benefits over the application dates of International Patent Application No. PCT/CN2024/071819 filed on January 11, 2024 and International Patent Application No. PCT/CN2024/111992 filed on August 14, 2024, the entire contents of which, including all figures and sequences, are incorporated herein by reference.

The development of RSV vaccines has taken decades, during which time it has experienced numerous failures and setbacks. Only recently have new discoveries guided researchers back on track.

Human RSV is an enveloped, negative-sense, single-stranded RNA virus belonging to the Paramyxoviridae family. Currently, RSV exists in two antigenic subgroups (group A and group B) determined by genetic differences in the G and F genes. During the same RSV epidemic period, both A and B viruses can co-spread. The RSV genome contains 10 genes encoding 11 proteins. Among these, there are three non-structural proteins (NS1, NS2, and M2-2) and eight structural proteins (SH, G, F, N, P, M, M2-1, and L). Of the three surface glycoproteins, G and F mediate the attachment and fusion of the virion with the respiratory epithelial membrane, which is crucial for viral infectivity and pathogenesis. In addition, the F glycoprotein also mediates the aggregation of neighboring cells, leading to the formation of specific syncytiosomes.

Both RSV G and F proteins possess antigenic determinants that induce the production of neutralizing antibodies. However, the G protein is antigenically variable and highly glycosylated, making its use as a broad-spectrum protective vaccine antigen challenging. In contrast, the F protein is highly conserved in RSV A and B subtypes, has a limited number of glycosylation sites, and >90% of highly effective human neutralizing antibodies target this protein, particularly the Ø epitope. The F protein exhibits two conformations on the virion surface: a metastable state designated pre-fusion (pre-F) and a stable state designated post-fusion (post-F). Pre-F is the active form of the F protein with six antigenic sites (Ø, I, II, III, IV, V) for neutralization. Sites Ø and V are only present on the pre-F protein, while the other sites are shared by both pre-F and post-F. During virional attachment and fusion with the host cell membrane, the pre-F protein can be readily triggered to fold back into its post-F form to facilitate membrane fusion; however, post-F cannot revert to its functional pre-F form. After structural configuration, the post-F protein loses its apical neutralizing sensitive sites – Ø and V, and neutralizing antibodies targeting other sites are less potent than those binding to sites Ø and V. Therefore, the site Ø-specific monoclonal antibody D25 exhibits 100-fold higher neutralizing activity than plivizumab. Accumulated preclinical and clinical data have revealed that stable pre-F proteins, particularly in their trimer state, can induce potent antibody responses in both animal models and humans. Therefore, trimer-stabilized pre-F proteins are the desired antigenic form for RSV vaccines. Several methods can be employed to stabilize the RSV F protein in this pre-fusion state by optimizing the pre-F protein structure. These methods include: introducing short linkers using disulfide bonds; and using cavity-filling replacements.

Extensive efforts have been made to optimize the pre-F protein structure to stabilize the RSV F protein in its pre-fusion state. Three types of strategies have been employed to stabilize the pre-fusion state: introduction of short linkers, disulfide bonds, and cavity-filling substitution. Due to the furin cleavage process in the peptide 27 (p27) region, a hydrophobic fusion peptide (FP) can subsequently be released to trigger a refolding process. Therefore, GS short linkers are often used to replace the p27-FP region between the F1 and F2 subunits of RSV F to prevent this release. Disulfide bonds (including intra- and inter-unit disulfide bonds) are introduced as additional forces in the pre-fusion conformation to enhance hinge movement and the refolding region. Furthermore, specific hydrophobic cavities near the binding sites of the D25 antibody (which are characteristic of the pre-fusion configuration) are other sites that are intended to be modified to enhance D25 recognition. Therefore, cavity filling replaces the prevailing sidechain configuration with minimal collisions to stabilize the cavity.

Although RSV F protein exists as a homotrimer, there is an equilibrium between the open and closed states of the pre-fusion F protein, a transient state caused by respiratory movements in the pre-fusion conformation. Each monomer can aggregate via the transmembrane domain (TM) and cytoplasmic tail (CT) to form the pre-fusion F trimer. Furthermore, whether the pre-fusion RSV F protein completely dissociates (monomers) depends on the presence of the TM domain. Therefore, the metastable pre-fusion F protein is anchored to the viral membrane via the TM and CT domains. However, it has also been reported that the unaggregated (monomer) state of the pre-fusion F protein can be secreted by deleting the TM and CT domains.

However, even without anchoring TM and CT in the pre-fusion F variant design, the original organism can be tethered by adding a C-terminal T4-phage fibroin trimerizing domain (T4 fold). Since the trimerization of the T4 fold stabilizes the trimer conformation and prevents original organism dissociation, it is expected to help maintain a more stable and sensitive binding site (antigenic site Ø) of the pre-fusion F protein (which is characteristic of specific antibodies with potent neutralizing capabilities).

Currently, the US FDA has approved two RSV vaccines (AREXVY and ABRYSVO for adults) developed by GSK and Pfizer, respectively. In addition, the FDA has also approved Pfizer's RSV vaccine for maternal immunization in pregnant women to protect their newborns from RSV infection. Both vaccines use soluble and trimer-stable pre-F protein as the antigen, in which T4 folds are fused to the C-terminus of the pre-F protein to maintain the trimer conformation, and the TM and CT regions of the RSV F protein are removed.

However, both vaccines are conventional protein submonovalent vaccines, with or without adjuvants. Their production processes, particularly in CMC (Consumer Control Center) processes, involve complex protein expression, purification, and analytical procedures, making them relatively expensive for most low- and middle-income countries (LMICs). Compared to protein submonovalent vaccines, mRNA vaccines (which are novel and based on vaccine platforms proven during the COVID-19 pandemic) offer several advantages, such as flexible antigen design, the ability to induce potent humoral and cellular immune responses, rapid development, and low manufacturing costs. Utilizing proprietary heat-stable lipid nanoparticle technology, we are developing highly effective and safe RSV mRNA vaccines to address unmet medical needs.

This invention provides immune components (e.g., RNA vaccines) that induce potent neutralizing antibodies against RSV antigens (e.g., respiratory syncytial virus (RSV) antigens). The term "RSV antigen" in this document encompasses RSV antigens encoded by the RNA of this invention (e.g., RSV F glycoprotein). It should be understood that the terms "RNA" and "RNA construct" are used interchangeably herein.

In some embodiments, the immunocomponent comprises a modified polynucleotide (e.g., messenger RNA (mRNA)) encoding a stable pre-fusion form of RSV F glycoprotein (e.g., hRSV F glycoprotein). In some embodiments, the modified polynucleotide is RNA (e.g., messenger RNA (mRNA)). In some embodiments, an RNA (e.g., having a 5' UTR, ORF, 3' UTR, and multiple (A) tails) encodes a pre-fusion RSV F glycoprotein.

The hRSV envelope contains three surface glycoproteins: F, G, and SH. G and F proteins are targets for protecting antigens and neutralizing antibodies. However, the F protein is more conserved across hRSV strains and types (A and B). The hRSV F protein is a well-conserved type I fusion glycoprotein among clinical isolates (including between the hRSV-A and hRSV-B antigenic subsets). The F protein transitions between a pre-fusion state and a more stable post-fusion state, thereby facilitating entry into target cells. The hRSV F glycoprotein is initially synthesized as a precursor protein for F0. hRSV F0 folds into a trimer, which is activated by furin cleavage into a mature pre-fusion protein comprising the FI and F2 subunits (Bolt et al., Vims Res., 68:25, 2000). Although the target of neutralizing monoclonal antibodies lies in the post-fusion conformation of the F protein, the neutralizing Ab response primarily targets the pre-fusion conformation of the F protein in naturally occurring hRSV-infected individuals (Magro M et al., Proc Natl Acad Sci USA 2012; 109(8):3089-94; Ngwuta JO et al., Sci Transl Med 2015; 7(309):309ral62). Consistent with this, the neutralizing immune response in animal models induced by the pre-fusion conformation of the hRSV F protein is greater than that observed with the post-fusion conformation of the hRSV F protein (McLellan et al., Science , 342:592-598, 2013). Therefore, stable pre-fusion hRSV F protein is a good candidate for inclusion in hRSV vaccines.

As discussed above, contrary to the common practice of removing both the TM and CT regions in RSV vaccines or only the CT region in RSV mRNA vaccines, the data revealed in this paper surprisingly demonstrate that retaining both the TM and CT regions in the pre-fusion F protein antigen structure optimizes RSV vaccine design. Therefore, in some phenotypes, this paper reveals modified polynucleotides (e.g., ribonucleotides or RNA (e.g., mRNA)) encoding the stable pre-fusion form of the immunogenic RSV F glycoprotein, wherein the modified polynucleotides include: (a) a first polynucleotide encoding the transmembrane (TM) domain of the RSV F glycoprotein; and (b) a second polynucleotide encoding the cytoplasmic tail (CT) region of the RSV F glycoprotein.

As used herein, the stable pre-fusion form of the RSV F protein (which exists in an unstable, high-energy state) includes mutations (e.g., stable mutations) to prevent the protein from transforming into its post-fusion conformation.

For example, in some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes one or more amino acid substitutions selected from the group consisting of: S46X (e.g., S46G), E92X (e.g., E92D), P102X (e.g., P102A), S215X (e.g., S215P), I379X (e.g., I379V), L373X (e.g., L373R), M447X (e.g., M447V), and K465X (e.g., K465Q), wherein X is any amino acid other than the original amino acid.

As used herein, in the context of an amino acid substitution/change representing the original amino acid at a specified position, "other than the original amino acid" means any of the 19 other protein amino acids that are different from the original amino acid. For example, E92X means that at position 92 (SEQ ID NO:1), X can be any of the 19 protein amino acids other than the original residual E. Therefore, necessaryly, different X in different mutations can refer to a different set of 19 amino acids, depending on the consistency of the original amino acid at the specified position.

In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of A149C and Y458C. In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of S155C and S290C. These substitutions of Cys can facilitate the introduction of additional/novel intrapeptide disulfide bonds.

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S190X (e.g., S190F) and V207X (e.g., V207L).

In some embodiments, the RSV F glycoprotein includes a truncated CT. In some embodiments, the truncated CT is substantially composed of or consists of a peptide sequence of KAR. In some embodiments, the truncated CT includes amino acid positions 4-24 of SEQ ID NO:16.

In some embodiments, any of the aforementioned modified polynucleotides further includes a third polynucleotide encoding an extracellular (EC) domain.

In some embodiments, any of the aforementioned modified polynucleotides further includes a fourth polynucleotide encoding the trimer domain of T4 fibroin.

In some embodiments, the RSV F glycoprotein encoded by the modified polynucleotide of any of the aforementioned states lacks the p27-fusion protein (p27-FP) domain. The RSV F glycoprotein may be replaced by a short linker (e.g., a GS linker) to replace the p27-fusion protein domain. Therefore, in some embodiments, the RSV F glycoprotein has its p27-FP domain replaced by a linker (e.g., a GS linker).

Some embodiments of the present invention provide compositions comprising modified polynucleotides (e.g., ribonucleotides or RNA) (e.g., immunogenic and/or vaccine compositions) that encode a stable pre-fusion form of the RSV F glycoprotein disclosed herein.

In some embodiments, when administered intramuscularly (IM) or intradermally (ID), the RSV RNA vaccines described herein are at least 10, 20, 40, 50, 100, 500, or 1,000 times superior to conventional vaccines (e.g., recombinant DS-Cav1 protein + aluminum adjuvant). These results are achieved even when administering significantly lower doses of RNA (e.g., mRNA) compared to conventional vaccine doses.

In some embodiments, the RSV RNA vaccine described herein elicits a balanced Th1 and Th2 immune response. The RSV RNA vaccine described herein can stimulate a balanced Th1/Th2 cellular response that helps clear RSV infection while avoiding inducing VERD (e.g., a reduced Th2 response compared to FI-RSV vaccines that induce VERD, see J Virol 89:11692-11705. doi:10.1128/JVI.02018-15). In some embodiments, the RSV RNA vaccine described herein elicits a human antibody IgG1/IgG4 ratio greater than approximately 1:1, 2:1, 3:1, 4:1, 5:1, or 10:1. In some embodiments, the RSV RNA vaccine disclosed herein induced a mouse antibody IgG2a/IgG1 ratio greater than approximately 1:1, 2:1, 3:1, 4:1, 5:1, or 10:1.

It should be understood that the immunocomposites (e.g., RNA vaccines) of this invention are not natural. In other words, the RNA polynucleotides encoding RSV antigens, as provided herein, do not exist in nature. It should also be understood that the RNA polynucleotides described herein are isolated from viral proteins and viral lipids that exist in nature. Therefore, as provided herein, immunocomposites including RNA formulated in lipid nanoparticles (e.g.) exclude viruses (i.e., the compositions are not viruses and do not contain viruses).

An antigen is a protein that can induce an immune response (e.g., cause the immune system to produce antibodies against the antigen). In some embodiments, unless otherwise stated, the use of the term "antigen" covers both immunogenic proteins and immunogenic fragments (immunogenic fragments that induce (or can induce) an immune response to RSV (e.g., hRSV). It should be understood that the term "protein" covers peptides and the term "antigen" covers antigenic fragments.

An exemplary sequence of the RNA of the RSV antigen and the RSV antigen of the present invention is provided in Table 7.

In some embodiments, the composition includes RNA encoding a pre-fusion form of an RSV (e.g., hRSV) F glycoprotein comprising a sequence that is at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to a fragment of SEQ ID NO:1 or thereof.

In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S46X (e.g., S46G), E92X (e.g., E92D), P102X (e.g., P102A), S215X (e.g., S215P), I379X (e.g., I379V), L373X (e.g., L373R), M447X (e.g., M447V), and K465X (e.g., K465Q), wherein X is any amino acid other than the original amino acid. In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: A149C and Y458C. In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S155C and S290C. In some embodiments, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S190X (e.g., S190F) and V207X (e.g., V207L).

DS-Cav1 is a normally soluble, disulfide-linked double-stranded protein that incorporates stable mutations of intrinsic disulfides (S155C, S290C), cavity-filling mutations (S190F, V207L), and native substitutions (P102A, I379V, M447V) to enhance its performance. The stable pre-fusion form of the RSV F protein disclosed herein, relative to SEQ ID NO:1, includes one or more amino acid substitutions selected from the group consisting of: S46X (e.g., S46G), E92X (e.g., E92D), S215X (e.g., S215P), L373X (e.g., L373R), K465X (e.g., K465Q), A149C, and Y458C, wherein X is any amino acid other than the original amino acid.

In one example, the stable pre-fusion forms of the RSV F protein disclosed herein include S46G, E92D, P102A, A149C, S155C, S190F, V207L, S215P, S290C, Y458C, and/or K465Q (e.g., any combination thereof). In another example, the stable pre-fusion forms of the RSV F protein disclosed herein include S46G, E92D, P102A, A149C, S155C, S190F, V207L, S215P, S290C, Y458C, K465Q, and/or F572A (e.g., any combination thereof). In one instance, the stable prefusion forms of RSV F proteins disclosed herein include S46G, E92D, P102A, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C and/or K465Q (e.g., any combination thereof). In one instance, the stable prefusion form of the RSV F protein disclosed herein includes S46G, E92D, P102A, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C and/or K465Q (e.g., any combination thereof), wherein the RSV F protein further includes a trimerizing domain of T4 cellulose located between the EC and TM domains.

In some embodiments, relative to SEQ ID NO:1, RSV F glycoprotein includes amino acid substitution F572A.

In some embodiments, the RSV F glycoprotein is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to fragments of SEQ ID NO:17, 18, 19, 20, 21, or 22. In some embodiments, the RSV F glycoprotein comprises, is substantially composed of, or is composed of the sequence of SEQ ID NO:17, 18, 19, 20, 21, or 22. In some embodiments, the RSV F glycoprotein comprises, is substantially composed of, or is composed of the sequence of SEQ ID NO:17. In some embodiments, the RSV F glycoprotein comprises, is substantially composed of, or is composed of the sequence of SEQ ID NO:18. In some embodiments, the RSV F glycoprotein comprises, is substantially composed of, or is composed of the sequence of SEQ ID NO:19. In some embodiments, the RSV F glycoprotein includes, is substantially composed of, or is composed of the sequence of SEQ ID NO:20. In some embodiments, the RSV F glycoprotein includes, is substantially composed of, or is composed of the sequence of SEQ ID NO:21. In some embodiments, the RSV F glycoprotein includes, is substantially composed of, or is composed of the sequence of SEQ ID NO:22. In some embodiments, the RSV F glycoprotein includes, is substantially composed of, or is composed of the sequence of SEQ ID NO:46.

It should be understood that any of the RNA-encoded antigens described in this article may or may not include a signal sequence.

The nucleic acid composition of this invention includes at least one open reading frame (ORF) of RNA that encodes an RSV antigen. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA (e.g., mRNA) further includes 5' UTR, 3' UTR, multiple (A) tails, and/or 5' cap analogues.

Nucleic acids are polymers of nucleotides (nucleotide monomers). Therefore, nucleic acids are also called polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with b-D-ribo conformation, a-LNA with a-L-ribo conformation (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-a-LNA with 2'-amino functionalization), vinyl nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof.

Messenger RNA (mRNA) is any RNA that encodes at least one protein (a naturally occurring, non-natural, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or in vitro. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequence described in this application may describe a "T" in a representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced by a "U". Thus, when each "T" in the DNA sequence is replaced by a "U", any DNA revealed and identified herein by a specific sequence identifier also reveals a corresponding RNA (e.g., mRNA) sequence that is inversely complementary or complementary to the DNA, and vice versa. When each "U" in an RNA sequence is replaced by a "T", any RNA revealed and identified by a specific sequence identification code in this paper also reveals a corresponding DNA sequence that is inversely complementary or complementary to the RNA.

An open reading frame (ORF) is a continuous DNA or RNA sequence that begins with a start codon (e.g., methionine (ATG or AUG)) and terminates with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. It should be understood that the sequences disclosed herein may further include additional elements (e.g., 5' and 3' UTRs), but these elements (unlike ORFs) do not necessarily need to be present in the RNA polynucleotides of this invention.

In some embodiments, the present invention's composition contains RNA encoding an RSV antigen variant. An antigen variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from the wild-type, native, or reference sequence. Compared to the native or reference sequence, the antigen/polypeptide variant may have substitutions, deletions, and/or insertions at certain positions within the amino acid sequence. Typically, the variant shares at least 50% identity with the wild-type, native, or reference sequence. In some embodiments, the variant shares at least 80% or at least 90% identity with the wild-type, native, or reference sequence.

The variant antigens/peptides encoded by the nucleic acids of this invention may contain amino acid variations that confer any of a number of desired properties (e.g., enhanced immunogenicity, enhanced expression, and/or improved stability or PK/PD properties in individuals). Variant antigens/peptides can be prepared using conventional mutagenesis techniques and analyzed as needed to determine whether they possess the desired properties. Analyses for determining expression levels and immunogenicity are well known in the art, and illustrative examples of such analyses are described in the Examples section. Similarly, the PK/PD properties of protein variants can be measured using art-recognized techniques (e.g., by measuring antigen expression over time in vaccinated individuals and/or by observing the durability of the induced immune response). The stability of proteins encoded by variant nucleic acids can be measured by analyzing thermal stability or stability after urea denaturation, or by computer-predicted measurement. Such experimental and computer-based methods are known in the industry. In some embodiments, the composition includes RNA or RNA ORF comprising a nucleotide sequence of any of the sequences provided herein (see, for example, Table 7), or comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleotide sequences provided herein.

The term "identity" refers to the relationship between sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids), as determined by sequence comparison. Identity also refers to the degree of sequence correlation between sequences, as determined by the number of matches between two or more amino acid or nucleic acid residue strings. Identity measures the percentage of identical matches between the smaller of two or more sequences, where alignment gaps (if present) are addressed by a specific mathematical model or computer program (e.g., an "algorithm"). Identity of related antigens or nucleic acids can be readily calculated using known methods. When applied to peptide or polynucleotide sequences, "percentage of similarity (%)" is defined as the percentage of candidate amino acids or nucleic acid residues (amino acid residues or nucleic acid residues) that are identical to those in the amino acid or nucleic acid sequence of the second sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of similarity. The methods and computer programs used for alignment are well-known in the industry. It should be understood that similarity depends on the calculation of the percentage of similarity, but can vary depending on the inclusion of vacancies and penalties in the calculation. Typically, a variant of a specific polynucleotide or polypeptide (e.g., an antigen) has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity with a specific reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. The tools used for alignment include BLAST kits (Stephen F. Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res . 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). General global alignment techniques are based on the dynamically programmed Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins . " J. Mol. Biol. 48:443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other best global alignment methods (including the Niederman-Onsch algorithm).

Therefore, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions, and covalent modifications, particularly the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this invention, relative to a reference sequence. For example, sequence tags or amino acids (e.g., one or more lysines) may be added to a peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues at the carboxyl and amino terminal regions of the amino acid sequence in a peptide or protein may be deleted, thereby providing a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) may be deleted alternatively, depending on the use of the sequence. In some embodiments, sequences used for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, polymerizing domains (e.g., folded regions), and such sequences can be substituted with substitution sequences to achieve the same or similar functions. In some embodiments, cavities in the protein core can be filled (e.g., by introducing larger amino acids) to improve stability. In other embodiments, buried hydrogen bond networks can be stabilized by substitution with hydrophobic residues. In still other embodiments, glycosylation sites can be removed and replaced with appropriate residues. These sequences are readily identifiable by those skilled in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminus or C-terminus) that may be deleted before being used, for example, in the preparation of RNA (e.g., mRNA) vaccines.

As is recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the RSV antigen of interest. For example, this document provides any protein fragment of a reference protein (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference antigen sequence but otherwise identical), provided that the fragment is immunogenic and confers a protective immune response against RSV. In addition to truncated variants identical to the reference protein, in some embodiments, the antigen contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided herein or in the reference sequences. The length of the antigen/antigenic polypeptide can range from about 4, 6, or 8 amino acids to the full-length protein.

In addition to other structural features (such as a 5'-cap or a 3'-poly(A) tail), naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including (but not limited to) the untranslated regions (UTRs) at their 5'-end (5' UTR) and/or their 3'-end (3' UTR). Both the 5' UTR and 3' UTR are elements of pre-mature mRNA that are normally transcribed from genomic DNA. Typically, characteristic structural features of mature mRNA (such as a 5'-cap and a 3'-poly(A) tail) are added to transcribed (pre-mature) mRNA during mRNA processing.

In some embodiments, the composition comprises an RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide having at least one modification and at least one 5' cap, and is configured within lipid nanoparticles. According to the manufacturer’s protocol, the following chemical RNA cap analogues can be used to simultaneously perform 5'-capping of the polynucleotide during in vivo transcription to generate a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [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). Vaccinia virus capping enzymes can be used to complete the 5'-capping of modified RNA after transcription to generate the "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). A cap 1 structure can be generated using both vaccinia virus capping enzymes and 2'-O-methyltransferases to produce: m7G(5')ppp(5')G-2'-O-methyl. A cap 2 structure can be generated from the cap 1 structure, followed by 2'-O-methylation of the 5'-last nucleotide using 2'-O-methyltransferases. A cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the 5'-last nucleotide using 2'-O-methyltransferases. The enzymes can be derived from recombinant sources.

The 3'-poly(A) tail is typically an adenine nucleotide segment added to the 3' end of a transcribed mRNA. In some cases, it comprises up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be essential for the stability of individual mRNAs.

In some embodiments, the composition includes a stabilizing element. The stabilizing element may include, for example, a histokines stem-loop. A stem-loop binding protein (SLBP) (32 kDa protein) has been identified. It binds to the histokines stem-loop at the 3' end of histokines signals in both the nucleus and cytoplasm. Its expression is regulated by the cell cycle; it peaks during S phase, when histokines mRNA levels also increase. This protein has been shown to be essential for efficient 3'-end processing of histokines mRNA precursors by U7 snRNP. Following processing, SLBP continues to bind to the stem-loop and then stimulates the translation of mature histone mRNA into histone in the cytoplasm. The RNA-binding domain of SLBP is conserved throughout metazoans and protozoa; its binding to the histone stem-loop depends on the loop's structure. The minimal binding site comprises at least three nucleotides 5' and two nucleotides 3' relative to the stem-loop.

In some embodiments, the RNA (e.g., mRNA) contains a coding region, at least one histostem loop, and, where appropriate, multiple (A) sequences or multiple adenosine monophosphate (MAP) signals. The multiple (A) sequences or MAP signals should generally enhance the expression of the encoded protein. In some embodiments, the encoded protein is not a histogenin, reporter gene protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selector protein (e.g., α-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

In some embodiments, RNA (e.g., mRNA) contains a combination of a poly(A) sequence or a polyadenylation signal with at least one histostem loop, which, even though both represent substitution mechanisms in nature, work synergistically to increase protein expression beyond what would be observed using individual elements. The synergistic effect of the combination of poly(A) and at least one histostem loop is independent of the order of the elements or the length of the poly(A) sequence.

In some embodiments, the RNA (e.g., mRNA) does not contain a histrin downstream element (HDE). A "histrin downstream element" (HDE) comprises a purine-rich polynucleotide segment of approximately 15 to 20 nucleotides at the 3' end of a naturally occurring stem-loop, representing the binding site of U7 snRNA involved in the processing of histrin mRNA precursors into mature histrin mRNA. In some embodiments, the nucleic acid does not contain introns.

RNA (e.g., mRNA) may or may not contain enhancer and/or promoter sequences, and may be modified or unmodified, or may be activated or inactivated. In some embodiments, histrin stem-loops are typically derived from histrin genes and consist of intramolecular base pairing of two adjacent portions or completely inversely complementary sequences separated by septa (composed of short sequences), forming a loop structure. Unpaired loop regions typically cannot pair with any of the bases in the stem-loop element. Because they are key components of many RNA secondary structures, they are more commonly found in RNA, but can also be present in single-stranded DNA. The stability of stem-loop structures generally depends on their length, the number of mismatches or protrusions, and the base composition of the paired regions. In some embodiments, oscillating base pairs (non-Watson-Crick base pairs) may be generated. In some embodiments, at least one histokinetic loop sequence comprises 15 to 45 nucleotides in length.

In some embodiments, one or more AU-rich sequences are removed from the RNA (e.g., mRNA). These sequences (sometimes called AURES) are unstable sequences found in the 3' UTR. AURES can be removed from the RNA vaccine. Alternatively, AURES can be retained in the RNA vaccine. Signal peptides

In some embodiments, the composition includes RNA (e.g., mRNA) with an ORF encoding a signal peptide fused to the RSV antigen. The signal peptide (comprising the N-terminal 15-60 amino acids of a protein) is typically required for transmembrane translocation along the secretory pathway, thereby universally controlling the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directly guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growth peptide chain across it for processing. ER processing produces mature proteins, in which the signal peptide is typically cleaved from the precursor protein by a host cell's ER-resident signaling peptidase, or remains unclew and serves as a membrane anchor. The signal peptide can also promote protein targeting to the cell membrane.

Signal peptides can be 15-60 amino acids long. For example, signal peptides can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids long. In some embodiments, the lengths of the signal peptides are 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25 or 15-20 amino acids.

Signal peptides derived from heterologous genes (which regulate the expression of genes other than RSV antigens in nature) are known in the industry and can test desired properties, and are then incorporated into the nucleic acid of this invention.

In some embodiments, the present invention 's compositions comprise RNA (e.g., mRNA) encoding an antigenic fusion protein. Therefore, one or more encoded antigens may comprise two or more proteins (e.g., proteins and/or protein fragments) conjoined together.

In some embodiments, such as the RNA (e.g., mRNA) vaccines provided herein, the scaffold portion encodes a fusion protein of an RSV antigen linked to the scaffold portion. In some embodiments, these scaffold portions endow the antigen with the desired properties encoded by the nucleic acids of the present invention. For example, the scaffold protein may, for example, improve the immunogenicity of the antigen by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or enabling the antigen to bind to a binding partner.

In some embodiments, the scaffold portion is a protein capable of self-assembling into highly symmetrical, stable, and structurally ordered protein nanoparticles with a diameter of 10–150 nm (a highly suitable size range for optimal interaction with various cells of the immune system). In some embodiments, viral proteins or viral particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold portion is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of -22 nm and lacks nucleic acids, thus being non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58–68). In some embodiments, the scaffold portion is composed of hepatitis B core antigen (HBcAg) that self-assembles into particles with a diameter of 24-31 nm, similar to the viral core acquired from the liver of a human infected with HBV. The generated HBcAg self-assembles into two different types of nanoparticles with diameters of 300 Å and 360 Å, corresponding to 180 or 240 atoms.

In some embodiments, a bacterial protein platform may be used. Non-limiting examples of such self-assembling proteins include ferritin, lumazine, and encapsulation proteins.

Ferritin is a protein whose main function is to store iron in cells. Ferritin is composed of 24 subunits, each composed of a tetra-α-helix bundle, which self-assemble into a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009;390:83-98). Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is composed of 24 identical primordia. In animals, ferritin light and heavy chains exist that can assemble individually or in different ratios into 24 subunit particles (Granier T. et al. J Biol Inorg Chem. 2003;8:105-111; Lawson D.M. et al. Nature. 1991;349:541-544). Ferritin self-assembles into thermo- and chemically stable nanoparticles. Therefore, ferritin nanoparticles are ideally suited for carrying and exposing antigens.

Dioxetine tetrahydropteridine synthase (LS) is also well-suited as a nanoparticle platform for antigen display. LS (which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin) is an enzyme found in a wide variety of organisms, including archaea, bacteria, fungi, plants, and fungi (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). LS monomers are 150 amino acids long and consist of a β-sheet and tandem α-helices on both sides. Several different quaternary structures of LS have been reported, illustrating its morphological versatility: from homopentamers to symmetrical assemblages of 12 pentamers forming a 150 Å diameter capsid. LS cages with more than 100 subunits have even been described (Zhang X. et al. J Mol Biol. 2006;362:753-770).

Encapsulating proteins (novel protein cage nanoparticles isolated from *Thermotoga maritima*) are also used as a platform for presenting antigens on the surface of self-assembled nanoparticles. The encapsulating proteins are assembled from 60 copies of identical 31 kDa monomers, which have thin and icosahedral T=1 symmetrical cage structures (internal and external diameters of 20 and 24 nm, respectively) (Sutter M. et al., *Nat Struct Mol Biol.* 2008, 15:939-947). Although the exact function of encapsulation proteins in Thermocystis aeruginosa is not yet fully understood, their crystal structure has recently been resolved and their function hypothesized as cellular compartments of encapsulation proteins (such as DyP (dye decolorizing peroxidase) and Flp (ferritin-like protein)) involved in oxidative stress responses (Rahmanpour R et al. FEBS J. 2013, 280:2097-2104).

Linker and Cleavable Peptide In some embodiments, the present invention encodes more than one polypeptide (referred to herein as a fusion protein) by mRNA. In some embodiments, the encoded polypeptide after post-translational cleavage comprises two fragments (e.g., F1 and F2 fragments), wherein these fragments can be linked together by a polypeptide linker. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker may be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker system is selected from the group consisting of: F2A linkers, P2A linkers, T2A linkers, E2A linkers, and combinations thereof. This family of self-cleavable peptide linkers (referred to as 2A peptides) has been described in the art (see, for example, Kim, JH et al. (2011) PLoS ONE 6: el8556). In some embodiments, the ligand system is the F2A ligand. In some embodiments, the ligand system is the GGGS ligand (which may contain 1-20, 1-10, 1-5, 5-15, or 5-10 repetitions of this sequence). In some embodiments, the ligand system is the GS ligand (which may contain 1-20, 1-10, 1-5, 5-15, or 5-10 repetitions of this sequence). In some embodiments, the fusion protein contains three domains with an insert ligand, having the following structure: domain-ligand-domain-ligand-domain.

Known cleavable linkers in the art can be used in conjunction with this invention. Illustrated examples of such linkers include: F2A linkers, T2A linkers, P2A linkers, and E2A linkers (see, for example, International Publication No. WO2017127750, which is incorporated herein by reference in its entirety). Those skilled in the art will understand that other art-recognized linkers may be suitable for use with the revealed RNA (e.g., the nucleic acid encoded by this invention). Those skilled in the art will also understand that other polycistronic RNAs (e.g., mRNAs that individually encode more than one antigen/peptide within the same molecule) may be suitable for the uses provided herein.

In some embodiments, sequence optimization involves codon optimization of the ORF of the antigen of the present invention. Codon optimization methods are known in the industry. For example, the ORF of any one or more of the sequences provided herein can be codon optimized. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeating codons or base strings that can impair gene construction or expression; customize transcription and translation control regions; insert or remove protein delivery sequences; remove/add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; add, remove, or reorganize protein domains; insert or remove restriction sites; modify ribosome-binding sites and mRNA degradation sites; regulate translation rates to allow proper folding of various protein domains; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the industry – unrestricted examples include services from GeneArt (Life Technologies)/DNA2.0 (Menlo Park CA) and/or proprietary methods. In some implementations, optimization algorithms are used to optimize open read frame (ORF) sequences.

In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with the naturally occurring or wild-type sequence ORF (e.g., the naturally occurring or wild-type mRNA sequence encoding the RSV antigen). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with the naturally occurring or wild-type sequence (e.g., the naturally occurring or wild-type mRNA sequence encoding the RSV antigen). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with the naturally occurring or wild-type sequence (e.g., the naturally occurring or wild-type mRNA sequence encoding the RSV antigen). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with the naturally occurring or wild-type sequence (e.g., the naturally occurring or wild-type mRNA sequence encoding the RSV antigen).

In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with naturally occurring or wild-type sequences (e.g., naturally occurring or wild-type mRNA sequences encoding RSV antigens).

In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with naturally occurring or wild-type sequences (e.g., naturally occurring or wild-type mRNA sequences encoding RSV antigens). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity with naturally occurring or wild-type sequences (e.g., naturally occurring or wild-type mRNA sequences encoding RSV antigens).

In some embodiments, the codon-optimized sequence encodes an antigen that has the same or higher immunogenicity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% higher) than an RSV antigen encoded by a non-codon-optimized sequence.

When transfected into mammalian host cells, the modified mRNA is stable for 12-18 hours or more (e.g., 24, 36, 48, 60, 72 or more) and can be expressed by mammalian host cells.

In some embodiments, codon-optimized RNA can be one with an enhanced G/C ratio. The G/C ratio of nucleic acid molecules (e.g., mRNA) can affect RNA stability. RNA with increased amounts of guanine (G) and/or cytosine (C) residues can be functionally more stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, International Patent Application No. WO2002098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modifications in the transcription region (International Patent Application No. WO2002098443 is incorporated herein by reference in its entirety). Due to the degeneracy of the genetic code, modification works by replacing existing codons with those that promote greater RNA stability without altering the resulting amino acids. This method is limited to the coding regions of RNA.

In some embodiments, the chemically modified nucleotides are derived from unmodified RNA (e.g., mRNA) comprising standard ribonucleotides composed of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the invention include standard nucleoside residues (e.g., those present in transcribed RNA, such as A, G, C, or U). In some embodiments, the nucleotides and nucleosides of the invention include standard deoxyribonucleosides (e.g., those present in DNA, such as dA, dG, dC, or dT). Chemical modification

In some embodiments, the present invention comprises RNA having an open reading frame encoding an RSV antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that may be standard (unmodified) or modified, as known in the art. In some embodiments, the nucleotides and nucleosides of the present invention comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may be contained in the sugar, backbone, or nucleobase portions of the nucleotides and/or nucleosides, as recognized in the art.

In some embodiments, the naturally occurring modified nucleotides or nucleotides of the present invention are as commonly known or recognized in the art. Such naturally occurring modified nucleotides and non-limiting examples of nucleotides can be found, in particular, in the widely recognized MODOMICS database.

In some embodiments, the non-natural modified nucleotides or nucleosides of the present invention are commonly known or recognized in the art. Non-limiting examples of such non-natural modified nucleotides and nucleosides can be found, in particular, in U.S. Applications PCT/US2012/058519, PCT/US2013/075177, PCT/US2014/058897, PCT/US2014/058891, PCT/US2014/070413, PCT/US2015/36773, PCT/US2015/36759, PCT/US2015/36771 or PCT/IB 2017/051367, all of which are incorporated herein by reference in their entirety.

Therefore, the nucleic acids (e.g., DNA nucleic acids and RNA nucleic acids (e.g., mRNA nucleic acids)) of this invention may include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

In some embodiments, the nucleic acids of the present invention (e.g., DNA nucleic acids and RNA nucleic acids (e.g., mRNA nucleic acids)) include various (one or more) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, specific regions of the nucleic acids contain one, two or more (as the case may be) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation compared to unmodified nucleic acids including standard nucleotides and nucleosides. In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced immunogenicity (e.g., reduced innate responses) compared to unmodified nucleic acids including standard nucleotides and nucleosides.

In some embodiments, nucleic acids (e.g., RNA (e.g., mRNA)) include non-natural modified nucleotides introduced during or after the synthesis of the nucleic acid to achieve a desired function or property. Modifications can be present at nucleotide links, purine or pyrimidine bases, or sugars. Modifications can be introduced via chemical synthesis or polymerase at the ends of the chain or anywhere in the chain. Any region of the nucleic acid can be chemically modified.

This invention provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof, combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). "Nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method (e.g., chemically, enzymatically, or recombinantly) to contain one or more modified or non-natural nucleosides. Nucleic acids may include one or more linked nucleoside regions. These regions may have variable backbone bonds. The bonds may be standard phosphodiester bonds, in which case the nucleic acid will include nucleotide regions.

The modified nucleotide base pairings encompass not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides including non-standard or modified bases, wherein the configuration of the hydrogen bond donor and acceptor allows for hydrogen bonding between non-standard and standard bases or between two complementary non-standard base structures (e.g., in nucleic acids having at least one chemical modification). One example of such non-standard base pairings is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base/sugar or linker may be incorporated into the nucleic acids of this invention.

In some embodiments, the modified nucleotides in the nucleic acid (e.g., RNA nucleic acid (e.g., mRNA nucleic acid)) include 1-methyl-pseudouridine (m1y), 1-ethyl-pseudouridine, 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y). In some embodiments, the modified nucleotides in the nucleic acid (e.g., RNA nucleic acid (e.g., mRNA nucleic acid)) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethyl-pseudouridine, 5-methylcytidine, and/or 5-methoxycytidine. In some embodiments, the polynucleotide comprises a combination of at least two (e.g., two, three, four, or more) of any of the modified nucleotides mentioned above (including, but not limited to, chemical modifications).

In some embodiments, the modified polynucleotide further includes chemical modification. In some embodiments, the chemical modification is the substitution of uridine in the polynucleotide with N1-methylpseuuridine; where applicable, all or substantially all of the uridine in the polynucleotide is substituted with N1-methylpseuuridine.

In some embodiments, mRNA is uniformly modified (e.g., completely modified, modified throughout the entire sequence) to obtain a specific modification. For example, all uridines in a polynucleotide can be replaced with N1-methylpseudouridine. Similarly, nucleic acids can be uniformly modified by replacing any type of nucleoside residue present in the sequence with modified residues (e.g., those described above).

The nucleic acid of the invention can be modified partially or completely along the entire length of the molecule. For example, in the nucleic acid of the invention or in its predetermined sequence region (e.g., in mRNA containing or excluding multiple (A) tails), one or more or all or predetermined types of nucleotides (e.g., purines or pyrimidines or any one or more or all of A, G, U, and C) can be uniformly modified. In some embodiments, all nucleotides X in the nucleic acid of the invention (or in its sequence region) are modified nucleotides, wherein X can be any of nucleotides A, G, U, and C, or combinations of A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

Nucleic acids may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content or relative to one or more types of nucleotides (i.e., any one or more of A, G, U, or C)) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% 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 ... 00%, 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%, and 95% to 100%). It should be understood that any remaining percentage is due to the presence of unmodified A, G, U, or C.

mRNA may contain a minimum of 1% and a maximum of 100% of modified nucleotides, or any intermediate percentage (e.g., at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90%) of modified nucleotides. For example, nucleic acids may contain modified pyrimidines (e.g., modified uracil or cytosine). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the nucleic acid is replaced by modified uracil (e.g., 5-substituted uracil). Modified uracil may be replaced by a compound having a single unique structure, or by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the nucleic acid is replaced by modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be replaced by a compound having a single unique structure, or by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures).

The untranslated region (UTR) of the mRNA of this invention may include one or more regions or portions that serve as or are used as untranslated regions. If the mRNA is designed to encode at least one antigen of interest, the nucleus may include one or more of these untranslated regions (UTRs). The wild-type untranslated region is a region that is transcribed but not translated into nucleic acid. In the mRNA, the 5' UTR begins at the transcription start site and continues to the start codon but does not contain the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal is received. Increasing evidence demonstrates that the UTR plays a regulatory role in the stability and translation of nucleic acid molecules. The regulatory features of the UTR can be incorporated into the polynucleotides of this invention to particularly enhance molecular stability. Specific features can also be incorporated to ensure controlled downregulation of transcripts in cases where they are misdirected to undesirable organ sites. Various 5' UTR and 3' UTR sequences are known and available in the industry.

The 5’ UTR is the mRNA region located 5’ directly upstream of the start codon (the first codon of the mRNA transcript translated from the ribosome). The 5’ UTR does not encode protein (it is non-coding). The native 5’ UTR is characterized by its role in the initiation of transcription. It possesses the signatures commonly known to be involved in processes that initiate the transcription of many genes via ribosome initiation (such as the Kozak sequence). The Kozak sequence has a shared CCR(A/G)CCAUGG (SEQ ID NO:15), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) (followed by another 'G'). The 5’ UTR is also known to form secondary structures involved in elongation factor binding.

In some embodiments of the present invention, the 5’ UTR is a heterologous UTR, that is, a UTR found in nature that binds to different ORFs. In another embodiment, the 5’ UTR is a synthetic UTR, that is, one that does not exist in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, such as UTRs that increase gene expression, and those that are completely synthesized. Exemplary 5’ UTRs include Xenopus xanthopod or human α-globin or β-globin (8278063; 9012219), human cytochrome b-245a polypeptide and hydroxysteroid (17b) dehydrogenase and tobacco erosion virus (US8278063, 9012219). Alternatively, the CMV Immediate Early 1 (IE1) gene (US20140206753, WO2013/185069) sequence GGGAUCCUACC (SEQ ID NO:30) (WO2014144196) can be used. In another embodiment, the 5' UTR of the TOP gene is the 5' UTR of the TOP gene lacking the 5' TOP motif (oligopyrimidine bundle) (e.g., WO/2015101414, WO02015101415, WO/2015/062738, WO2015024667, WO2015024667); 5' UTR elements derived from the ribosomal protein Large 32 (L32) gene (WO/2015101414, WO02015101415, WO/2015/062738), 5' UTR elements derived from the 5' UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or 5' UTR elements derived from ATP5A1 can be used. UTR element (WO2015024667). In some embodiments, an internal ribosome entry site (IRES) is used instead of the 5' UTR.

In some embodiments, the 5’ UTR of the present invention includes SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42 or 43. In some embodiments, the 5’ UTR of the present invention includes SEQ ID NO:4.

This article also discloses modified polynucleotides including a 5' untranslated region (UTR) comprising a nucleic acid sequence having a secondary structure substantially identical to SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 or fragments thereof, and, where applicable, at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 or fragments thereof. In some embodiments, the 5' UTR comprises a nucleic acid sequence at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% identical to SEQ ID NO:4. Secondary structures may include one or more (e.g., all) of the following characteristics: 1) having a length of approximately 50 nucleotides; 2) having binding sites for translation initiation factors and ribosomes; 3) having GC-rich hairpin regions that stabilize secondary structures; 4) having short AT-rich regions to allow for rapid ribosome passage; 5) having GC-rich sequences adjacent to the Kozak sequence to allow for efficient binding of ribosomes to the AUG start codon; and/or 6) lacking repressive domains for translation (e.g., lacking non-canonical start codons).

In some embodiments, the modified polynucleotide further includes a 3’ UTR. In some embodiments, the 3’ UTR includes the nucleic acid sequence of SEQ ID NO:5 or 44.

This paper demonstrates that mRNAs possessing the 5’UTRs (e.g., Δ1, Δ4, Δ6, and Δ7 5’UTRs) disclosed herein produce higher antibody titers than WT 5’UTRs, indicating that the mRNA can induce stronger protein (e.g., antigen) expression. Therefore, in some embodiments, the modified polynucleotide further includes a nucleic acid sequence encoding a polypeptide (e.g., antigen). In some embodiments, the antigen is a viral antigen (e.g., hRSV antigen). In some embodiments, the antigen induces higher antibody titers when expressed in a host mammal (e.g., human) compared to the same antigen encoded by a control polynucleotide lacking the 5’UTR.

The 3’ UTR is the mRNA region directly downstream (3’) of the self-termination codon (the codon of the mRNA transcript that sends the translation termination signal). The 3’ UTR does not encode protein (it is non-coding). It is known that natural or wild-type 3’ UTRs contain segments containing adenosine and uridine. These AU-rich imprints are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) (SEQ ID NO:28) nonmers.

Most proteins that bind to AREs are known to destabilize messengers, but literature demonstrates that members of the ELAV family (most notably HuR) increase mRNA stability. HuR binds to all three types of AREs. Modifying the HuR-specific binding site to the 3' UTR of a nucleic acid molecule induces HuR binding and thereby stabilizes in vivo signaling.

The introduction, removal, or modification of 3' UTR-rich AU elements (AREs) can be used to regulate the stability of the nucleic acids (e.g., RNA) of this invention. When modifying specific nucleic acids, one or more copies of AREs can be introduced to make the nucleic acids of this invention less stable, thereby reducing translation and decreasing the production of the resulting proteins. Similarly, AREs can be identified, removed, or mutated to increase intracellular stability, thereby increasing translation and the production of the resulting proteins. The nucleic acids of this invention can be used to perform transfection experiments in relevant cell lines, and protein production can be analyzed at various time points after transfection. For example, cells can be transfected using different ARE-modified molecules, and the produced proteins can be analyzed at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection using an ELISA kit for the relevant proteins.

3’ UTRs can be heterologous or synthetic. Regarding 3’ UTRs, globin UTRs (including Xenopus β-globin UTRs and human β-globin UTRs) are known in the industry (8278063, 9012219, US20110086907). By selectively grafting two sequentially human β-globin 3’ UTRs from head to tail, modified β-globin nucleic acids (e.g., mRNA) have been developed (with enhanced stability in some cell types) and are well-known (US2012/0195936, WO2014/071963). Additionally, α2-globin, α1-globin, UTRs, and their mutants are also known in the industry (WO2015101415, WO2015024667). Other 3' UTRs described in non-patented literature in mRNA include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other illustrative 3' UTRs include the 3' UTRs of bovine or human growth hormone (wild-type or modified) (WO2013/185069, US20140206753, WO2014152774), rabbit β-globin, and hepatitis B virus (HBV). The 3' UTRs of α-globin and viral VEEV are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014144196) is used. In some embodiments, the 3' UTRs of human and mouse ribosomal proteins are used. Other examples include rps9 3 UTR (W02015101414), FIG4 (W02015101415), and human albumin 7 (W02015101415).

In some embodiments, the 3’ UTR of the present invention includes SEQ ID NO:5 or 44.

Those familiar with this technique will understand that a heterologous or synthetic 5' UTR can be used with any desired 3 UTR sequence. For example, a heterologous 5' UTR can be used with a synthetic 3 UTR as well as with a heterologous 3 UTR.

Non-UTR sequences can also be used as regions or subregions within nucleic acids. For example, introns or portions of intron sequences can be incorporated into regions of the nucleic acids of this invention. The inclusion of intron sequences can increase protein production and nucleic acid content.

Combinations of features may be contained within flanking regions and may be included within other features. For example, an ORF may flank a 5’ UTR (which may contain a strong Kozak transcription start signal) and/or a 3’ UTR (which may contain an oligo(dT) sequence for template addition of a multi-A tail). The 5’ UTR may include a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes (e.g., the 5’ UTR described in US Patent Application Publication No. 20100293625 and PCT/US2014/069155 (incorporated herein by reference in its entirety)).

It should be understood that any UTR from any gene can be incorporated into a region of nucleic acid. Furthermore, multiple wild-type UTRs from any known gene can be utilized. Artificial UTRs providing variants that are not wild-type regions are also within the scope of this invention. Such UTRs, or portions thereof, can be oriented in the same orientation as the transcript from which they are selected, or their orientation or position can be altered. Thus, 5' or 3' UTRs can be inverted, shortened, or lengthened, and can be prepared using one or more other 5' or 3' UTRs. As used herein, when referring to a UTR sequence, the term "altered" means that the UTR has been altered in some way relative to a reference sequence. For example, a 3' UTR or 5' UTR can be altered relative to a wild-type or natural UTR by changes in orientation or position (as taught above), or by incorporating additional nucleotides, deleting nucleotides, exchanging nucleotides, or transposing nucleotides. Any of the variations that produce a "modified" UTR (3' or 5') includes the variant UTR.

In some embodiments, double, triple, or quadruple UTRs (e.g., 5’ UTR or 3’ UTR) may be used. As used herein, a “double” UTR is two copies of the same UTR encoded in a tandem or substantially tandem manner. For example, a 3’ UTR of bis-β-globin may be used as described in US Patent Publication 20100129877, the contents of which are incorporated herein by reference in their entirety.

Patterned UTRs are also within the scope of this invention. As used herein, “patterned UTR” refers to UTRs that reflect repetitive or alternating patterns, such as ABABAB or AABBBAABBAABB or ABCABCABC or variations thereof repeated once, twice or more. In these patterns, each letter (A, B or C) represents a different UTR at the nucleotide level.

In some embodiments, the flanking regions are selected from transcript families that share common functions, structures, features, or properties with their proteins. For example, the polypeptide of interest may belong to a protein family that is expressed in a specific cell, tissue, or at a particular time during development. A UTR from any of these genes may exchange with any other UTR from the same or different protein families to produce a new polynucleotide. As used herein, "protein family" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, feature, location, origin, or mode of expression.

The non-translated area may also include translated enhancement sub-elements (TEEs). As a non-limiting example, a TEE may include those described in U.S. Application No. 20090226470 (which are incorporated herein by reference in their entirety) and those known in the art.

RNA can be transcribed into cDNA containing the polynucleotides described herein using the in vivo extracellular transcription (IVT) system. In vivo extracellular transcription of RNA is known in the art and described in International Publication WO/2014/152027, which is incorporated herein by reference in its entirety.

In some embodiments, an unamplified, linearized DNA template is used to generate RNA transcripts in an in vivo transcription reaction. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, cDNA is formed by reverse transcription of RNA polynucleotides (e.g., but not limited to, RSV mRNA). In some embodiments, plasmid DNA templates are used to transfect cells (e.g., bacterial cells (e.g., E. coli, e.g., DH-1 cells)). In some embodiments, transfected cells are cultured to replicate plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template contains an RNA polymerase promoter (e.g., a T7 promoter located at and operatively linked to the 5' end of the gene of interest). In some embodiments, the in vivo exotranscriptional template encodes a 5' untranslated (UTR) region containing an open reading frame and encodes a 3' UTR and multiple (A) tails. The specific nucleic acid sequence composition and length of the in vivo exotranscriptional template depend on the mRNA encoded by the template.

The "5' untranslated region" (UTR) refers to the mRNA region that does not encode a polypeptide, located directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome). During the generation of the RNA transcript, the 5' UTR may include a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in the vaccine of this invention.

The "3' untranslated region" (UTR) refers to the non-coding region of the mRNA of the polypeptide located directly downstream (i.e., 3') from the termination codon (i.e., the codon of the mRNA transcript that sends the termination translation signal).

An "open reading frame" is a continuous segment of DNA that begins with a start codon (such as methionine (ATG)) and ends with a stop codon (such as TAA, TAG, or TGA) and encodes a polypeptide.

A "multiple (A) tail" is an mRNA region downstream (e.g., directly downstream, i.e., 3') of a 3' UTR containing multiple, consecutive monoadenosine monophosphates (MAPs). A multiple (A) tail can contain 10 to 300 MAPs. For example, a multiple (A) tail can contain 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 MAPs. In some embodiments, a multiple (A) tail contains 50 to 250 MAPs. In relevant biological environments (e.g., in cells, in living organisms), (e.g.) in the cytoplasm, the (A) tail protects mRNA from enzymatic degradation and facilitates transcription termination and/or the export and translation of mRNA from the cell nucleus.

In some embodiments, nucleic acids contain 200 to 3,000 nucleotides. For example, nucleic acids may contain 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

The in vivo extracorporeal transcription system typically includes a transcription buffer, nucleoside triphosphates (NTPs), RNase inhibitors, and polymerases.

NTP can be manufactured internally, either from a supplier or synthesized as described herein. NTP can be selected from (but is not limited to) those described herein (including natural and non-natural (modified) NTP). Any number of RNA polymerases or variants can be used in the methods of this invention. The polymerase can be selected from (but is not limited to) bacteriophage RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase) and/or mutant polymerases (e.g., polymerases capable of incorporating modified nucleic acids and/or modified nucleotides (including chemically modified nucleic acids and/or nucleotides)). Some embodiments exclude the use of DNases.

In some embodiments, RNA transcripts are capped by enzymatic capping. In some embodiments, the RNA includes a 5' cap (e.g., cap-0 or m7G(5')ppp(5')N1mpNp cap).

Solid-phase chemical synthesis . This invention relates to the fabrication of nucleic acids, wholly or partially, using solid-phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis can be used to introduce site-specific chemical modifications into nucleic acid sequences.

Liquid-phase chemical synthesis. The nucleic acid of this invention can be synthesized in the liquid phase by sequentially adding monomer structural units.

Combinations of synthetic methods. The synthetic methods discussed above each have their own advantages and limitations. Attempts have been made to combine these methods to overcome these limitations. Such combinations are within the scope of this invention. Solid-phase or liquid-phase chemical synthesis combined with enzymatic ligation provides an efficient method for generating long-chain nucleic acids (which cannot be obtained by chemical synthesis alone).

The ligation of nucleic acid regions or subregions can also be achieved using ligases to assemble nucleic acids. DNA or RNA ligases promote intermolecular linkages at the 5' and 3' ends of polynucleotide chains by forming phosphodiester bonds. Nucleic acids (e.g., chimeric polynucleotides and/or round nucleic acids) can be prepared by ligating one or more regions or subregions. DNA fragments can be joined by ligase-catalyzed reactions to produce recombinant DNA with different functions. Two oligodeoxynucleotides (one with a 5' phospho group and the other with a free 3' hydroxyl group) are used as the acceptor of DNA ligases. Purification

The purification of nucleic acids described herein may include (but is not limited to) nucleic acid removal, quality assurance, and quality control. Removal may be performed using methods known in the industry, such as (but not limited to) AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA™ oligo-T trapping probes (EXIQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods (such as (but not limited to) strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)). When used in connection with nucleic acids (e.g., "purified nucleic acids"), the term "purified" means that which has been separated from at least one contaminant. A "contaminant" is any substance that renders another substance unqualified, impure, or inferior. Therefore, purified nucleic acids (such as DNA and RNA) exist in a form or setting different from those found in the autothermal environment or different from those that existed before they were subjected to treatment or purification methods.

Quality assurance and/or quality control checks can be performed using methods such as (but not limited to) gel electrophoresis, UV absorbance or analytical HPLC.

In some embodiments, nucleic acids can be sequenced by methods including (but not limited to) reverse transcriptase-PCR.

In some embodiments, the nucleic acids of this invention can be quantified in exosomes or when derived from one or more bodily fluids. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSL), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal cavity fluid, pericardial fluid, lymph, chyme, bile, interstitial fluid, menstrual blood, pus, sebum, vomitus, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, lavage fluid from sinuses, bronchopulmonary aspirate, coelomic fluid, and umbilical cord blood. Alternatively, exosomes can be extracted from any of the following groups of organs: lungs, heart, pancreas, stomach, intestines, bladder, kidneys, ovaries, testes, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

Analysis can be performed using antigen-specific probes, blood cell counting, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof. Immunohistochemical methods (e.g., enzyme-linked immunosorbent assay (ELISA)) can also be used to separate exosomes. Alternatively, exosomes can be separated using particle size chromatography, density gradient centrifugation, differential centrifugation, nanofiltration, immunosorbent assay (IPA), affinity purification, microfluidic separation, or combinations thereof.

These methods enable researchers to monitor the amount of remaining or delivered nucleic acids in real time. This is because, in some embodiments, the nucleic acids of this invention differ from the endogenous form due to structural or chemical modifications.

In some embodiments, nucleic acids can be quantified using methods such as (but not limited to) ultraviolet-visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is the NANODROP® spectrometer (Thermo Fisher, Waltham, MA). Quantified nucleic acids can be analyzed to determine if they are of appropriate size and to check for nucleic acid degradation. Nucleic acid degradation can be checked using methods such as (but not limited to) agarose gel electrophoresis, HPLC-based purification methods (such as (but not limited to) strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE)).

In some embodiments, the RNA of the invention (e.g., mRNA) is directed into lipid nanoparticles ( LNPs ). Lipid nanoparticles typically include ionizable cationic lipids, non-cationic lipids, sterol and PEG lipid components, and the nucleic acid charge of interest. The lipid nanoparticles of this invention can be produced using components, compositions, and methods commonly known in the art, see, for example, PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491, all of which are incorporated herein by reference in their entirety. In some instances, the components, compositions and methods described in International Publications WO2023104114 and WO2022166213 can be used to generate the lipid nanoparticles disclosed herein, which are incorporated herein by reference in their entirety.

The vaccine of the present invention is typically formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles include at least one ionizable cationic lipid, at least one phospholipid, at least one sterol and/or at least one polyethylene glycol (PEG) modified lipid. In some embodiments, the LNP includes a mixture of lipids containing the following lipids: (1) about 10%-50%, about 20%-60%, about 30%-50% or about 40% (moles percentage) of ionizable cationic lipids; (2) about 30%-70% (e.g., about 30%-60%, about 40-60% or about 50%) (moles percentage) of sterol lipids; (3) about 5%-30% (e.g., about 5-15% or about 10%) (moles percentage) of phospholipids; or (4) about 0%-5% (e.g., about 1-3% or about 2%) (moles percentage) of occult lipids or PEG-modified lipids.

In some embodiments, the lipid nanoparticles include ionizable cationic lipids with a molar ratio of 40%, 45%, 50%, or 55%. In some embodiments, the ionizable lipid is one of the lipids disclosed in International Patent Publication No. WO2023104114A2, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is selected from the group consisting of lipid 2, lipid 4, lipid 5, and lipid 8 (disclosed in International Patent Publication No. WO2023104114A2). In some embodiments, the ionizable cationic lipid is lipid 4.

Ionizable cationic lipid 2 has the following structure: Ionizable cationic lipid 4 has the following structure: Ionizable cationic lipid 5 has the following structure: Furthermore, ionizable cationic lipid 8 has the following structure: .

In some embodiments, the steroid lipid is cholesterol. In some embodiments, the phospholipid is 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC). In some embodiments, the PEG-modified lipid is 1,2-dimyristyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000). In some embodiments, the mixture of lipids includes ionizable cationic lipids, cholesterol, DSPC, and DMG-PEG2000. In some embodiments, the molar ratio of ionizable cationic lipids, cholesterol, DSPC, and DMG-PEG2000 is approximately 40:approximately 48:approximately 10:approximately 2.

In some embodiments, the average diameter of the LNP is less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, or less than 60 nm; (e.g., about 40 nm-70 nm, about 50-70 nm, about 55 nm-65 nm, or about 60 nm).

In some embodiments, the polydispersity index (PDI) of LNP is about 0.01 to about 0.15 or about 0.05 to about 0.10.

In some embodiments, the zeta potential of the LNP is about 1.00 mV to about 5.00 mV or about 1.50 mV to about 4.00 mV.

This document provides compositions (e.g., pharmaceutical compositions ), methods, kits, and reagents for the prevention or treatment of RSV in humans and other mammals. The compositions provided herein can be used as therapeutic or preventative agents. They can be used as agents for the prevention and/or treatment of RSV infection. In some embodiments, an RSV vaccine containing RNA, as described herein, can be administered to an individual (e.g., a mammalian individual (e.g., a human individual)) and the RNA polynucleotide can be translated in vivo to produce an antigenic polypeptide (antigen).

An "effective amount" of a combination (e.g., including RNA) is based on (at least in part) the target tissue, target cell type, mode of administration, physical properties of the RNA (e.g., length, nucleotide composition, and/or degree of modification of nucleosides), other components of the vaccine, and other determinants (e.g., individual age, weight, height, sex, and general health). Typically, an effective amount of the combination provides an induced or enhanced immune response that varies depending on the antigen produced in an individual's cells. In some embodiments, an effective amount of a combination containing an RNA polynucleotide with at least one chemical modification is more effective than a combination containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production can be demonstrated by increased cell transfection (the percentage of cells transfected with RNA vaccines), increased protein translation and/or expression from polynucleotides, decreased nucleic acid degradation (as demonstrated by, for example, increased duration of protein translation from modified polynucleotides), or altered antigen-specific immune responses in host cells.

The term "medical composition" refers to a combination of an active agent and an inert or active carrier (which makes the composition particularly suitable for in vivo or in vitro diagnostic or therapeutic use). A "medically acceptable carrier" does not cause undesirable physiological effects upon administration to an individual or onto the body. The carrier in a medical composition must be "acceptable," meaning it is compatible with the active ingredient and can stabilize it. One or more solubilizers may be used as medical carriers to deliver the active agent. Examples of medically acceptable carriers include (but are not limited to) biocompatible mediators, adjuvants, additives, and diluents to achieve a composition usable in a dosage form. Other examples of transporters include colloidal silica oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical transporters and diluents, as well as pharmaceutical excipients using them, are described in Remington’s Pharmaceutical Sciences.

In some embodiments, the present invention’s compositions (including polynucleotides and the polypeptides they encode) can be used to treat or prevent RSV infection. The compositions can be administered preventively or therapeutically to healthy individuals as part of an active immunization program, either during the incubation period, early in the course of infection, or during active infection after the onset of symptoms. In some embodiments, the amount of RNA provided to cells, tissues, or individuals can be an effective amount for immune prophylaxis.

The combination may be administered in combination with other preventive or therapeutic compounds. As a non-limiting example, the preventive or therapeutic compound may be an adjuvant or an enhancer. As used herein, when referring to a preventive combination (e.g., a vaccine), the term "enhancer" refers to an additional administration of the preventive (vaccine) combination. The enhancer (or enhancer vaccine) may be administered after an early administration of the preventive combination. The administration time between the initial administration of the preventive combination and the enhancer may be (but is not limited to) 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, or 20 minutes. 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months Months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the time interval between the initial administration of the preventive composition and the administration of the enhancer may be (but is not limited to) 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some implementations, the compound can be administered intramuscularly ( IM ), intranasally, or intradermally, similar to the administration of inactivated vaccines known in the industry.

Depending on the prevalence of infection or the degree or level of medical inadequacy, the combination can be used in various environments. As a non-limiting example, RNA vaccines can be used to treat and/or prevent a variety of infectious diseases. RNA vaccines have superior properties because they produce much higher antibody titers, better neutralizing immunity, produce a longer-lasting immune response, and/or produce a response earlier than commercially available vaccines.

This article provides pharmaceutical compositions comprising RNA and/or complexes, which may be combined, as appropriate, with one or more pharmaceutically acceptable excipients. RNA may be formulated or administered alone or in combination with one or more other components. For example, immunocomposites may include other components (including, but not limited to, adjuvants).

In some embodiments, the immune composition does not contain an adjuvant (it is adjuvant-free).

RNA may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the vaccine composition includes at least one additional active substance (e.g., a therapeutic-active substance, a preventative-active substance, or a combination of both). The vaccine composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceuticals (e.g., vaccine compositions) can be found (e.g.) in Remington : The Science and Practice of Pharmacy , 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety).

In some embodiments, the immune composition is administered to humans, human patients, or individuals. For the purposes of this invention, the phrase "active ingredient" generally refers to an RNA vaccine or a polynucleotide contained therein (e.g., an RNA polynucleotide encoding an antigen (e.g., an mRNA polynucleotide)).

Formulations of the vaccine compositions described herein can be prepared using any method known in the field of pharmacology or developed below. Generally, such preparation methods involve binding an active ingredient (e.g., mRNA polynucleotide) with an adjuvant and/or one or more other excipients, and then (as needed and/or desired) segmenting, shaping, and/or packaging the product into desired single- or multiple-dose units.

The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any other components in the pharmaceutical composition of the present invention will vary depending on the identity, size, and/or condition of the individual being treated and, more specifically, on the route of administration of the composition. For example, the composition may include 0.1% to 100% (e.g., 0.5% to 50%, 1-30%, 5-80%, at least 80%) (w/w) of the active ingredient.

In some embodiments, one or more excipients are used to modulate RNA to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g. from accumulated excipients); (4) alter biological distribution (e.g., target specific tissues or cell types); (5) increase the translation of encoded proteins in vivo; and/or (6) alter the release characteristics of encoded proteins (antigens) in vivo. In addition to conventional adjuvants (such as any and all solvents, dispersion media, diluents, or other liquid media, dispersion or suspension additives, surfactants, isotonic agents, thickeners or emulsifiers, preservatives), adjuvants may include (but are not limited to) lipids, liposomes, lipid nanoparticles, polymers, liposome complexes, core-shell nanoparticles, peptides, proteins, RNA-transfected cells (e.g. for transplantation into individuals), hyaluronidase, nanoparticle mimics, and combinations thereof.

This document provides information on the administration of immunocomposites (e.g., RNA vaccines), methods, kits, and reagents for the prevention and/or treatment of RSV infection in humans and other mammals. Immunocomposites can be used as therapeutic or preventative agents. In some embodiments, immunocomposites are used to provide preventative protection against RSV infection. In some embodiments, immunocomposites are used to treat RSV infection. In some embodiments, immunocomposites are used to activate immune effector cells (e.g., activated peripheral blood mononuclear cells (PBMCs) in vitro, followed by infusion (reinfusion) into an individual).

An individual can be any mammal (including non-human primates and human individuals). Typically, the individual refers to a human individual.

In some embodiments, an effective amount of an immune composition (e.g., an RNA vaccine) is administered to an individual (e.g., a mammalian individual (e.g., a human individual)) to induce an antigen-specific immune response. The RNA encoding the RSV antigen is expressed and translated in vivo to produce the antigen, which then stimulates the individual's immune response.

The term "treatment" refers to therapeutic treatment and preventative or preventive measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or symptoms. For the purposes of this invention, beneficial or anticipated clinical outcomes include (but are not limited to) relief of symptoms, reduction of the severity of disease or infection, stabilization (i.e., non-deterioration) of disease status, delay or slowing of disease progression, and improvement or mitigation of disease status (e.g., lung pathology score), whether detectable or undetectable. Those requiring treatment include those who already have symptoms or symptoms, those who are predisposed to having such symptoms or symptoms, and those who wish to prevent such symptoms or symptoms.

Prophylactic protection against RSV (e.g., hRSV) can be achieved after administration of the present invention's immune composition (e.g., an RNA vaccine). The immune composition can be administered once, twice, three times, four times, or more, but a single administration of the vaccine (followed by a single booster, if applicable) may be sufficient. While less desirable, the immune composition may be administered to infected individuals to achieve a therapeutic response. Administration may need to be adjusted accordingly.

This invention provides a method for inducing an immune response against RSV in an individual. In some embodiments, the method involves administering an immune complex to an individual comprising RNA (e.g., mRNA) having an open reading frame encoding RSV F glycoprotein, thereby inducing an immune response against a respiratory viral antigen in the individual, wherein the antiantigen antibody titer in the individual increases after vaccination relative to a conventional vaccine against the antigen administered at a prophylactic effective dose. "Antiantigen antibody" refers to a serum antibody that specifically binds to an antigen.

The preventive effective dose is the clinically acceptable dose that is effective in preventing viral infection. In some embodiments, the effective dose is the dose listed in the vaccine packaging insert. As used herein, conventional vaccines refer to vaccines other than the mRNA vaccine of this invention. For example, conventional vaccines include (but are not limited to) live microbial vaccines, killed microbial vaccines, submonosome vaccines, protein antigen vaccines, DNA vaccines, virus-like particle (VLP) vaccines, etc. In exemplary embodiments, conventional vaccines are vaccines that have been approved by regulatory agencies and/or registered by national drug regulatory agencies (such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA)).

In some embodiments, the antiantigen antibody titer of an individual after vaccination increases by 1 to 10 log compared to the antiantigen antibody titer of an individual who has received a prophylactic effective dose of a conventional vaccine against RSV or who has not been vaccinated. In some embodiments, the antiantigen antibody titer of an individual after vaccination increases by 1, 2, 3, 4, 5, or 10 log compared to the antiantigen antibody titer of an individual who has received a prophylactic effective dose of a conventional vaccine against RSV or who has not been vaccinated.

Other embodiments of the present invention provide a method for inducing an immune response against RSV in an individual. This method involves administering to an individual an immune composition (e.g., an RNA vaccine) comprising an RNA polynucleotide containing an open reading frame encoding an RSV antigen, thereby inducing an immune response against RSV in the individual, wherein the individual's immune response is equivalent to that of an individual administered a conventional vaccine against RSV at a dose 2 to 100 times that of the immune composition.

In some embodiments, the immune response of an individual is equivalent to that of an individual who received a conventional vaccine at twice the dose of the present invention's immunizing complex. In some embodiments, the immune response of an individual is equivalent to that of an individual who received a conventional vaccine at three times the dose of the present invention's immunizing complex. In some embodiments, the immune response of an individual is equivalent to that of an individual who received a conventional vaccine at 4, 5, 10, 50, or 100 times the dose of the present invention's immunizing complex. In some embodiments, the immune response of an individual is equivalent to that of an individual who received a conventional vaccine at doses ranging from 10 to 1000 times the dose of the present invention's immunizing complex. In some embodiments, the individual’s immune response is equivalent to that of an individual who has been given a conventional vaccine at a dose of 100 to 1000 times that of the immune composition of the present invention.

In other embodiments, the immune response is evaluated by measuring the antibody titer of an individual. In other embodiments, the ability of serum or antibodies from an immunized individual to neutralize viral uptake or reduce RSV conversion of human B lymphocytes is tested. In other embodiments, the ability to promote a stable T-cell response is measured using an industry-recognized technique.

Other embodiments of the invention provide a method for inducing an immune response against RSV in an individual by administering an immune complex (e.g., an RNA vaccine) comprising RNA having an open reading frame encoding RSV antigens to the individual, thereby inducing an immune response against RSV antigens in the individual, wherein the induction of the immune response is advanced by 2 days to 10 weeks compared to the immune response induced in individuals receiving a prophylactic effective dose of a conventional vaccine against RSV. In some embodiments, an immune response is induced in individuals receiving a prophylactic effective dose of a conventional vaccine at a dose 2 to 100 times higher than that of the immune complex of the invention.

In some implementations, the immune response in an individual is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier than that induced in an individual receiving a preventative effective dose of a conventional vaccine.

Immunocomponents (such as RNA vaccines) can be administered via any route that produces a therapeutically effective outcome. These include (but are not limited to) intradermal, intramuscular, intranasal, and/or subcutaneous administration. This invention provides methods for administering RNA vaccines to individuals in need. The exact amount required may vary depending on the individual's species, age and general condition, disease severity, specific compendium, mode of administration, mode of activity, and so on. RNA is typically formulated in dose units to facilitate administration and achieve dose uniformity. However, it should be understood that the total daily dose of RNA should be determined by the attending physician within reasonable medical judgment. The specific therapeutic efficacy, preventative efficacy, or appropriate imaging agent dosage for any given patient depends on a number of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific combination used; the patient's age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in medical technology.

As provided herein, the effective amount of RNA can be as low as 20 μg, (for example) as a single dose or as two 10 μg doses. In some embodiments, the effective amount of RNA is a total dose of 20 μg-300 μg or 25 μg-300 μg. For example, the effective amount of RNA can be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg. In some embodiments, the effective amount of RNA is a total dose of 25 μg to 300 μg. In some embodiments, the effective amount is a total dose of 50 μg to 100 μg. In some embodiments, the effective dose is a total dose of 20 μg. In some embodiments, the effective dose is a total dose of 25 μg. In some embodiments, the effective dose is a total dose of 50 μg. In some embodiments, the effective dose is a total dose of 75 μg. In some embodiments, the effective dose is a total dose of 100 μg. In some embodiments, the effective dose is a total dose of 150 μg. In some embodiments, the effective dose is a total dose of 300 μg.

The RNA described in this article can be formulated into the dosage forms described in this article (e.g., intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous)).

Vaccine Efficacy : Some aspects of this invention provide formulations of immune components (e.g., RNA vaccines), wherein the RNA is formulated in an effective amount to induce an antigen-specific immune response in an individual (e.g., to induce antibodies against RSV antigens). "Effective amount" refers to the dose of RNA that effectively induces an antigen-specific immune response. Methods for inducing an antigen-specific immune response in an individual are also provided herein. As used herein, an immune response to the vaccine or LNP of this invention is a humoral and/or cellular immune response in response to one or more RSV proteins present in the vaccine. For the purposes of this invention, a "humoral" immune response refers to an immune response mediated by antibody molecules (including, for example, secreted (IgA) or IgG molecules), while a "cellular" immune response is mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+). T cell (e.g., CTL) and/or other leukocyte-mediated immune responses. An important aspect of cellular immunity involves antigen-specific responses via cytolytic T cells (CTLs). CTLs possess specificity for peptide antigens presented along with proteins encoded by the major histocompatibility complex (MHC) and expressed on the cell surface. CTLs help induce and promote the destruction or infection of these intracellular microorganisms. The lysis of microbial cells. Another aspect of cellular immunity involves antigen-specific responses via helper T cells. Helper T cells help stimulate and concentrate the activity of nonspecific effector cells targeting peptide antigens and their surface MHC molecules. Cellular immune responses also result in the production of intercytokines, chemokines, and other molecules produced by activated T cells and/or other leukocytes (including those derived from CD4+ and CD8+ T cells).

In some embodiments, antigen-specific immune responses are characterized by measuring the titer of anti-RSV antigen antibodies produced in an individual given an immune composition as provided herein. Antibody titer is a measure of the amount of antibodies (e.g., antibodies against a specific antigen (e.g., anti-hRSV F glycoprotein) or an epitope of an antigen) in an individual. Antibody titer is typically expressed as the reciprocal of the maximum dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA), for example, is a common assay used to determine antibody titer.

In some embodiments, antibody titer is used to assess whether an individual has been infected or to determine whether immunization is necessary. In some embodiments, antibody titer is used to measure the strength of an autoimmune response, determine whether booster immunization is needed, determine the effectiveness of a previous vaccine, and identify any recent or previous infection. According to the present invention, antibody titer can be used to measure the strength of an immune response induced by an immune complex (e.g., an RNA vaccine) in an individual.

In some embodiments, the anti-RSV antigen antibody titer produced in the individual increases by at least 1 log relative to the control. For example, the anti-RSV antigen antibody titer produced in the individual may increase by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to the control. In some embodiments, the anti-RSV antigen antibody titer produced in the individual increases by 1, 1.5, 2, 2.5, or 3 log relative to the control.

In some embodiments, the titer of anti-RSV antigen antibodies produced in individuals increases by 1-3 log relative to the control. For example, the titer of anti-RSV antigen antibodies produced in individuals may increase by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to the control.

In some embodiments, the titer of anti-RSV antigen antibodies produced in individuals is at least 2-fold higher than that of the control. For example, the titer of anti-RSV antigen antibodies produced in individuals may be at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than that of the control. In some embodiments, the titer of anti-RSV antigen antibodies produced in individuals is 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than that of the control. In some embodiments, the titer of anti-RSV antigen antibodies produced in individuals is 2-10-fold higher than that of the control. For example, compared to the control, the titer of antibodies against respiratory viral antigens produced in individuals can increase by 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times.

In some embodiments, the control is the antibody titer against RSV antigens produced in individuals who have not been administered an immune composition (e.g., an RNA vaccine). In some embodiments, the control is the antibody titer against respiratory virus antigens produced in individuals administered a recombinant or purified protein vaccine. Recombinant protein vaccines typically contain protein antigens produced in heterologous expression systems (e.g., bacteria or yeast) or purified from a large number of pathogenic organisms.

In some embodiments, the efficacy of the immune complex (e.g., an RNA vaccine) is measured in a mouse model. For example, the immune complex can be administered to a mouse model and the induction of neutralizing antibody titers in the mouse model can be analyzed. Viral challenge studies can also be used to evaluate the efficacy of the vaccine of the present invention. For example, the immune complex can be administered to a mouse model, the mouse model can be challenged with a virus, and the survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., intercytokine response)) of the mouse model can be analyzed.

In some embodiments, the RSV RNA vaccine disclosed herein elicits a balanced Th1 and Th2 immune response. In some embodiments, the RSV RNA vaccine disclosed herein elicits an IgG2a/IgG1 ratio in mice greater than approximately 1:2, 1:1, 4:1, 5:1, or 10:1.

In some implementations, the effective dose of an immune complex (e.g., an RNA vaccine) is a reduced dose compared to the standard nursing dose of a recombinant tissue protein vaccine. As used herein, "standard of nursing" refers to medical or psychological treatment guidelines and can be general or specific. "Standard of nursing" specifies appropriate treatment based on scientific evidence and collaboration among medical professionals involved in treating a given condition. It is a diagnostic and treatment protocol that physicians/clinicians should follow for a particular type of patient, condition, or clinical situation. As provided in this article, "standard nursing dose" refers to the dose of recombinant or purified protein vaccine, live attenuated or inactivated vaccine or VLP vaccine that a physician/clinician or other healthcare professional will administer to an individual to treat or prevent RSV infection or related symptoms while following standard nursing guidelines for the treatment or prevention of RSV infection or related symptoms.

In some implementations, the anti-RSV antigen antibody titer produced in individuals administered an effective amount of the immunizing composition is equivalent to the anti-RSV antigen antibody titer produced in control individuals administered a standard nursing dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine or a VLP vaccine.

Vaccine efficacy can be evaluated using standard analyses (e.g., see Weinberg et al., J. Infect Dis. 2010 1 June; 201(11): 1607-10). For example, vaccine efficacy can be measured by double-blind, randomized, clinically controlled trials. Vaccine efficacy can be expressed as the reduction in the proportion of disease incidence (AR) between the unvaccinated (ARU) and vaccinated (ARV) study groups and can be calculated using the following formulas from the relative risk (RR) of disease in the vaccinated group: efficacy = (ARU - ARV)/ARU × 100; and efficacy = (1-RR) × 100.

Similarly, standardized analyses can be used to evaluate vaccine efficacy (see, for example, Weinberg et al., J Infect Dis. 2010 1 June; 201(11): 1607-10). Vaccine efficacy is an evaluation of how a vaccine (which has been shown to have high efficacy) reduces disease in a population. This metric assesses the net balance of benefits and adverse effects of a vaccination program (not just the vaccine itself) under natural field conditions rather than in controlled clinical trials. Vaccine efficacy is proportional to vaccine potency (efficacy) but is also affected by the level of immunization in the target group in the population and by other non-vaccine-related factors such as hospitalization, outpatient visits, or costs in the ‘real world’. For example, a retrospective case-control analysis can be used, in which vaccination rates are compared in a cohort of infected cases and in appropriate controls. Vaccine effectiveness can be expressed as the rate difference, where the odds ratio (OR) of infection occurring after vaccination is: effectiveness = (1 - OR) × 100.

In some implementations, the efficacy of the immunocomplex (e.g., an RNA vaccine) relative to unvaccinated controls is at least 60%. For example, the efficacy of the immunocomplex relative to unvaccinated controls may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100%.

Elimination immunity. Elimination immunity refers to a unique immune status that protects a host from infection by an effective pathogen. In some embodiments, the effective amount of the immunocompound of the present invention is sufficient to provide elimination immunity in an individual for at least one year. For example, the effective amount of the immunocompound of the present invention is sufficient to provide elimination immunity in an individual for at least two, three, four, or five years. In some embodiments, the effective amount of the immunocompound of the present invention is sufficient to provide elimination immunity in an individual at a dose at least five times lower than the control. For example, the effective amount may be sufficient to provide elimination immunity in an individual at a dose at least 10, 15, or 20 times lower than the control.

Detectable antigens. In some embodiments, such as when measured in the serum of an individual 1-72 hours after administration, the effective amount of the immunocompound of the invention is sufficient to produce a detectable amount of RSV antigen.

Antibody titer. Antibody titer is a measure of the amount of antibodies (e.g., antibodies against a specific antigen, such as anti-RSV antigen) in an individual. Antibody titer is usually expressed as the reciprocal of the maximum dilution that provides a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay used to determine antibody titer.

In some embodiments, as measured in the serum of an individual 1-72 hours after administration, the effective amount of the immunocompound of the present invention is sufficient to produce 1,000-10,000 neutralizing antibody titers against RSV antigen. In some embodiments, as measured in the serum of an individual 1-72 hours after administration, the effective amount is sufficient to produce 1,000-5,000 neutralizing antibody titers against RSV antigen.

In some embodiments, such as those measured in the serum of an individual 1–72 hours after administration, the effective amount is sufficient to produce 5,000–10,000 neutralizing antibody titers against RSV antigen.

In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50.

In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units/mL (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL.

In some embodiments, the anti-RSV antigen antibody titer produced in the individual increases by at least 1 log relative to the control. For example, the anti-RSV antigen antibody titer produced in the individual may increase by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 log relative to the control.

In some embodiments, the titer of anti-RSV antigen antibodies produced in the individual is at least 2-fold higher than that of the control. For example, the titer of anti-RSV antigen antibodies produced in the individual is at least 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than that of the control.

In some embodiments, the geometric mean (which is the nth root of the product of n numbers) is often used to describe proportional growth. In some embodiments, the geometric mean is used to characterize the antibody titer produced in an individual.

Control groups could be, for example, individuals who have not been vaccinated or individuals who have been given a live attenuated virus vaccine, an inactivated virus vaccine, or a protein subunit vaccine.

Example 1: Evaluation of the effect of CT on vaccine design. To enhance the expression and immunogenicity of human RSV F glycoprotein, different modifications were introduced and evaluated based on previous findings. Figure 1 is a schematic diagram of the different designs between wild-type RSV F protein and the RSV F variant described in this paper. DS-Cav1 is a normally soluble, disulfide-linked double-stranded protein (containing stable mutations of in vivo disulfides (S155C, S290C), cavity-filling mutations (S190F, V207L), and naturally occurring substitutions (P102A, I379V, M447V) to enhance its expression) and a trimer with an additional C-terminal T4 fold to form RSV F protein. It has been used in in vivo as a fusion pre-F glycoprotein in assays and as a positive control vaccine containing an aluminum oxide adjuvant in mouse immunogenicity studies. Compared to wild-type RSV F protein, DS-Cav1 lacks the transmembrane domain and cytoplasmic tail region.

Based on the site mutation strategy of DS-Cav1 and the advantages of DS2-Cav1 and SC-DM over DS-Cav1 in previous studies, Lead-2 was first designed as a vaccine candidate. Lead-2 is a codon-optimized mRNA chain encoding the membrane-bound F protein. In addition to the mutation sites of DS-Cav1 (S155C, S190F, V207L, S290C), it also incorporates interprotopic (A149C, Y458C) disulfide-stabilizing mutations, phenotype-enhancing mutations (S46G, E92D, S215P, K465Q), and stability-increasing mutations (S215P). Simultaneously, the p27-FP region is replaced by the GS linker from Lead-2 to enhance the stability of the two subunits of the F protein. Although the cytoplasmic tail (CT) region of the RSV F protein has been reported to be necessary for the assembly and budding of infectious RSV, the effect of CT on the expression of the F protein in eukaryotic cells remains poorly investigated. To evaluate the effect of the cytoplasmic tail (CT) region on the expression of the RSV F protein, CT F572A substitution and CT truncation (leaving KAR 3 residues) were introduced into Lead-2 cells, generating Lead-2 F572A and Lead-2 ΔCT, respectively.

Example 2. In Vivo Screening for the Effects of CT : In vivo tests using Lead-2, Lead-2 F572A, and Lead-2 ΔCT were conducted to investigate the effects of CT on F protein expression. 2 µg of various mRNAs were transfected into A549 cells using lipofectamine MessengerMax. After 24 or 48 hours, cells were harvested and lysed, and Western blotting analysis was performed on 10 µg of whole-cell lysate using a commercial monoclonal rabbit anti-human RSV (A2) F glycoprotein IgG antibody that identifies total F proteins without conformational specificity. As shown in Figure 2, the results confirmed that Lead-2 and its variants can induce the expression of RSV F glycoproteins with the correct molecular weight. Qualitatively, Lead-2 exhibited a relatively higher level of RSV F glycoprotein compared to Lead-2 F572A and Lead-2 ΔCT.

To quantitatively compare the expression levels in A549 cells, whole cell lysates were continuously diluted and loaded into ELISA plates pre-coated with palivizumab (PVZ). Pre-fusion and post-fusion F glycoproteins in the cell lysates were then detected using D25 and 4D7 antibodies, respectively. The results clearly showed that Lead-2 and its variants only expressed F protein in the pre-fusion phase. As previously reported, neither F572A substitution nor CT truncation enhanced protein production; conversely, Lead-2 with intact CT showed higher expression levels in the first 24 hours (Figure 3). At 48 hours post-transfection, the concentration of F protein in the cell lysates was too low to demonstrate differences between the groups.

The next experiment measured the expression levels of pre-fusion and post-fusion RSV F on the cell surface of Lead-2 and its variants. Similarly, A549 cells were transfected with mRNA encoding Lead-2 or its variants, and then, 24 hours post-transfection, the expression of pre-fusion RSV F glycoprotein on the cell surface was measured by flow cytometry using D25 (for pre-fusion RSV F glycoprotein) and 4D7 (for post-fusion RSV F glycoprotein) antibodies. The data illustrated in Figure 4 show that Lead-2 and its variants induced relatively low levels of post-fusion F protein on the cell surface, consistent with the ELISA data above. Furthermore, neither F572A substitution (Lead-2 F572A) nor CT truncation (Lead-2 ΔCT) increased the surface expression levels of pre-fusion F protein in A549 cells and 293F cells. Conversely, Lead-2 with intact CT showed comparable or even higher expression on the cell surfaces of different cell lines.

In previous studies, the omission of the CT domain in RSV vaccines was a standard design. However, the results above surprisingly found that intact CT actually promoted higher expression levels of the RSV pre-F protein. Therefore, intact CT was used in the following vaccine design.

Example 3. Optimization of mRNA Vaccine Candidates. Previous studies have revealed a positive correlation between the expression levels of the pre-fusion form of RSV F glycoprotein, particularly the pre-fusion trimer form, and the immunogenicity of RSV vaccines in animals. In this example, various mRNA lead designs were screened to evaluate the effects of different features (e.g., mutation, specific modification, truncation, structural changes) on the expression levels of both pre-fusion and trimer pre-fusion F glycoproteins.

Based on the performance of Lead-2 in the screening above, three other vaccine candidates were designed and named Lead-5, Lead-6 and Lead-7 for optimization. All three lead molecules are codon-optimized mRNA-encoding membrane-bound, single-stranded (Lead-5, Lead-6) or disulfide-linked double-stranded (Lead-7) F proteins. These include intracellular (S155C, S290C) and intercellular (A149C, Y458C) disulfide-stabilized mutations, expression-enhancing mutations (S46G, E92D, S215P, L373R, K465Q), stability-increasing mutations (S215P), naturally occurring substitutions (P102A, I379V, M447V) to enhance expression levels, and cavity-filling mutations (S190F, V207L). Additionally, a T4 fold is inserted between the extracellular and transmembrane domains (Lead-6). (Figure 5).

Subsequently, Lead-2, 5, 6, and 7 were tested in vitro and in vivo to compare the performance and immunogenicity of different candidates, and based on the screening findings above, all of them possessed complete CT. For in vitro screening, 1 µg of each mRNA (Lead-2, 5, 6, 7) was transfected into A549 cells using lipofectamine MessengerMax. After 24 hours, the cells were harvested and lysed, and then 10 µg of whole-cell lysate was subjected to Western ink dot analysis. As shown in Figure 6, the results confirmed that all leads could induce the expression of RSV F glycoproteins with the correct molecular weight. Qualitatively, Lead-5 and Lead-6 showed relatively higher levels of RSV F glycoprotein expression, and Lead-7 showed two bands, which was due to the cleavage of native p27 in the cell.

To quantitatively compare the expression levels of different leads in A549 cells, whole cell lysates were serially diluted and loaded into ELISA plates pre-coated with palizumab (PVZ). Pre-fusion F glycoprotein, pre-trimeric pre-fusion F glycoprotein, and post-fusion F glycoprotein in the cell lysates were then detected using D25, AM14, and 4D7 antibodies, respectively. Results showed that Lead-5 induced the highest levels of pre-fusion trimeric F glycoprotein, followed by Lead-7 and Lead-6, while Lead-2 only showed basal levels of pre-fusion F trimeric glycoprotein, serving as a control (untransfected blank A549 cells). Regarding the total pre-fusion F glycoprotein content, Lead-2, 6, and 7 showed similar levels, while Lead-5 still exhibited a higher level. It should be noted that post-fusion F glycoprotein was not detected in any of the cell lysate samples from these four leads, indicating that all leads stabilize the pre-fusion conformation of RSV F glycoprotein in cells (Figure 7).

The next experiment evaluated the expression of pre-fusion RSV F, pre-fusion RSV F trimer, and post-fusion RSV F on the cell surface of these four lead compounds, which can provide some predictive information for immunogenicity in animals. Similarly, A549 cells were transfected with either no (control) or lead-2, 5, 6, and 7 mRNA samples. Twenty-four hours post-transfection, the expression of pre-fusion RSV F glycoprotein on the cell surface was measured by flow cytometry using D25 (for pre-fusion RSV F glycoprotein), AM14 (for trimer pre-fusion RSV F glycoprotein), and 4D7 (for post-fusion RSV F glycoprotein). The data, shown in Figure 8, confirm that all lead compounds induced relatively low levels of post-fusion F protein on the cell surface, consistent with the ELISA data above. Furthermore, Lead-5, Lead-6, and Lead-7 induced significantly higher levels of pre-fusion F protein compared to Lead-2. Regarding the surface expression levels of the pre-fusion F trimer, a similar pattern to that of pre-fusion F was observed. The similarity in expression levels between pre-fusion F and its trimer demonstrates that almost all pre-fusion F proteins expressed on the cell surface by Lead-5, 6, and 7 exhibit a trimer conformation. In conclusion, Lead-5, 6, and 7 demonstrate satisfactory levels of pre-fusion F and its trimer on the cell surface, thus all possessing strong potential for further screening through in vivo immunogenicity studies.

Example 4. In vivo immunogenicity study of optimized vaccine candidates ( mice ) The immunogenicity of RSV mRNA vaccine candidates was then evaluated using in vivo analysis. Mice were immunized according to the timetable shown in Table 1. Six-week-old BALB/c mice (n = 6/group) were immunized intramuscularly (IM) using lead mRNA or DS-Cav1 protein formulated in RTU LNP with aluminum adjuvant as a positive control or PBS as a negative control. In a previous study, an equivalent dose of the aluminum adjuvant DS-Cav1 protein was also used as a positive control for various RSV mRNA vaccines. Lead mRNA and DS-Cav1 protein + aluminum oxide adjuvant were administered at 3-week intervals (day 0 and day 21), and mouse serum was collected on days 0, 21, 35, and 49. Table 1. Immunogenicity Study Design in Mice Group Number of mice vaccine Dosage (µg mRNA or protein / mouse ) Dosage frequency Investment channels G1 6 Lead-2 2.0 2 im G2 6 Lead-2 10.0 2 im G3 6 Lead-5 2.0 2 im G4 6 Lead-5 10.0 2 im G5 6 Lead-6 2.0 2 im G6 6 Lead-6 10.0 2 im G7 6 Lead-7 2.0 2 im G8 6 Lead-7 10.0 2 im G9 6 DS-Cav1 Protein + Alum Adju 10.0 2 im G10 6 PBS N/A 2 im

Serum IgG antibody titers against pre- and post-fusion RSV F glycoprotein were determined using ELISA. At day 49 (Figure 9), the RSV F lead compound exhibited a clear dose-dependent characteristic. At a high dose (10.0 µg), all mRNA lead compounds showed equivalent or 2-3 times higher pre-fusion F-specific antibody titers compared to the control vaccine containing DS-Cav1 protein and aluminum adjuvant. Furthermore, the post-fusion F-specific antibody titers induced by Lead-5, 6, and 7 were significantly higher than those induced by DS-Cav1 protein and aluminum adjuvant (Figure 9). At low doses (2.0 µg), Lead-2, 5, 6, and 7 exhibited similar levels of pre-fusion F-specific antibody titers, and similar levels of post-fusion F-specific antibody titers (Figure 9). Overall, Lead-2, 5, 6, and 7 showed similar efficacy in inducing pre-fusion F-specific antibodies, and similar efficacy in inducing post-fusion F-specific antibodies, with Lead-7 showing the best performance in serum antibody titers. However, ELISA can only measure antibody levels of pre- or post-fusion proteins, not serum neutralizing antibodies induced by various mRNA vaccine leads. Neutralizing antibody analysis is more comprehensive and significant for evaluating vaccine efficacy.

Next, human RSV microneutralization assays were used to detect RSV-specific neutralizing antibodies in mouse serum. In the RSV A subline Long, the positive control vaccine showed only an NT50 of 384, while various mRNA vaccine leads induced significantly higher levels of neutralizing antibodies, except for Lead-2 (which induced even lower levels of neutralizing antibodies compared to the DS-Cav1 protein + aluminum adjuvant control vaccine). Among Lead-5, 6, and 7, at low doses, Lead-7 showed the highest NT50 (3.6-fold increase), followed by Lead-6 (2.4-fold increase) and Lead-5 (0.8-fold increase); however, at high doses, Lead-6 showed the highest NT50 (approximately 13-fold increase compared to the DS-Cav1 protein + aluminum adjuvant control vaccine), followed by Lead-7 (approximately 7.7-fold increase) and Lead-5 (approximately 6.2-fold increase) (Figure 10). To evaluate the cross-protection of the lead vaccine, RSV subtype 18537 was also used in a micro-neutralization analysis. The data in Figure 10 confirm that Lead-6 exhibited the most potent neutralizing antibody at both low (5.4-fold increase) and high (30-fold increase) doses, followed by Lead-7 (1.5-fold increase at low dose and 11-fold increase at high dose) and Lead-5 (1.8-fold increase at low dose and 8.2-fold increase at high dose). Surprisingly, Lead-6 outperformed Lead-5 and Lead-7 in neutralizing antibody titers in both RSV A and B sublines. The RSV-specific neutralizing antibody results compared to the overall antibody titer indicate that this novel Lead-6 design can exhibit more epitopes on the pre-fusion F protein or have a more stable pre-fusion F conformation to induce neutralizing antibodies. In summary, the data above confirms that Lead-6 can induce the highest levels of neutralizing antibodies against both RSV A and RSV B sublines, making it a potential final candidate for RSV mRNA vaccines.

Since the ratio of IgG2a and IgG1 serum titers is correlated with the Th1 and Th2 immune response profiles, and the Th2-biased immune response can play a role in vaccine-enhanced respiratory disease (VERD) that occurred during the early development of RSV vaccines, the serum levels of IgG subclasses targeting RSV Pre-F proteins were evaluated. The results in Figure 11 show that all mRNA vaccine leads induced similar levels of IgG1 and IgG2a titers in mouse serum, exhibiting a clear dose-dependent characteristic, indicating that the immunization of mRNA vaccine leads elicits a balanced Th1 and Th2 immune response. However, recombinant DS-Cav1 protein with an aluminum adjuvant primarily induced high levels of IgG1, thus indicating a Th2-biased immune response. IgG subtype analysis data revealed that all mRNA vaccine leads can induce a balanced Th1 and Th2 immune response, thus indicating a low risk of VERD.

To further determine the binding epitope of serum antibodies to RSV F protein, we evaluated the ability of immune sera from vaccinated mice to competitively bind to pre-fusion or post-fusion F protein against AM14 or 4D7 monoclonal antibodies. 4D7 specifically recognizes the post-fusion F protein, while AM14 recognizes the pre-fusion F protein trimer. As shown in Figure 12, all vaccinated mouse sera exhibited high and dose-dependent inhibitory activity against the pre-fusion F protein when competing with AM14. At high doses (10 μg), all sera immunized with the mRNA lead showed significantly stronger competitive effects against AM14 compared to sera from the DS-Cav1 protein + aluminum adjuvant control group. In contrast, serum from mice immunized with mRNA vaccines did not show significant competitive effects against 4D7 for binding to the post-fusion F protein. These data suggest that the antigen expressed in tissues of mice immunized with mRNA vaccines primarily exhibits the pre-fusion F protein trimer conformation and very little of the post-fusion conformation. Serum from mice inoculated with recombinant DS-Cav1 protein showed weak competitiveness against 4D7 for binding to the post-fusion F protein, indicating that the recombinant protein induces more post-fusion F-specific antibodies than the mRNA vaccine lead. In summary, all mRNA vaccine leads primarily expressed the RSV F protein antigen in its pre-fusion trimer conformation and induced high levels of antibodies with the AM14-binding epitope, with almost no detectable serum antibodies against the 4D7-binding epitope.

To evaluate cellular immune responses following mRNA vaccine immunization, the frequency of antigen-specific T cell subsets was measured in spleen cells of immunized mice by intracellular cytokine staining and flow cytometry after in vivo restimulation with a peptide pool of RSV F protein, as described in Materials and Methods. As shown in Figure 13, all mRNA vaccines induced dose-dependent RSV F-specific CD4 ⁺ and CD8 ⁺ T cell responses, as measured by the expression of IL-2, IFN-γ, or TNF-α in CD4 ⁺ and CD8 ⁺ T cell subsets. Generally, the percentage of activated CD8 ⁺ T cells was higher than that of CD4 ⁺ T cells. In activated CD4 ⁺ T cells, TNF-α was the most expressed intercytokine; however, in CD8 ⁺ T cells, the expression levels of TNF-α and IFN-γ were similar. In both CD4 ⁺ and CD8 ⁺ T cells, IL-2 production was weaker compared to other intercytokines. Among Lead-2, 5, 6, and 7, Lead-5 exhibited a relatively higher T-cell response than the other leads. In contrast to mRNA vaccines, animals immunized with the aluminum adjuvant DS-Cav1 protein showed extremely low to undetectable T-cell responses (Figure 13). In conclusion, these data indicate that all mRNA vaccine leads can induce potent T-cell responses that promote complete RSV clearance.

In summary, the data above confirm that Lead-5, 6, and 7 exhibit considerable immunogenicity in mice. Given the importance of neutralizing antibody titer for prophylactic RSV vaccines, Lead-6, which induced the highest neutralizing antibody titer in animals, was selected as the final vaccine candidate. Although Lead-6 was not superior to Lead-5 and Lead-7 in pre-F specific antibody titer and pre-F specific IgG subclass titer, the neutralizing antibody titer induced by Lead-6 was significantly higher than that induced by Lead-5 and Lead-7, indicating the generation of potent neutralizing antibodies targeting novel epitopes of the F protein through antigenic structural differences.

Example 5. Comparison of Lead-6 and a commercial RSV vaccine: The differences between Lead-6, the final candidate in the third round of screening and evaluation, and a commercial RSV mRNA vaccine, Moderna mRNA-1345, in terms of cellular expression levels, stability, and immunogenicity.

To further investigate the efficaciousness of Lead-6, 1 µg of Lead-6 mRNA and Moderna's RSV mRNA vaccine mRNA-1345 (MOD-1: the MRNA-1345 F protein sequence has the same 5'UTR, 3'UTR, and multiple A binding as used in our mRNA lead as described in this invention. MOD-1 ORF refers to the sequence disclosed in patent application WO2021155243A1.) were transfected into A549 cells. At 24, 48, and 72 hours post-transfection, the levels of pre-fusion F, pre-fusion F trimer, and post-fusion F proteins were measured by flow cytometry. The data in Figure 14A confirm that Lead-6 exhibits similar or better performance kinetics than MOD-1 for pre-fusion F and pre-fusion F trimers. Regarding post-fusion F performance, Lead-6 shows lower-order but similar kinetics. Normalizing the 24-hour performance levels of each mRNA, the relative MFI kinetic data in Figure 14B show that, particularly for pre-fusion F protein trimers, Lead-6 exhibits the highest increase at 48 hours and the lowest decrease at 72 hours, indicating that Lead-6 may have an advantage over MOD-1 in performance stability. However, all mRNAs show a similar decay rate to F proteins from 48 to 72 hours.

To compare the immunogenicity between Lead-6 and mRNA1345 in vivo, 6-week-old BALB/c mice (n = 6/group) were immunized intramuscularly (IM) with Lead-6 or MOD-1 mRNA modulated in RTU LNP or PBS as a negative control, according to the timetable shown in Table 2. The mRNA vaccine and PBS were administered at 3-week intervals (day 0 and day 21), and mouse serum was collected on days 0, 21, and 35. Table 2. Mouse Immunogenicity Study Design Group Number of mice vaccine Dosage ( µg mRNA/ mouse ) Dosage frequency Investment and Pathways G1 6 Lead-6 1.0 2 im G2 6 Lead-6 5.0 2 im G3 6 MOD-1 1.0 2 im G4 6 MOD-1 5.0 2 im G5 6 PBS N/A 2 im

Serum IgG antibody titers against RSV F glycoprotein before and after fusion were determined by ELISA using the same method as the second round of screening described in Materials and Methods. Regarding serum antibody titers on day 35 (Figure 15), Lead-6 exhibited a significant dose-dependent characteristic, while MOD-1 showed a relatively weak dose response. At high doses (5.0 µg), Lead-6 exhibited pre-fusion F-specific antibody titers similar to those of MOD-1. Furthermore, the post-fusion F-specific antibody titers induced by Lead-6 were approximately twice that of MOD-1. At low doses (1.0 µg), Lead-6 exhibited lower pre-fusion F-specific antibody titers and similar post-fusion F-specific antibody titers compared to MOD-1 (Figure 15). Overall, at high doses, Lead-6 demonstrated similar efficacy to MOD-1 in serum antibody titers, particularly for pre-fusion F-specific antibodies.

Next, the titer of RSV-specific neutralizing antibodies in mouse serum was measured using a human RSV microneutralization assay. In the RSV A subline Long, all mRNA vaccines induced high levels of neutralizing antibodies. At low doses (1.0 µg), Lead-6 showed a relatively higher NT50 (2.3-fold increase) compared to MOD-1; unexpectedly, at high doses (5.0 µg), Lead-6 significantly outperformed MOD-1 in NT50, with an approximately 5.5-fold increase compared to MOD-1 (Figure 16A). To evaluate the cross-protection of the mRNA vaccines, the RSV B subline 18537 was also used in the microneutralization assay. The data in Figure 16B confirm that Lead-6 induced the most potent neutralizing antibodies at both low and high doses (1.5-fold increase compared to MOD-1). In summary, these data demonstrate that Lead-6 induces neutralizing antibodies against higher concentrations of RSV A and RSV B strains compared to MOD-1, further demonstrating the superiority of Lead-6 as a final candidate for RSV mRNA vaccines.

Based on the in vivo immunogenicity screening described above, the serum levels of IgG subclasses (IgG2a and IgG1) against RSV Pre-F protein were further evaluated. The results in Figure 17 show that all mRNA vaccines induced similar levels of IgG1 and IgG2a titers in mouse serum; however, Lead-6 showed a significant dose-dependent characteristic, while the dose response of MOD-1 was not significant. Notably, at low doses, the IgG1 titers of Lead-6 and MOD-1 were relatively lower than those of IgG2a, but the difference became smaller at high doses. In summary, the IgG subtype analysis data revealed that the Lead-6 and MOD-1 mRNA vaccines can induce a balanced Th1 and Th2 immune response, thus indicating a low risk of VERD.

Finally, cellular immune responses between Lead-6 and MOD-1 were compared using the same methods described in Materials and Methods. As shown in Figure 18, all mRNA vaccines induced RSV F-specific CD4 ⁺ and CD8 ⁺ T cell responses, as measured by the expression of IL-2, IFN-γ, or TNF-α in CD4 ⁺ and CD8 ⁺ T cell subsets. However, dose-dependent responses to Lead-6 and MOD-1 were not significant. TNF-α was consistently the most expressed intercytokine in activated CD4 ⁺ T cells, while the expression levels of TNF-α and IFN-γ were similar in CD8 ⁺ T cells. IL-2 production was weaker in both CD4 ⁺ and CD8 ⁺ T cells compared to other intercytokines. The CD4 ⁺ T cell response patterns between Lead-6 and MOD-1 are quite similar, with Lead-6 inducing slightly higher levels of TNF-α ⁺ CD4 ⁺ T cells (Figure 18A). In contrast, the CD8 ⁺ T cell response of Lead-6 is slightly weaker than that of MOD-1, especially at high doses (5.0 µg) (Figure 18B). Considering the crucial role of T cells in viral clearance and immunopathological progression during RSV infection, and the safety profile of the mRNA-1345 vaccine in the clinical trial (NCT05127434), it can be concluded that the Lead-6 vaccine can elicit strong cellular immunity against RSV while preventing VERD formation.

In summary, the data above confirm that Lead-6 exhibits similar or even better performance in terms of cellular stability and immunogenicity in mice compared to mRNA vaccines expressing the same antigen ORF as mRNA-1345. Given the importance of neutralizing antibody titer for prophylactic RSV vaccines, the Lead-6 mRNA vaccine is expected to be successful in future clinical trials. Therefore, Lead-6 was selected as the final candidate for an RSV mRNA vaccine and renamed Lead-6A for use in subsequent experiments.

Example 6. Efficacy, safety, and immunogenicity of candidate vaccines in an RSV/A2 cotton rat model. A challenge study was then conducted to test the efficacy of Lead-6A in preventing RSV infection and disease in immunized cotton rats ( Sigmodon hispidus ) after intranasal RSV/A2 virus challenge. As described in Table 3, female cotton rats (6-8 weeks old) were randomly assigned to 6 groups (6 animals/group). Table 3. Group assignment in the challenge study using the cotton rat model of RSV/A2 . Group animal numbers vaccine Dosage Volume Dosage frequency Investment and Pathways 1 6 Lead-6A (Attack on Virus) 5.0 µg 62.5 µL Day 0, Day 28 IM injection 2 6 Lead-6A (Attack on Virus) 16.0 µg 200 µL Day 0, Day 28 IM injection 3 6 Using RSV A2 virus for pre-attack (attack) 1.0 x ^10⁵ pfu 100 µL Day 0 IN 4 6 FI-RSV batch number 100 (anti-virus) 1:100 100 µL Day 0, Day 28 IM injection 5 6 Unvaccinated (challenge) Blank LNP 100 µL Day 0, Day 28 IM injection 6 6 Unvaccinated (no challenge to virus) Blank LNP 100 µL Day 0, Day 28 IM injection

Cotton-fed mice were immunized twice by intranasal administration on days 0 and 28 with low (Group 1) and high (Group 2) doses of Lead-6A (5 μg or 16 μg mRNA/dose, respectively). Group 3 animals were infected once by intranasal administration of RSV/A2 strain at ^10⁵ pfu/animal (IN) on day 0. Back titration was performed to confirm the RSV A2 infection dose. Group 4 animals were vaccinated with formalin-inactivated RSV (FI-RSV) batch number 100. A mediator control (blank LNP) was administered via intranasal administration to unvaccinated, negative control animals (Groups 5 and 6).

Three weeks after the second immunization (day 49), animals in groups 1 through 5 were challenged with RSV/A2 at ^10⁵ pfu/animal via IN. Back titration was performed to confirm the RSV/A2 challenge dose. Blood samples were collected on days 0, 28, 49, and 54 (terminal bleeding). Lung and nasal tissues were collected at the end of the study for viral titration and histopathological analysis. Morbidity and mortality were monitored throughout the study.

Animal body weight was monitored on days 0, 28, 49, and 54. No weight loss was observed in any of the 36 animals throughout the study period. Physical and clinical signs (behavioral activity, stress signs, posture, fur appearance, food intake, excretion, respiratory status, and mortality) were monitored in all animals. No atypical appearances or behaviors were observed. Furthermore, no deaths or morbidities occurred. Therefore, the test drug administration protocol showed no significant toxicity in cotton-ear mice.

On day 54 (5 days after RSV challenge), RSV viral titers in the lungs and noses of all animals were measured (Figure 23). Animals vaccinated twice with blank LNP and challenged with RSV/A2 (Group 5) served as controls for maximum viral replication and were compared with other vaccinated groups. Animals previously infected with RSV (Group 3) served as positive controls for protection. Animals vaccinated with blank LNP but not challenged (Group 6) showed no virus presence. Animals vaccinated with FI-RSV vaccine (Group 4) showed partial lung protection (reduced to ~1.4 Log ₁₀ pfu). Animals vaccinated with Lead-6A vaccine at two different doses (Groups 1 and 2) showed complete protection of lung tissue (no detectable viral load). Animals vaccinated with FI-RSV batch 100 vaccine (Group 4) showed no protection of the nose. Animals vaccinated with the Lead-6A vaccine showed almost complete protection of the nose at low doses (Group 1, only 1 of 6 animals had detectable virus in the nose) and protection at high doses (no detectable viral load) (Group 2).

Next, RSV neutralizing antibodies against RSV/A2 were measured in serum samples obtained from all animals before the vaccine booster (day 28) and before RSV/A2 challenge (day 49). Control animals infected with RSV/A2 on day 0 (group 3) produced moderate levels of neutralizing antibodies on day 28, which remained unchanged on day 49 (~8 Log ₂ ). Animals vaccinated with FI-RSV (group 4) or vaccinated with blank LNP (groups 5 and 6) showed no detectable induction of neutralizing antibodies. Animals vaccinated with Lead-6A vaccine showed dose-dependent induction of neutralizing antibodies (groups 1 and 2). When measured on day 28, the levels were comparable to those of animals previously infected with RSV (~8-9 Log ₂ ), but increased further to the highest levels after booster immunization (12 and 14 Log ₂ for 5 µg and 16 µg doses, respectively) (Figure 24).

Finally, lung histopathology was assessed in all animals during the experiment. Animals vaccinated with FI-RSV batch number 100 showed the highest scores across all measured parameters (Group 4). Animals previously infected with RSV/A2 on day 0 (Group 3) and then challenged, and animals vaccinated with blank LNP (Group 5) and challenged with RSV/A2, showed low but detectable peribronchitis, perivasculitis, interstitial pneumonia, and alveolitis. Animals vaccinated with Lead-6A vaccine (Groups 1 and 2) showed the lowest lung histopathology, with all scores equivalent to those of uninfected animals in Group 6 (Figure 25).

In summary, immunization with the Lead-6A vaccine induced a strong, dose-dependent neutralizing antibody response against RSV/A2 in cotton rats. Primary immunization with 5 µg and 16 µg doses of Lead-6A induced neutralizing antibody levels similar to those induced by RSV infection (titers ranging from 7.9 to 9.3 _Log² on day 28 in groups 1, 2, and ₃ ), which further increased with lead-6A boosters (titers of 12.5 and 14.3 Log² on day 49 in groups 1 and 2, respectively). Lead-6A immunization also provided complete lung protection at both doses, complete nasal protection at a high dose (16 µg), and near-complete protection at a low dose (5 µg) (no detectable viral load in the nose in 5 of 6 animals), as confirmed by viral load suppression. Lead-6A vaccination conferred eliminating immunity in the lungs, as lung histology showed no difference between uninfected control animals and lead-6A-treated mice challenged with RSV/A2 virus. In contrast, the FI-RSV batch 100 vaccine exhibited lung histopathology consistent with vaccine-enhanced respiratory disease (VERD). The Lead-6A vaccine also demonstrated a favorable safety profile, with no deaths or illnesses occurring during the study period. Therefore, administration of the vaccine to mice showed no significant toxicity. In summary, this study demonstrates that Lead-6A possesses beneficial safety, efficacy, and immunogenicity characteristics in a wiry cotton rat model challenged with RSV/A2.

Following in vivo administration, this novel design of the vaccine elicits potent RSV-neutralizing antibodies against the RSV F antigen and a robust T-cell response. Due to the robust T-cell response, the mRNA vaccine candidate is expected to contribute to the complete clearance of RSV, which was not observed in RSV protein subunit vaccines. Simultaneously, this novel design of the mRNA vaccine also induces higher levels of IgG antibodies against the pre-F antigen (a Th1/Th2 balanced rather than Th2-biased immune response profile). The predominant antigenic form expressed in vivo by the mRNA vaccine candidate is the trimerized pre-F protein rather than the post-F protein, as confirmed in competitive ELISA using epitope-specific antibodies. Compared to mRNA vaccines encoding the same ORF as mRNA-1345 (which is the first commercially available mRNA RSV vaccine targeting the pre-F antigen), the final candidate of this invention demonstrated superior performance in neutralizing antibody titer and was selected as the final candidate for RSV mRNA vaccine, internally renamed Lead-6A. Furthermore, inoculation with Lead-6A in cotton-ear mice induced potent neutralizing antibodies, resulting in complete protection of the lungs (as confirmed by viral load suppression and the absence of lung histopathology) and complete protection of the nose (high dose) or near-complete protection (low dose) (as confirmed by viral load suppression after intranasal RSV/A2 challenge). In addition, the vaccine of this invention is thermally stable at room temperature for at least one week, which greatly facilitates the transportation, storage and vaccination of large quantities of LMIC.

Example 7. Comparison between Lead-6/6A and commercial RSV vaccines: Computer design and optimization of 5'UTR ( non-translated region ) . Based on the 5'UTR sequence of the chicken β-globin gene and following design principles and rules, candidate 5'UTR sequences were designed by computer.

1.1 Design Principles and Rules: 1) Length approximately 50 bp; 2) Contains translation initiation factors and ribosome binding sites; 3) Contains GC-rich hairpin regions to stabilize the structure; 4) Contains short AT-rich fragments to enable rapid ribosome passage; 5) Contains GC-rich and Kozak sequences to enable efficient ribosome binding to AUG; 6) Excludes inhibitory structural domains (e.g., non-canonical start codons), etc.

1.2 Based on the principles and rules, we designed four different 5'UTRs. The candidate 5'UTR sequences are listed in Table 4: Table 4. Sequences of 5' UTRs . Name Sequence (5' --- 3') Chi β-G WT ACCAGCGUGCUAUCCCCACGGGAGCAAGAGCCCAGACCUCCUCCGUACCGACAGCCACACGCUACCCUCCAACCGCCGCC (SEQ ID NO:35) Chi β-G Δ1 GACAAUUACAAUAGUCAACGAGCUAGACAAGCUACGUCGACAGACCGCCACC (SEQ ID NO:36) Chi β-G Δ4 GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO:4) Chi β-G Δ6 GACAAUUACAAUAGUCACGAGCUAGACAAGCUACGUCGACAGACCGCCACC (SEQ ID NO:37) Chi β-G Δ7 GACAAUUAAGAAUCAAGCGAGCUAGACAAGCUCGCUACGUCAACCGCCACC (SEQ ID NO:38)

2. Evaluation of the function of the newly designed 5'UTR 2.1 mRNA architecture To evaluate the effect of the newly designed 5'UTR on protein expression, the UTR was selected and colonized into a conventional mRNA expression vector, and the spike protein (S2P) was used as the target gene (GOI). The architecture design is shown in Figure 19.

2.2 In vivo animal studies To compare the protein expression activities of different 5'UTRs (Δ1, Δ4, Δ6, Δ7 and WT), lipid nanoparticles (LNPs) encapsulating mRNA with different 5'UTRs were injected into mice via intramuscular injection ( im ) and the antibody titers against S2P in mouse serum were tested (Figures 20 and 21).

Mouse serum was collected at indicated time points and antibody titers were detected by ELISA. The overall titers of mRNAs with Δ1, Δ4, Δ6, and Δ7 5'UTRs were higher than those with WT 5'UTRs (Figure 21 and Table 5). To further analyze the differences between each group, the antibody titers of all mRNAs with Δ1, Δ4, Δ6, and Δ7 5'UTRs were normalized to the day 7 antibody titers of mRNAs with WT 5'UTRs. It was found that the overall titers of mRNAs with Δ4 5'UTRs were higher than those of other groups, indicating that mRNAs with Δ4 5'UTRs can induce stronger antigen/protein expression (Figure 22 and Table 6). Table 5. Mouse antibody titers. Group Day 7 Day 14 Day 21 pT7-Chi β-G Δ1 -S2P 5440 23040 17920 pT7-Chi β-G Δ4-S2P 10240 25600 23040 pT7-Chi β-G Δ6-S2P 5760 17920 20480 pT7-Chi β-G Δ7-S2P 5760 12800 19200 pT7-Chi β-G (WT) -S2P 4000 11520 6400 Table 6. Multiples of mouse antibody titer compared to WT titer on day 7 . Group Day 7 Day 14 Day 21 pT7-Chi β-G Δ1 -S2P 1.36 5.76 4.48 pT7-Chi β-G Δ4-S2P 2.56 6.4 5.76 pT7-Chi β-G Δ6-S2P 1.44 4.48 5.12 pT7-Chi β-G Δ7-S2P 1.44 3.2 4.8 pT7-Chi β-G (WT) -S2P 1 2.88 1.6

3. Conclusion Based on the results, in the above examples, we used chicken β-globin Δ4 5'UTR in the mRNA backbone to enhance target gene expression. Table 7 . Lead-2 Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 8) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYCVNKQEGQS LYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 17) Multiple A tails 125 nt Lead-2 F572A Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 9) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYCVNKQEGQS LYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAASN (SEQ ID NO: 18) Multiple A tails 125 nt Lead-2 ΔCT Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 10) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKAR (SEQ ID NO: 19) Multiple A tails 125 nt Lead-5 Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 11) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQS LYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 20) Multiple A tails 125 nt Lead-6 / Lead-6A Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) GC C GC UGCU GC CAGCAAC (SEQ ID NO: 45) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFY DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN AA A A SN (SEQ ID NO: 46) Multiple A tails 125 nt Lead-6 variant Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 12) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPL VFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 21) Multiple A tails 125 nt Lead-7 Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 13) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQS LYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 22) Multiple A tails 125 nt MOD-1 Chemistry N1-Methylpseudouridine cap ^m7 G(5')ppp(5')N1 _m pNp 5' UTR GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (SEQ ID NO: 4) The ORF of mRNA (excluding the termination codon) (SEQ ID NO: 14) 3' UTR GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (SEQ ID NO: 5) Corresponding amino acid sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLY (SEQ ID NO: 23) Multiple A tails 100 nt

The following experimental details are provided for illustrative purposes only and do not constitute a limitation in any respect. On the other hand, the details set forth herein are part of the general description of embodiments of the invention and are therefore to be understood as being able to be combined with any other form or one or more embodiments of the invention as generally described above.

Materials and Methods: Cell lines, viruses, and animal A549 and 293F cell lines were obtained from ATCC (Manassas, VA, USA) and maintained according to ATCC's instructions. Mycoplasma contamination was routinely checked during the culture process.

RSV strain A, Long (catalog number: VR-26, ATCC), and strain B, 18537 (catalog number: VR-1580, ATCC), were ordered from ATCC and propagated in Hep-2 cells in our BSL-2 laboratory. Both strains were used for serum neutralization analysis.

Female BALB/c mice aged 5-7 weeks were obtained from Shanghai Lingchang Biological Technology (Shanghai, China) and used for immunogenicity studies. Mice were housed in a specific pathogen-free (SPF) environment (constant temperature (21-22℃) and humidity (40-70%)) at the Laboratory Animal Center of Shanghai Jiao Tong University. Ear tags were used for identification. All in vivo experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.

The design, plasmid construction, and extraction of genes encoding each vaccine lead were based on the previously reported principle of stable RSV pre-fusion F[18] and further modified. Additional modifications are illustrated in Figures 1 and 5. The vaccine mRNA (containing Lead-2, Lead-2 F572A, Lead-2 ΔCT, Lead-5, Lead-6, Lead-7, and MOD-1) contained a modified chicken β-globin 5'-untranslated region (UTR), a codon-optimized gene encoding the RSV F antigen, a chicken β-globin 3'-UTR, and a 125-nucleotide multi-A tail. Gene DNA was then synthesized at Azenta Life Sciences (Suzhou, Jiangsu Province, China) and subselected into a pT7-2G-6.0 plasmid vector with an upstream T7 RNA polymerase promoter. The sequence-verified plasmids were amplified in approximately 100 mL of Stbl3 E. coli culture and extracted using a commercial endotoxin-free plasmid extraction kit (catalog number: 740420.50, MN, Germany) following the manufacturer’s instructions.

DS-Cav1 and its fusion-restructured protein, fused with the C-terminal trimeric domain of T4 fibroin (T4 fold) and its His tag (amino acid sequence in the sequence listing section), were expressed in CHO cells and purified using a HisTrap FF Crude column (Cytiva, MA, USA) from GenScript Biotech Corporation (Nanjing, Jiangsu Provine, China). Both proteins were diluted in 1×PBS (pH 7.2). Purity was greater than 90% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with endotoxin levels controlled to be less than 2 EU/mg. Store protein aliquots at -60℃ to -80℃ to avoid repeated freezing and thawing cycles.

To generate a DS-Cav1/Alum Adju (Rehydragel LV, Catalogue No.: 26050, Miragen, USA) recombinant protein vaccine for mouse immunization, 750 µL of DS-Cav1 protein (320 µg/mL) was dropwise added to 1.65 mL of diluted Al(OH) ₃ (1164 µg/mL) in a 15 mL tube with gentle shaking. The final concentrations of DS-Cav1 and Al(OH) ₃ in the adjuvant vaccine were 100 µg/mL and 800 µg/mL, respectively.

The detection antibodies were generated in Biointron as human monoclonal antibodies D25 and AM14, a humanized mouse monoclonal antibody palizumab (PVZ), and a mouse monoclonal antibody 4D7. The variable regions of the heavy and light chains were synthesized and subselected into eukaryotic expression vectors containing the constant regions of the heavy and light chains. Plasmids encoding both the heavy and light chains were co-transfected into a suspension of CHO cells cultured in serum-free FreeStyle CHO expression medium (GIBCO, Waltham, MA, USA). After 6 days, the supernatant was harvested and purified using a Protein A resin FF column (Genscript, Nanjing, Jiangsu Province, China). After purification, the antibody buffer was replaced with PBS.

After quantification, D25 and AM14 antibodies were labeled using YF488 (UELandy, Suzhou, Jiangsu Province, China) or biotin (Invitrogen, Waltham, MA, USA), and 4D7 was labeled using YF640 (UELandy, Suzhou, Jiangsu Province, China) or biotin. The labeled antibodies were used for ELISA and flow cytometry analysis.

mRNA vaccine production utilizes in vivo extracellular transcription (IVT) to generate mRNAs encoding Lead-2, Lead-2 F572A, Lead-2 ΔCT, Lead-5, Lead-6, Lead-7, and MOD-1. In short, plasmids, including the 5'-UTR, open reading frame (ORF), 3'-UTR, and multiple A regions, are linearized by digestion with BspQ-I (NEB, MA, USA) at 50°C for 1.5 hours. After purification, the linearized plasmid DNA is used as a template to generate mRNA via in vivo extracellular transcription (IVT). The reaction system contained IVT buffer, _MgCl₂ (6 mM), a mixture of ribonucleoside triphosphates (6 mM each), yeast inorganic pyrophosphatase (2 U/mL), RNase inhibitor (1000 U/mL), and T7 RNA polymerase (5000 U/mL) (all from Hongene Biotech, Shanghai, China). Urate triphosphate was replaced with N1-methyl-pseudouridine triphosphate (Hongene Biotech, Shanghai, China) to enhance ORF protein expression. Turbo DNAase (Invitrogen, MA, USA) was used to remove the DNA template from the reaction system.

The transcribed mRNA was capped using the following components: vaccinia capping system (500 U/ml), capping buffer, GTP (0.5 mM), S-adenosylmethionine (0.128 mM), RNase inhibitor (667 U/ml), and 2'-O-methyltransferase (2500 U/ml) (all from Hongene Biotech, Shanghai, China). The capping reaction was performed by incubating mRNA with the vaccinia virus capping enzyme (Hongene Biotech, Shanghai, China) at 37 ± 2 °C for 90 min, which added a 7-methylguanylic acid cap (cap 0) to the 5' end of the mRNA. Then, the mRNA was purified by precipitation with LiCl (Invitrogen, MA, USA), followed by analysis by agarose gel electrophoresis, capillary electrophoresis, and liquid chromatography-mass spectrometry.

To prepare the mRNA-LNP complex, RTU LNPs were prepared by rapidly mixing the ethanol and aqueous phases using a microfluidic device (INano™ L system, Micro & Nano). The aqueous phase contained citrate buffer (pH 6.0, 50 mM). The ethanol phase included ionizable cationized lipid 4 (Immorna Biotechnology, Hangzhou, Zhejiang Province, China), cholesterol (Jiangsu Southeast Nanomaterials, Huaian, Jiangsu Province, China), 1,2-distearyl-sn-glycerol-3-phosphate choline (Jiangsu Southeast Nanomaterials), and 1,2-dimyristyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (SINOPEG, Xiamen, Fujian Province, China). RTU LNPs were assembled using four lipid components mixed in a molar ratio of 40:48:10:2.0, and characterized by particle size, polydispersity index, and zeta potential. The average diameter of these RTU LNPs was approximately 60 nm, with a polydispersity index of 0.05–0.10 and a zeta potential of 1.50–4.00 mV. The ionizable cationic lipids used in the RTU LNPs are described in International Patent Application PCT/CN2021/119577. The RTU formulations are described in International Patent Application PCT/CN2022/137326.

After concentration using NanoDrop One C (Thermo Fisher Scientific, USA), the mRNA sample was adjusted to 1 µg/µL and stored at -60°C to -80°C. RTU LNPs were stored at 4°C protected from light. Before administration, the mRNA component (vial A) and RTU LNP dispersion (vial B) were equilibrated to room temperature for approximately 30 minutes. The RTU LNP dispersion was drawn from vial B using a syringe and added to the mRNA vaccine in vial A. The required volume of RTU LNP depends on the amount of mRNA (determined by a final mRNA complexation efficiency of 50 µg/mL). The vials were then inverted for approximately 30 seconds to thoroughly mix and obtain the reconstituted vaccine (which appears as a white to off-white suspension). The suspension was incubated at room temperature for 10 minutes and then used for animal immunization.

One day prior to transfection , A549 or 293F cells were trypsinized or repeatedly pipetted, counted, and seeded at 5 × ^10⁵ cells (A549) or 1 × ^10⁶ cells (293F) per well in 6-well plates. The plates were prepared in 5 mL DMEM medium supplemented with 10% FBS + 1% penicillin-streptomycin (A549) or 5 mL Transpro CD01 medium supplemented with 6 mM L-glutamic acid (GIBCO, Waltham, MA, USA) (293F) (Duoning Biotechnology, Shanghai, China). The cells were then cultured overnight in a humidified incubator at 37°C and 5% _CO2 .

On the day of transfection, dilute the mRNA sample to an appropriate volume of Opti-MEM medium (volume can be based on the amplification and shrinkage of 1.0 µg mRNA diluted in 125 µL Opti-MEM medium), and dilute lipofectamine MessengerMax (Invitrogen, MA, USA; for 1.0 µg mRNA diluted in 125 µL Opti-MEM medium, volume can be based on the amplification and shrinkage of 7.5 µL liposomes) in another tube using an equal volume of Opti-MEM medium. After incubating at room temperature for 10 minutes, add the diluted mRNA to the diluted MessengerMax reagent and mix gently. The mRNA-lipid complex was then incubated at room temperature for 5 minutes, followed by loading into cells and gently rotating the plate. Subsequently, the cells were cultured in a humidified incubator at 37°C and 5% _CO2 for 24 hours.

Cell surface staining and flow cytometry analysis: A549 cells or 293F cells were harvested 24 hours after transfection for cell surface staining and flow cytometry analysis. In short, 5 × ^10⁵ cells (A549) or 1 × ^10⁶ cells (293F) were deposited in U-bottom 96-well plates, washed once with PBS, and then resuspended in 200 µL PBS. 1 µL of Zombie UV™ dye (Biolegend, San Diego, CA, USA) reconstituted in DMSO was added, and the cells were incubated in the dark at room temperature for 30 minutes to distinguish between live and dead cells. Subsequently, the cells were washed twice with 300 µL PBS supplemented with 2% BSA (PBS/2% BSA). Cells were resuspended in 200 µL PBS/2% BSA and incubated at 4°C in the dark for 30 minutes using either YF488-conjugated D25+ and YF640-conjugated 4D7 or YF488-conjugated AM14+ and YF640-conjugated 4D7 (2 µg/mL each). Cells were then precipitated and washed twice with 300 µL PBS/2% BSA. Finally, cells were resuspended in 200 µL PBS/2% BSA and analyzed using a CytoFLEX flow cytometer (Beckman Coulter). Data were processed using CytoExpert software (version 2.4).

The sandwich-type enzyme-linked immunosorbent assay (ELISA) involves quantifying the proteins using the Pierce™ BCA protein assay kit (Thermo, USA), adjusting the total protein concentration of different cell lysates to the same level, and then analyzing the expression levels of pre- or post-fusion proteins in the cell lysates using a standard sandwich-type ELISA protocol. In short, 1 µg/mL palizumab antibody in 100 µL of ELISA coating buffer (35 mM _NaHCO3 , 15 mM _Na2CO3 ) is coated overnight on a 96-well flat-bottom plate (MaxiSorp ELISA plate, NUNC, USA) at 4°C. After washing three times with PBS (PBST) containing 0.5% Tween 20, the coated plates were blocked for 1 h at 37°C with 100 μL of ELISA blocking buffer (PBST + 2% BSA) per well. After washing three times with PBS-T, the cell lysate sample was added to the designated wells at 10 µg total protein per well, and the standard protein (DS-Cav1 and fusion) was serially diluted 2-fold from 2 µg/mL and transferred to the designated wells, followed by incubation at 37°C for 1 h. After washing five times with PBS-T, the plates were incubated at 37°C for 1 h with biotin-conjugated D25 (for pre-fusion F), biotin-conjugated AM14 (for pre-fusion F trimer), or biotin-conjugated 4D7 (for post-fusion F) (all at a concentration of 2 µg/mL in the blocking buffer). After washing three times, the plates were incubated at 37°C for 20 min with HRP-conjugated streptavidin (A0303, Beyotime), followed by three washes. Next, all plates were incubated at 37°C with TMB single-component acceptor solution (Solarbio Life Sciences, Beijing, China) for 7 min, and then the reaction was stopped using ELISA termination solution (Solarbio Life Sciences, Beijing, China). The absorbance at 450 nm (OD450) was read on a microplate reader (Varioskan™ LUX, Thermo, USA).

After quantification using the Pierce™ BCA protein analysis kit (Thermo, USA), the total protein concentration of different cell lysates was adjusted to the same level, and then the expression level of total F protein in the cell lysates was analyzed using the standard Western ink dot method. In short, cell lysates containing 60 µg of total protein or 1 µg of recombinant proteins (DS-Cav1 and post-fusion) were loaded into each lane of an SDS-PAGE gel. After electrophoresis, the proteins in the gel were transferred to a PVDF membrane, followed by blocking with 5% skim milk emulsion in PBS-T for 1 h. After washing three times with PBS-T, the PVDF membrane was incubated at room temperature with rabbit anti-RSV-F protein IgG antibody diluted 1:1000 in PBS-T (which can identify non-conformity-specific total F protein (SinoBiological, Beijing, China)) for 2 h. After three vigorous washes, the PVDF membrane was detected at room temperature with goat anti-rabbit IgG H&L-HRP diluted 1:5000 in PBS-T (Abcam, Cambridge, UK) for 1 h. After three washes, the PVDF membrane was incubated with ECL receptor (ShareBio, Shanghai, China) for 1 min to generate a signal, which was then captured by ChemoDoc (BioRad, Hercules, CA, USA).

Mouse immunization was conducted at the Laboratory Animal Center of Shanghai Jiao Tong University. For the second round of selection, 60 SPF-grade female BALB/c mice (5-7 weeks old) were randomly assigned to 10 groups. According to the predetermined immunization schedule shown in Table 1, mice were immunized at 3-week intervals using either 10 μg of DS-Cav1 protein formulated with Alum Adju, or 2 μg or 10 μg of candidate mRNA vaccine in LNP formulation. For the third round of comparison, 30 SPF-grade female BALB/c mice (5-7 weeks old) were randomly assigned to 5 groups. According to the predetermined immunization schedule shown in Table 2, mice were immunized at 3-week intervals using either 1.0 μg or 5.0 μg of candidate mRNA vaccine in LNP formulation.

All procedures related to animal handling, care, and treatment in this study were performed in accordance with guidelines approved by the Society’s Animal Care and Use Committee. Each animal received intramuscular immunization with both vaccines on day 0 and day 21. Blood samples were collected on days 0, 21, 35, and 49 for the second round of selection experiments, and on days 0, 21, and 35 for the third round of selection control experiments. Serum preparation was performed as described below. Furthermore, spleens were harvested from all mice on day 49 (second round of selection experiments) or day 35 (third round of control experiments) to isolate spleen cells for T-cell response measurement.

Throughout the study, all animals were examined for their appearance and physical characteristics, behavior, posture, diet, fur, response to stimuli, glandular secretions, excretion, respiratory status, and mortality. Weight was also monitored weekly.

Serum preparation: Mouse blood samples were collected in Eppendorf tubes and kept on ice for 1 hour. After centrifugation at 1,500 g for 10 min at 4°C, the supernatant was immediately transferred to new tubes and stored at a temperature below -70°C.

ELISA is used to quantify antibody titers against RSV F protein. In short, 0.5 μg/mL of recombinant RSV F protein in its pre-fusion conformation (DS-Cav1) and 3 μg/mL of its post-fusion conformation (post-fusion) are pre-coated in 96-well clarified polystyrene microplates (MaxiSorp ELISA plate, NUNC, USA) at 4°C and incubated overnight. After washing three times with PBST, the pre-coated plates are blocked for 1 h at 37°C with 100 μL of ELISA blocking buffer (PBST + 2% BSA + 15% goat serum) per well. Serum samples were serially diluted 2-fold in blocking buffer, transferred to coated plates, and incubated at 37°C for 1 h. After washing, the plates were incubated at 37°C for 1 h with HRP-conjugated rabbit anti-mouse IgG (H+L) (Abcam, Cambridge, UK), goat anti-mouse IgG1 antibody (Jackson Immunoresearch, PA, USA), or goat anti-mouse IgG2a antibody (Jackson Immunoresearch, PA, USA) (for Pre-F) and biotinylated rabbit anti-mouse IgG (H+L) antibody (Abcam, Cambridge, UK) (for Post-F). All plates were washed three times with PBST, and plates used for Post-F were incubated at 37°C for 20 min with HRP-conjugated streptovir (A0303, Beyotime, Shanghai, China). All plates were incubated at 37°C with TMB single-component substrate solution (Solarbio Life Sciences, Beijing, China) for 7 min, and the reaction was terminated using ELISA termination solution (Solarbio Life Sciences, Beijing, China). The absorbance at 450 nm (OD450) was read using a microplate reader (Varioskan™ LUX, Thermo, USA). The endpoint titer was defined as the reciprocal of the endpoint dilution at which the serum sample's OD450 signal was greater than or equal to 2.1 times the background signal.

RSV virus transmission: RSV A long (ATCC VR-26) and RSV B 18537 (ATCC VR-1580) were transmitted to near-converged Hep-2 cells. Two × ^10⁶ Hep-2 cells were seeded in 10 cm DMEM dishes containing 10 mL of 10% FBS and incubated overnight at 37°C. On the second day, Hep-2 cells were infected with RSV at an MOI of 0.1–0.01. Five days after infection, the medium was collected and clarified by centrifugation (1,000 × g) for 10 min. The supernatant was mixed with PEG solution (10% final concentration) and incubated with stirring at 4°C for 2 h. Subsequently, RSV was precipitated by centrifugation at 4°C (3,250 × g) for 45 min. The RSV precipitate was then suspended in DMEM containing 3% sucrose, aliquoted, and stored in the gas phase of liquid nitrogen.

RSV plaque analysis measures the number of focal units (FFU) of transmitted RSV. In short, 2 × ^10⁴ cells/well of Vero-E6 cells were seeded in 96-well microplates and incubated overnight at 37°C in a humidified incubator with 5% _CO₂ . A 2-fold serially diluted RSV sample in PBS containing 5% FBS was then incubated for 1 h at 37°C. The Vero-E6 cell culture medium was then replaced with serum-free DMEM, followed by the addition of 100 μL of serially diluted RSV to infect the Vero-E6 cells. Two hours later, the medium was replaced with 150 μL of DMEM supplemented with 5% FBS, 1% methylcellulose, and 1% penicillin and streptomycin, and the cells were cultured for another 3 days. The medium was then removed, and the cells were fixed in 4% paraformaldehyde at room temperature for 60 min. The fixed cells were then washed three times with PBST and blocked with PBST containing 5% BSA. Viral plaques were stained at 37°C for 1 h with biotinylated anti-RSV-F antibody (catalog number: 11049-R302-B, SinoBiological, Beijing, China) diluted 1:10,000 in PBST/5% BSA. After washing three times with PBST, cells were incubated for 20 min at 37°C with HRP-conjugated streptavidin (A0303, Beyotime) diluted 1:40,000 in PBST/5% BSA. Unbound antibodies were removed by washing three times with PBST. Finally, plaques were visualized by incubating at room temperature with TrueBlue peroxidase receptor (KPL, Seracare) for 20 min. Plaques at different dilutions in each well were counted using an iSpot EliSpot FluoroSpot reader (AID, Strassberg, Germany).

Serum microneutralization analysis was performed similarly to plaque analysis. Briefly, 2 × ^10⁴ Vero cells/well were seeded into 96-well microplates and incubated overnight at 37°C in a humidified incubator with 5% _CO₂ . Mouse serum was heat-inactivated and serially diluted 3-fold. The diluted serum was mixed with RSV at a final concentration of 150 FFU/well and incubated at 37°C for 1 h. The serum-RSV mixture was added to each well of a 96-well microplate with Vero cell growth and the plates were incubated at 37°C for 2 h. The culture medium was then removed. After adding 150 µL of DMEM supplemented with 5% FBS, 1% methylcellulose, and 1% penicillin and streptomycin, the cells were cultured at 37°C for 3 days. The cells were then fixed and stained with biotinylated anti-RSV-F antibody, followed by HRP-conjugated streptomycin. Plaques were visualized by incubation with TrueBlue peroxidase and counted using an iSpot EliSpot FluoroSpot reader, as described in the RSV plaque analysis section above. The half-maximum neutralizing titer ( _NT50 ) was calculated by performing four-parameter curve fitting on the plaque count using GraphPad Prism 8.3.0 software.

Competitive ELISA analyzes serum samples by performing competitive binding assays of F protein with AM14 or 4D7 antibodies before or after fusion. The following combinations of F protein and antibody were tested: AM14/pre-fusion and 4D7/post-fusion. In short, 100 µL of 2 μg/mL recombinant DS-Cav1 protein (against AM14 antibody) diluted in ELISA coating buffer and 0.125 μg/mL recombinant Post-F protein (against 4D7 antibody) diluted in ELISA coating buffer (35 mM _NaHCO3 , 15 mM _Na2CO3 ) were pre-coated overnight in 96-well clarified polystyrene microplates at 4 _° C. After washing three times with PBST, the coated plates were blocked for 1 h at 37°C with 100 μL ELISA blocking buffer/well. Serum samples were serially diluted 3-fold in the blocking buffer, transferred to the coated plates (50 μL/well), and incubated at 37°C for 1 h. 10 μg/mL biotin-labeled antibodies (Biotin-AM14, Biotin-4D7) were also loaded into designated wells as 100% competitive controls. Wells without serum and antibodies were designated as 0% competitive controls. After washing, the plates were incubated at 37°C with 12.5 ng/mL biotin-AM14 or 6.25 ng/mL biotin-4D7 for 30 min. All plates were washed three times with PBST and then incubated at 37°C for 20 min with HRP-conjugated streptavidin (1:10,000 dilution, A0303, Beyotime). After washing, the plates were incubated at 37°C with TMB single-component acceptor solution (Solarbio Life Sciences, Beijing, China) for 7 min, and the reaction was terminated with ELISA termination solution (Solarbio Life Sciences, Beijing, China). The absorbance at 450 nm (OD450) was read on a microplate reader (Varioskan™ LUX, Thermo, USA). The percentage of competition was determined by converting the raw OD450 values to signals from 0% and 100% competitive control wells.

Splenic cell isolation was performed by isolating fresh spleens from immunized female SPF BALB/c mice or control mice. The spleen cells were gently crushed in 1 mL PBS in a culture dish using the plunger of a syringe through a 70-μm cell filter (BD Falcon, USA). The spleen cells were then collected in 15 mL cone-shaped centrifuge tubes. After washing, the red blood cells were lysed using erythrocyte lysate buffer according to the manufacturer's instructions (BasalMedia, Shanghai, China). After washing twice with PBS, the spleen cells were resuspended in 0.5 mL of RPMI-1640 medium supplemented with 10% FBS and 1× penicillin-streptomycin, counted, and cultured for intracellular cytokine staining.

Intracellular cytokine staining and flow cytometry analysis: Fresh spleen cells (2 × ^10⁶ cells in 200 μL of medium) were seeded into 96-well round-bottom microplates and restimulated with a 1.25 μg/mL pool of peptides spanning the full length of the RSV F protein (a pool of 139 15-meric peptides with 11 overlapping amino acids) and a 1.25 μg/mL pool of CD28 monoclonal antibody + CD49d monoclonal antibody. The plates were incubated at 37°C in a humidified incubator with 5% _CO₂ for 2 h, followed by overnight treatment with a protein transport inhibitor mixture.

Cells were washed with Dulbecco's phosphate-buffered saline (DPBS) and stained with LIVE/DEAD™ fixative light green dead cell stain for 30 min. Cells were then washed with 200 μL of fluorescently activated cell sorting (FACS) wash buffer and incubated with fluorescently labeled primary antibodies for 30 min to stain cell surface proteins. The antibodies contained anti-mouse CD3 APC-Vio 770 (pure REA641), anti-mouse CD4 VioBlue (pure REA604), and anti-mouse CD8 PerCP (pure REA601) (0.5 μL/well). Cells were then washed with FACS wash buffer and incubated for 30 min using fixation/permeabilization working solution (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Cells were then washed twice with permeabilization buffer and incubated for 30 min with fluorescently labeled primary antibodies to detect intracellular cytokine expression. The antibodies included anti-mouse IFN-γ FITC (pure REA638), anti-mouse TNF-α PE (pure REA636), and anti-mouse IL-2 APC (pure REA665) (0.5 μL/well). Finally, cells were washed with 200 μL of permeabilization buffer and then resuspended in 200 μL of PBS for flow cytometry analysis using a CytoFlex flow cytometer (Beckman, CA, USA). Data were analyzed using CytExpert software (Beckman, CA, USA).

Animal experiments on cotton-haired hamster immunization and RSV challenge were conducted by Sigmovir Biosystems, Inc. (Rockville, MD, USA). The study used a cotton-haired hamster model challenged with RSV/A2. Thirty-six (36) inbred female cotton-haired hamsters aged 6 to 8 weeks were maintained and treated under veterinary supervision in accordance with National Institutes of Health guidelines and the animal research protocol approved by the Sigmovir Committee on Animal Care and Use (IACUC Protocol #15). The hamsters were individually housed in transparent polycarbonate cages and provided with standard rodent food (Harlan #7004) and tap water.

Female cotton-fed rats were randomly assigned to 6 groups (Table 3) and immunized on day 0 with two different doses (5 µg and 16 µg) of candidate vaccines administered intramuscularly, followed by booster doses of the same vaccine 4 weeks later. Control groups consisted of animals infected with RSV/A2 on day 0, animals vaccinated with formalin-inactivated RSV (FI-RSV) vaccine on days 0 and 28 and boosted, or animals vaccinated with blank LNP mediator on days 0 and 28 as a control group. On day 49 (3 weeks after booster), animals were challenged with RSV/A2 at an inoculum dose of ^10⁵ plaque-forming units (pfu) per animal. All animals were euthanized on day 54 (5 days post-infection) to analyze viral load in the nose and lungs, lung histopathology, and quantification of serum neutralizing antibodies against RSV/A2.

Throughout the study, all animals were examined for their appearance and physical characteristics, behavior, posture, diet, fur, response to stimuli, glandular secretions, excretion, respiratory status, and mortality. Body weight was also monitored.

RSV lung and rhinovirus titration was performed by centrifugation to clarify homogenates of cotton rat lungs and noses and diluting them in Eagle's Minimum Essential Medium (EMEM). The diluted homogenates were used to infect two-well plates, forming Hep-2 monolayers. After incubation at 37°C in a 5% _CO2 incubator for one hour, the wells were covered with 0.75% methylcellulose medium. After four days of incubation, the covers were removed, and the cells were fixed with 0.1% crystal violet for one hour, followed by rinsing and air-drying. Plaques were counted, and viral titer was expressed as plaque-forming units per gram of tissue. Viral titer was calculated as the geometric mean plus standard error for all animals in the given time group.

RSV neutralizing antibody assay (60% PRNT) : Heat-inactivated cotton rat serum was diluted 1:10 with EMEM and further serially diluted 1:4. The diluted serum was incubated with RSV/A2 (25-50 pfu) at room temperature for 1 hour and seeded in duplicate onto a Hep-2 monolayer in 24-well plates. After incubation at 37°C in a 5% _CO2 incubator for 1 hour, the wells were covered with 0.75% methylcellulose medium. After 4 days of incubation, the covers were removed, and the cells were fixed and stained with 0.1% violet for 1 hour, then rinsed and air-dried. The statistical program " plqrd.manual.entry " was used to determine the corresponding reciprocal neutralizing antibody titer at the endpoint of a 60% reduction in the virus control. The geometric mean ± standard error was calculated for all animals in the given time group.

Lung histopathology was performed on the lungs of cotton-ear mice, which were inflated to their normal volume with 10% neutral buffered formalin and then immersed in the same fixative. After fixation, the lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Four parameters of pneumonia were assessed: peribronchial pneumonia (PB, peribronchial inflammatory cell infiltration), perivascular pneumonia (PV, perivascular inflammatory cell infiltration), interstitial pneumonia (IP, inflammatory cell infiltration and alveolar wall thickening), and alveolitis (A, cells within the alveolar cavities). Slides were blinded using a 0-4 severity scale. The scores were then converted to a 0-100% histopathological scale.

Statistical analysis measures statistical significance between groups using multiple t -tests or two-factor ANOVA and multiple comparison tests, as illustrated in the brief description of the graph. All statistical tests are performed using GraphPad Prism (version 8.3.0, GraphPad Software, Boston, MA).

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Sequence Amino acid sequence of wild-type RSV F glycoprotein (SEQ ID NO: 1) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKS LYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

Amino acid sequence of DS-Cav1 heavy tissue protein (SEQ ID NO: 2) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLHHHHHH

Amino acid sequence of the fused reorganized protein (SEQ ID NO: 3) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLS NGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHHHHHH

5' UTR RNA sequence ( SEQ ID NO:4) GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC

RNA sequence of 3' UTR (SEQ ID NO:5) GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA

5' UTR RNA sequence ( SEQ ID NO:6) GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (completely modified by N1-methylpseudouridine)

3' UTR RNA sequence ( SEQ ID NO:7) GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (completely modified by N1-methylpseudouridine)

Lead-2 mRNA ORF (SEQ ID NO:8)

Lead-2 F572A mRNA ORF (SEQ ID NO:9)

Lead-2 ΔCT mRNA ORF (SEQ ID NO:10)

Lead-5 mRNA ORF (SEQ ID NO:11)

Lead-6 / 6A mRNA ORF (SEQ ID NO:45) GC C GC UGCU GC CAGCAAC

Lead-6 variant mRNA ORF (SEQ ID NO:12)

Lead-7 mRNA ORF (SEQ ID NO:13)

MOD-1 mRNA ORF (SEQ ID NO:14)

The RNA sequence of the Kozak common sequence (SEQ ID NO:15) (gcc)gccRccAUGG

The amino acid sequence of the cytoplasmic tail region of hRSV F glycoprotein (SEQ ID NO:16) KARSTPVTLSKDQLSGINNIAFSN

The amino acid sequence of the transmembrane (TM) domain of RSV F glycoprotein (SEQ ID NO:24) IMITTIIIVIIVILLSLIAVGLLLYC

The amino acid sequence of the cytoplasmic tail region (CT) of RSV F glycoprotein (SEQ ID NO:25) KARSTPVTLSKDQLSGINNIAFSN

RNA sequence of the TM domain of RSV F glycoprotein (SEQ ID NO:26) AUUAUGAUCACCACCAUUAUUAUUGUGAUCAUCGUGAUCCUGCUGUCUCUGAUUGCUGUGGGCCUGCUGCUGUACUGC

RNA sequence of the CT domain of RSV F glycoprotein (SEQ ID NO:27) AAAGCCAGGUCCACCCCUGUGACACUGAGCAAGGACCAGCUGUCCGGCAUCAAUAACAUUGCUUUCAGCAAC. The mRNA sequences of all lead compounds can be combined with the listed 5'UTR and 3'UTR without N1-methylpseudoruridine. In some cases, to reduce innate immune responses against the mRNA molecule and improve mRNA expression, N1-methylpseudoruridine will be used instead of uridine in the mRNA molecule. Variations in RSV vaccine lead compounds:

5' UTR RNA sequence ( SEQ ID NO:4) GACAAUUACAAUACAAGCGAGCUAGACAAGCUCGCUAGACCGCCACC (completely modified by N1-methylpseudouridine)

3' UTR RNA sequence ( SEQ ID NO:5) GCACCAGCACCAAAGAUCACGGAGCACCUACAACCAUUGCAUGCACCUGCAGAAAUGCUCCGGAGCUGACAGCUUGUGACAAAUAAAGUUCAUUCAGUGACACUCA (completely modified by N1-methylpseudouridine)

Lead-2 mRNA ORF (SEQ ID NO:8) AUGGAGCUGCUGAUCCUCAAGGCCAACGCCAUCACCACCAUCCUGACCGCCGUGACCUUCUGCUUCGCCAGCGGGCAGAACAUCACCGAGGAGUUCUACCAGAGCACCUGCAGUGCCGUGAGCAAGGGCUACCUGGGCGCCCUCAGGACAGGCUGGUACACCUCCGUGAUCACCAUCGAGCUCUCCAACAUCAAGGAGAA CAAGUGCAACGGGACCGACGCCAAGGUGAAGCUGAUCAAGCAGGAGCUGGACAAGUACAAGAAUGCCGUGACCGACCUGCAGCUGCUGAUGCAGAGCACCCCUGCCACCGGCUCCGGCUCCGCCAUUUGCAGCGGGGUGGCCGUGUGCAAGGUGCUCCACCUGGAGGGCGAGGUCAACAAGAUCAAGUCCGCCCUGCUCAG CACCAACAAGGCCGUGGUCUCCCUGAGCAACGGCGUCUCCGUGCUGACCUUCAAGGUCCUGGACCUGAAGAACUACAUCGACAAGCAGCUCCUGCCCAUCCUGAACAAGCAGUCCUGCAGCAUCCCCAACAUCGAGACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUCCUGGAGAUCACCCGGGAGUUCUCCGUGA ACGCCGGGGUGACCACCCCUGUGAGCACCUACAUGCCGACCAACAGCGAGCUGCUCUCCCUGAUCAACGACAUGCCCAUCACCAAUGAUCAGAAGAAGCUGAUGUCCAAUAAUGUGCAGAUCGUGCGACAGCAGAGCUACAGCAUCAUGUGCAUCAUCAAGGAAGAGGUGCUGGCCUACGUGGUGCAGCUGCCUCUGUAUG GCGUGAUCGACACACCCUGCUGGAAGCUGCAUACCUCACCCCUGUGCACAACAAACACAAAGGAGGGGAGCAAUAUUUGUCUGACAAGGACAGAUAGAGGCUGGUACUGAUAACGCAGGCAGCGUGUCCUUCUUCCCACAGGCAGAGACCUGCAAAGUGCAGUCAAACAGGGUGUUUUGUGACACAAUGAACAGCCUG ACUCUGCCCAGUGAGAUCAACCUGUGCAAUGUGGAUAUCUUUAAUCCAAAGUACGACUGUAAGAUCAUGACUUCCAAAACCGACGUGUCUUCCUCAGUGAUCACCAGCCUGGGCGCCAUCGUGAGCUGCUAUGGCAAAACCAAGUGUACUGCCAGCAAUAAGAACAGAGGGAUCAUCAAAACCUUCUCAAACGGCUGCGAC UAUGUGUCCAAUAAAGGCAUGGACACAGUGUCUGUGGGGAAUACCCUGUAUUGCGUGAAUAAGCAGGAAGGCCAGUCUCUGUACGUGAAAGGAGAGCCCAUCAUCAACUUCUAUGACCCACUGGUGUUUCCAUCUGAUGAGUUUGAUGCCAGCAUCUCACAGGUGAAUGAGAAAAUUAAUCAGAGCCUGGCUUUCAUCAGGAAGUCUGACGAGCUGCUGCAUAACGUGAAUGCUGGCAAAAGCACCACAAAUAUUAUGAUCACCACCAUUAUUAUUGUGAUCAUCGUGAUCCUGCUGUCUCUGAUUGCUGUGGGCCUGCUGCUGUACUGCAAAGCCAGGUCCACCCCUGUGACACUGAGCAAGGACCAGCUGUCCGGCAUCAAUAACAUUGCUUUCAGCAAC (Completely modified by N1-methylpseudouridine)

Lead-5 mRNA ORF (SEQ ID NO:11) AUGGAGCUGCUGAUCCUCAAGGCCAACGCCAUCACCACCAUCCUGACCGCCGUGACCUUCUGCUUCGCCAGCGGGCAGAACAUCACCGAGGAGUUCUACCAGAGCACCUGCAGUGCCGUGAGCAAGGGCUACCUGGGCGCCCUCAGGACAGGCUGGUACACCUCCGUGAUCACCAUCGAGCUCUCCAACAUCAAGGAGAA CAAGUGCAACGGGACCGACGCCAAGGUGAAGCUGAUCAAGCAGGAGCUGGACAAGUACAAGAAUGCCGUGACCGACCUGCAGCUGCUGAUGCAGAGCACCCCUGCCACCGGCUCCGGCUCCGCCAUUUGCAGCGGGGUGGCCGUGUGCAAGGUGCUCCACCUGGAGGGCGAGGUCAACAAGAUCAAGUCCGCCCUGCUCAG CACCAACAAGGCCGUGGUCUCCCUGAGCAACGGCGUCUCCGUGCUGACCUUCAAGGUCCUGGACCUGAAGAACUACAUCGACAAGCAGCUCCUGCCCAUCCUGAACAAGCAGUCCUGCAGCAUCCCCAACAUCGAGACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUCCUGGAGAUCACCCGGGAGUUCUCCGUGA ACGCCGGGGUGACCACCCCUGUGAGCACCUACAUGCCGACCAACAGCGAGCUGCUCUCCCUGAUCAACGACAUGCCCAUCACCAAUGAUCAGAAGAAGCUGAUGUCCAAUAAUGUGCAGAUCGUGCGACAGCAGAGCUACAGCAUCAUGUGCAUCAUCAAGGAAGAGGUGCUGGCCUACGUGGUGCAGCUGCCUCUGUAUG GCGUGAUCGACACACCCUGCUGGAAGCUGCAUACCUCACCCCUGUGCACAACAAACACAAAGGAGGGGAGCAAUAUUUGUCUGACAAGGACAGAUAGAGGCUGGUACUGAUAACGCAGGCAGCGUGUCCUUCUUCCCACAGGCAGAGACCUGCAAAGUGCAGUCAAACAGGGUGUUUUGUGACACAAUGAACAGCAGG ACUCUGCCCAGUGAGGUGAACCUGUGCAAUGUGGAUAUCUUUAAUCCAAAGUACGACUGUAAGAUCAUGACUUCCAAAACCGACGUGUCUUCCUCAGUGAUCACCAGCCUGGGCGCCAUCGUGAGCUGCUAUGGCAAAACCAAGUGUACUGCCAGCAAUAAGAACAGAGGGAUCAUCAAAACCUUCUCAAACGGCUGCGAC UAUGUGUCCAAUAAAGGCGUGGACACAGUGUCUGUGGGGAAUACCCUGUAUUGCGUGAAUAAGCAGGAAGGCCAGUCUCUGUACGUGAAAGGAGAGCCCAUCAUCAACUUCUAUGACCCACUGGUGUUUCCAUCUGAUGAGUUUGAUGCCAGCAUCUCACAGGUGAAUGAGAAAAUUAAUCAGAGCCUGGCUUUCAUCAGGAAGUCUGACGAGCUGCUGCAUAACGUGAAUGCUGGCAAAAGCACCACAAAUAUUAUGAUCACCACCAUUAUUAUUGUGAUCAUCGUGAUCCUGCUGUCUCUGAUUGCUGUGGGCCUGCUGCUGUACUGCAAAGCCAGGUCCACCCCUGUGACACUGAGCAAGGACCAGCUGUCCGGCAUCAAUAACAUUGCUUUCAGCAAC (Completely modified with N1-methylpseudouridine)

Lead-6/6A mRNA ORF (SEQ ID NO:45) GC C GC UGCU GC CAGCAAC (completely modified with N1-methylpseudouridine)

Lead-6 variant mRNA ORF (SEQ ID NO:12) AUGGAGCUGCUGAUCCUCAAGGCCAACGCCAUCACCACCAUCCUGACCGCCGUGACCUUCUGCUUCGCCAGCGGGCAGAACAUCACCGAGGAGUUCUACCAGAGCACCUGCAGUGCCGUGAGCAAGGGCUACCUGGGCGCCCUCAGGACAGGCUGGUACACCUCCGUGAUCACCAUCGAGCUCUCCAACAUCAAGGAGAACAAGUGCAACGG GACCGACGCCAAGGUGAAGCUGAUCAAGCAGGAGCUGGACAAGUACAAGAAUGCCGUGACCGACCUGCAGCUGCUGAUGCAGAGCACCCCUGCCACCGGCUCCGGCUCCGCCAUUUGCAGCGGGGUGGCCGUGUGCAAGGUGCUCCACCUGGAGGGCGAGGUCAACAAGAUCAAGUCCGCCCUGCUCAGCACCAACAAGGCCGUGGUCUCCC UGAGCAACGGCGUCUCCGUGCUGACCUUCAAGGUCCUGGACCUGAAGAACUACAUCGACAAGCAGCUCCUGCCCAUCCUGAACAAGCAGUCCUGCAGCAUCCCCAACAUCGAGACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUCCUGGAGAUCACCCGGGAGUUCUCCGUGAACGCCGGGGUGACCACCCCUGUGAGCACCUACAUG CUGACCAACAGCGAGCUGCUCUCCCUGAUCAACGACAUGCCCAUCACCAAUGAUCAGAAGAAGCUGAUGUCCAAUAAUGUGCAGAUCGUGCGACAGCAGAGCUACAGCAUCAUGUGCAUCAUCAAGGAAGAGGUGCUGGCCUACGUGGUGCAGCUGCCUCUGUAUGGCGUGAUCGACACACCCUGCUGGAAGCUGCAUACCUCACCCCUGUGC ACAACAAACACAAAGGAGGGGAGCAAUAUUUGUCUGACAAGGACAGAUAGAGGCUGGUACUGUGAUAACGCAGGCAGCGUGUCCUUCUUCCCACAGGCAGAGACCUGCAAAGUGCAGUCAAACAGGGUGUUUUGUGACACAAUGAACAGCAGGACUCUGCCCAGUGAGGUGAACCUGUGCAAUGUGGAUAUCUUUAAUCCAAAGUACGACUG UAAGAUCAUGACUUCCAAAACCGACGUGUCUUCCUCAGUGAUCACCAGCCUGGGCGCCAUCGUGAGCUGCUAUGGCAAAACCAAGUGUACUGCCAGCAAUAAGAACAGAGGGAUCAUCAAAACCUUCUCAAACGGCUGCGACUAUGUGUCCAAUAAAGGCGUGGACAGUGUCUGUGGGGAAUACCCUGUAUUGCGUGAAUAAGCAGGAAG GCCAGUCUCUGUACGUGAAAGGAGAGCCCAUCAUCAACUUCUAUGACCCACUGGUGUUUCCAUCUGAUGAGUUUGAUGCCAGCAUCUCACAGGUGAAUGAGAAAAUUAAUCAGAGCCUGGCUUUCAUCAGGAAGUCUGACGAGCUGCUGAGCGCCAUCGGCGGCUACAUCCCCGAGGCCCCUAGAGACGGCCAAGCCUACGUGAGAAAAGACGGGGAGUGGGUGCUGCUGUCCACAUUCCUGCAUAACGUGAAUGCUGGCAAAAGCACCACAAAUAUUAUGAUCACCACCAUUAUUAUUGUGAUCAUCGUGAUCCUGCUGUCUCUGAUUGCUGUGGGCCUGCUGCUGUACUGCAAAGCCAGGUCCACCCCUGUGACACUGAGCAAGGACCAGCUGUCCGGCAUCAAUAACAUUGCUUUCAGCAAC (Completely modified with N1-methylpseudouridine)

Lead-7 mRNA ORF (SEQ ID NO:13) AUGGAGCUGCUGAUCCUCAAGGCCAACGCCAUCACCACCAUCCUGACCGCCGUGACCUUCUGCUUCGCCAGCGGGCAGAACAUCACCGAGGAGUUCUACCAGAGCACCUGCAGUGCCGUGAGCAAGGGCUACCUGGGCGCCCUCAGGACAGGCUGGUACACCUCCGUGAUCACCAUCGAGCUCUCCAACAUCAAGGAGAACAAGUGCAACGGGAC CGACGCCAAGGUGAAGCUGAUCAAGCAGGAGCUGGACAAGUACAAGAAUGCCGUGACCGACCUGCAGCUGCUGAUGCAGAGCACCCCUGCCACCAACAACCGGGCUCGGCGCGAGCUGCCCCGCUUCAUGAACUACCUGAACAAUGCCAAGAAGACCAAUGUGACCCUCUCCAAGAAGCGGAAGCGCAGGUUUCUGGGCUUCCUCCUGGGCU CCGGCUCCGCCAUUUGCAGCGGGGUGGCUGUGCAAGGUGCUCCACCUGGAGGGCGAGGUCAACAAGAUCAAGUCCGCCCUGCUCAGCACCAACAAGGCCGUGGUCUCCCUGAGCAACGGCGUCCGUGCUGACCUUCAAGGUCCUGGACCUGAAGAACUACAUCGACAAGCAGCUCCUGCCCAUCCUGAACAAGCAGUCCUGCAGCAUCCCC AACAUCGAGACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUCCUGGAGAUCACCCGGGAGUUCUCCGUGAACGCCGGGGUGACCACCCCUGUGAGCACCUACAUGCUGACCAACAGCGAGCUGCUCUCCCUGAUCAACGACAUGCCCAUCACCAAUGAUCAGAAGAAGCUGAUGUCCAAUAAUGUGCAGAUCGUGCGACAGCAGAGCUACAGC AUCAUGUGCAUCAUCAAGGAAGAGGUGCUGGCCUACGUGGUGCAGCUGCCUCUGUGUAUGGCGUGAUCGACACACCCUGCUGGAAGCUGCAUACCUCACCCCUGUGCACAACAAACACAAAGGAGGGGAGCAAUAUUUGUCUGACAAGGACAGAUAGAGGCUGGUACUGUGAUAACGCAGGCAGCGUGUCCUUCUUCCCACAGGCAGAGACCUGCAA AGUGCAGUCAAACAGGGUGUUUUGUGACACAAUGAACAGCAGGACUCUGCCCAGUGAGGUGAACCUGUGCAAUGUGGAUAUCUUUAAUCCAAAGUACGACUGUAAGAUCAUGACUUCCAAAACCGACGUGUCUUCCUCAGUGAUCACCAGCCUGGGCGCCAUCGUGAGCUGCUAUGGCAAAACCAAGUGUACUGCCAGCAAUAAGAACAGAGGGA UCAUCAAAACCUUCUCAAACGGCUGCGACUAUGUGUCCAAUAAAGGCGUGGACACAGUGUCUGUGGGGAAUACCCUGUAUUGCGUGAAUAAGCAGGAAGGCCAGUCUCUGUACGUGAAAGGAGAGCCCAUCAUCAACUUCUAUGACCCACUGGUGUUUCCAUCUGAUGAGUUUGAUGCCAGCAUCUCACAGGUGAAUGAGAAAAUUAAUCAGAGCCUGGCUUUCAUCAGGAAGUCUGACGAGCUGCUGCAUAACGUGAAUGCUGGCAAAAGCACCACAAAUAUUAUGAUCACCACCAUUAUUAUUGUGAUCAUCGUGAUCCUGCUGUCUCUGAUUGCUGUGGGCCUGCUGCUGUACUGCAAAGCCAGGUCCACCCCUGUGACACUGAGCAAGGACCAGCUGUCCGGCAUCAAUAACAUUGCUUUCAGCAAC

The corresponding DNA sequence of Lead-2 mRNA ORF (SEQ ID NO:29)

The corresponding DNA sequence of Lead-2 F572A mRNA ORF (SEQ ID NO:30)

The corresponding DNA sequence of Lead-2 ΔCT mRNA ORF (SEQ ID NO:31)

The corresponding DNA sequence of Lead-5 mRNA ORF (SEQ ID NO:32)

The corresponding DNA sequence of the Lead-6/6A mRNA ORF (SEQ ID NO:47)

The corresponding DNA sequence of the Lead-6 variant mRNA ORF (SEQ ID NO:33)

The corresponding DNA sequence of Lead-7 mRNA ORF (SEQ ID NO:34)

5'- UTR, Chi β-G WT (SEQ ID NO:35) ACCAGCGUGCUAUCCCCACGGGAGCAAGAGCCCAGACCUCCUCCGUACCGACAGCCACACGCUACCCUCCAACCGCCGCC

5'- U TR, Chi β-G Δ1 (SEQ ID NO:36) GACAAUUACAAUAGUCAACGAGCUAGACAAGCUACGUCGACAGACCGCCACC

5'-UTR, Chi β-G Δ6 (SEQ ID NO:37) GACAAUUACAAUAGUCACGAGCUAGACAAGCUACGUCGACAGACCGCCACC

5'- UTR, Chi β-G Δ7 (SEQ ID NO:38) GACAAUUAAGAAUCAAGCGAGCUAGACAAGCUCGCUACGUCAACCGCCACC

The corresponding DNA sequence of 5'-UTR, Chi β - G Δ4 (SEQ ID NO:39) is : GACAATTACAATACAAGCGAGCTAGACAAGCTCGCTAGACCGCCACC

5'-UTR, corresponding DNA sequence of Chi β -G WT (SEQ ID NO:40) ACCAGCGTGCTATCCCCACGGGAGCAAGAGCCCAGACCTCCTCCGTACCGACAGCCACACGCTACCCTCCAACCGCCGCC

The corresponding DNA sequence of 5'-UTR, Chi β -G Δ1 (SEQ ID NO:41) is : GACAATTACAATAGTCAACGAGCTAGACAAGCTACGTCGACAGACCGCCACC

The corresponding DNA sequence of 5'-UTR, Chi β -G Δ6 (SEQ ID NO:42) is : GACAATTACAATAGTCACGAGCTAGACAAGCTACGTCGACAGACCGCCACC

The corresponding DNA sequence of 5'-UTR, Chi β -G Δ7 (SEQ ID NO:43) is : GACAATTAAGAATCAAGCGAGCTAGACAAGCTCGCTACGTCAACCGCCACC

Corresponding DNA sequence of 3' UTR (SEQ ID NO:44) GCACCAGCACCAAAGATCACGGAGCACCTACAACCATTGCATGCACCTGCAGAAATGCTCCGGAGCTGACAGCTTGTGACAAATAAAGTTCATTCAGTGACACTCA

Lead-6A amino acid sequence (SEQ ID NO:46) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKN YIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETC KVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFY DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN AA A A SN.

Figure 1 shows a schematic diagram of RSV F glycoprotein variants (encoded by mRNA Lead-2, Lead-2 F572A and Lead-2 ΔCT), wild-type RSV F glycoprotein, and quasi-DS-Cav1-T4 fold reorganized protein.

Figure 2 shows the in vivo cellular expression levels of mRNA Lead-2, Lead-2 F572A, and Lead-2 ΔCT in A549 cells, measured by Western blot at 24 and 48 hours after transfection. β-actin was used as a loading control. The control group represents untransfected blank A549 cells.

Figure 3 shows the in vivo cellular expression levels of pre-F and post-F mRNA Lead-2, Lead-2 F572A, and Lead-2 ΔCT proteins in A549 cells, measured by ELISA at 24 and 48 hours after transfection. Whole cell lysates were used for ELISA measurements using D25 (human IgG1 monoclonal antibody specifically recognizing site Φ of the pre-F glycoprotein) and 4D7 (mouse IgG2a monoclonal antibody specifically recognizing site I of the post-F glycoprotein). The control group represents untransfected blank A549 cells.

Figures 4A-4B show the in vivo cell surface expression levels of mRNA Lead-2, Lead-2 F572A, and Lead-2 ΔCT in A549 cells (A) or 293F cells (B) measured by flow cytometry 24 hours after transfection. The normalized MFI of pre-F is shown in the figure below.

Figure 5 shows a schematic diagram of RSV F glycoprotein variants (encoded by mRNA Lead-2, Lead-5, Lead-6, Lead-7 and MOD-1), wild-type RSV F glycoprotein, and quasi-DS-Cav1-T4 folded reorganized protein.

Figure 6 shows the in vivo cellular expression levels of mRNAs with different characteristics in A549 cells, measured by Western ink dot assay 24 hours after transfection. β-actin was used as a loading control. The control group represents untransfected blank A549 cells.

Figure 7 shows the in vivo cellular expression levels of pre-F protein, pre-F protein trimer, and post-F protein with different mRNA characteristics in A549 cells, measured by ELISA 24 hours after transfection. Whole cell lysate was used for ELISA measurement. D25, human IgG1 monoclonal antibody specifically recognizing site Φ of pre-F glycoprotein. AM14, human IgG1 monoclonal antibody specifically recognizing pre-F glycoprotein trimer. 4D7, mouse IgG2a monoclonal antibody specifically recognizing site I of post-F glycoprotein. N.D., not detected. The control group represents untransfected blank A549 cells.

Figures 8A-8B show the in vivo cell surface expression levels of pre-F protein, pre-F protein trimer, and post-F protein with different mRNA characteristics in A549 cells, measured by flow cytometry 24 hours after transfection. The relative MFI of pre-F, pre-F trimer, and post-F, as well as the pre-F/post-F relative MFI ratio and the pre-F/post-F relative MFI ratio of the trimer, are shown in Figure 8B. The control group represents untransfected blank A549 cells.

Figure 9 shows the anti-pre-F and anti-post-F antibody titers in BALB/c mice serum on day 49 after administration of mRNA vaccines with different characteristics. Each dot represents a mouse serum sample. The height of the bar in the graph indicates the geometric mean titer ± 95% CI.

Figure 10 shows the RSV neutralizing antibody titers against RSV A (A2 substrain) and RSV B (18537 substrain) in BALB/c mice serum on day 49 after administration of mRNA vaccines with different characteristics. The half-maximum neutralizing titer ( _NT50 ) was calculated using a four-parameter curve fitting of plaque counts with GraphPad Prism 8.3.0 software. Each point represents one mouse serum sample. The height of the bar in the graph indicates the geometric mean titer ± 95% CI. p-values compared to the DS-Cav1 proteome were calculated using multiple t -tests.

Figure 11 shows the titers of anti-pre-F IgG subclasses (IgG2a and IgG1) on day 49 after administration of mRNA vaccines with different characteristics to BALB/c mice. Each dot represents a mouse serum sample. The height of the bar in the graph indicates the geometric mean titer ± 95% CI.

Figure 12 shows the antibody competitive ELISA of BALB/c mice serum on day 49 after administration of mRNA vaccines with different characteristics. The presence of AM14 or 4D7 competitive antibodies in serum obtained 4 weeks after the second dose was analyzed. 10 μg/mL biotin-conjugated AM14 or 4D7 antibodies were also added to the wells as a 100% competitive condition. Wells containing neither serum nor antibodies were set as a 0% competitive condition. The OD450 values of the mRNA vaccine group were normalized to the OD450 values of the 100% competitive group. The percentages of AM14 and 4D7 competition were compared with the DS-Cav1 proteome using a multiple t -test with Graph Pad Prism 8.3.0 software. Each point represents a mouse serum sample.

Figures 13A-13B show the T cell response in mouse spleen cells obtained on day 49 after administration of mRNA vaccines with different characteristics to BALB/c mice. The proportions of CD4+ T cells (A) and CD8+ T cells (B) producing IFN-γ (▼), IL-2 (●), TNF-α (■), or IFN ^- γ plus TNF-α (▲) after RSV ^F peptide stimulation are shown. Each dot represents a mouse spleen sample. The height of the bars in the graph indicates the geometric mean ± 95% CI. Compared with the DS-Cav1 proteome by two-way ANOVA and Dunnett's multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0001.

Figures 14A-14B show the in vivo cell surface expression levels of pre-F protein, pre-F protein trimer, and post-F protein, representing mRNAs with different characteristics, in A549 cells as measured by flow cytometry at 24, 48, and 72 hours after transfection. The percentages of pre-F positive (D25), pre-F trimer positive (AM14), and post-F positive (4D7) cells are shown in Figure 14A. The relative MFI kinetics (MFI normalized to each mRNA at 24 hours) of pre-F, pre-F trimer, and post-F are shown in Figure 14B. The control group represents untransfected blank A549 cells.

Figure 15 shows the anti-pre-F and anti-post-F antibody titers in BALB/c mice serum on day 35 after administration of mRNA vaccines with different characteristics. Each dot represents a mouse serum sample. The height of the bar in the graph indicates the geometric mean titer ± 95% CI.

Figure 16 shows the RSV neutralizing antibody titers against RSV A (A2 substrain) and RSV B (18537 substrain) in BALB/c mice serum on day 35 after administration of mRNA vaccines with different characteristics. The median neutralizing titer ( _NT50 ) was calculated using four-parameter curve fitting of plaque counts with GraphPad Prism 8.3.0 software. The height of the bar in the graph indicates the geometric mean titer ± 95% CI. p-values were calculated using multiple t -tests.

Figure 17 shows the titers of anti-pre-F IgG subclasses (IgG2a and IgG1) on day 35 after administration of mRNA vaccines with different characteristics to BALB/c mice. Each dot represents a mouse serum sample. The height of the bar in the graph indicates the geometric mean titer ± 95% CI.

Figures 18A-18B show the T cell response in mouse spleen cells obtained on day 35 after administration of mRNA vaccines with different characteristics to BALB/c mice. The proportions of CD4+ T cells (A) and CD8+ T cells (B) producing IFN-γ (▼), IL-2 (●), TNF-α (■), or IFN-γ ^plus TNF-α (▲) after RSV ^F peptide stimulation are shown. Each dot represents a mouse spleen sample. The height of the bars in the graphs indicates the geometric mean ± 95% CI. Comparisons between Lead-6 and MOD-1 were performed using two-way ANOVA and Tukey's multiple contrast assays; *p < 0.05, **p < 0.01.

Figure 19 illustrates the structure of mRNA. A typical mRNA structure includes: a 5' cap, a 5' untranslated region (5' UTR), a target gene, a 3' untranslated region, and a multi-A tail.

Figure 20 illustrates the in vivo study program. Each mouse received 2 µg of mRNA via intramuscular injection. Serum was collected on days 7, 14, and 21.

Figure 21 shows the antibody titer in mice.

Figure 22 shows the relative fold increase in mouse antibody titer. Normalization of mouse antibody titer was achieved using Chi β-G (WT) antibody titer on day 7.

Figures 23A-23B illustrate the protection provided by the Lead-6A mRNA RSV vaccine against RSV/A2 challenge in cotton-fed mice. Raw cotton-fed mice were immunized twice (days 0 and 28) with either 5 μg Lead-6A vaccine (Group 1) or 16 μg (Group 2) mRNA/dose, or twice with formalin-inactivated RSV (FI-RSV) vaccine batch 100 (Group 3), or once with ^10⁵ pfu/animal RSV/A2 (batch 092215 SSM) (Group 4) (day 0). Two control groups (Groups 5 and 6) included unvaccinated cotton-fed mice. On day 49, all animals (except one of the unvaccinated groups (Group 6)) were challenged with ^10⁵ pfu/animal RSV/A2 (batch 092215 SSM). RSV viral load was determined by plaque analysis of homogenates of lung tissue (Fig. 23A) or nasal tissue (Fig. 23B) isolated 5 days after challenge and expressed as plaque-forming units (pfu)/gram of tissue Log _10. For all groups, viral titer was calculated as geometric mean ± standard error (SE).

Figure 24 illustrates the induction of neutralizing antibodies against RSV A2 by the Lead-6A mRNA RSV vaccine in cotton-fed mice. Raw cotton-fed mice were immunized twice (days 0 and 28) with either 5 μg Lead-6A vaccine (Group 1) or 16 μg (Group 2) mRNA/dose; or twice with formalin-inactivated RSV (FI-RSV) vaccine batch 100 (Group 3); or once with ^10⁵ pfu/animal RSV/A2 (batch 092215 SSM) (Group 4) (day 0). Two control groups included unvaccinated cotton-fed mice (Groups 5 and 6). Serum samples collected 4 weeks after the first immunization and 3 weeks after the second immunization (days 28 and 49) were used to determine the neutralizing antibody titers of strain-matched RSV/A2 using a 60% plaque reduction neutralization titer assay (60% PRNT). The geometric mean ± standard error (SE) of all groups at the given time points was calculated and expressed as _Log² titer.

Figure 25 shows the lung histopathological scores of cotton-fed rats. Original cotton-fed rats were immunized twice (days 0 and 28) with either 5 μg Lead-6A vaccine (Group 1) or 16 μg (Group 2) mRNA/dose, or twice with formalin-inactivated RSV (FI-RSV) vaccine batch 100 (Group 3), or once with ^10⁵ pfu/animal RSV/A2 (batch 092215 SSM) (Group 4) (day 0). Two control groups included unvaccinated cotton-fed rats (Groups 5 and 6). All animals (except one in the unvaccinated group (Group 6)) were challenged on day 49 with ^10⁵ pfu/animal RSV/A2 (batch 092215 SSM) and sacrificed on day 54 (5 days post-infection). Lung histopathology was performed and scored on all animals, as described in the endpoint analysis section. Mean scores ± standard error (SE) for peribronchial inflammation (PB), perivascular inflammation (PV), interstitial pneumonia (IP), and alveolitis (A) are presented for all groups.

Claims (52)

A modified polynucleotide (e.g., ribonucleotide or RNA) encoding a stable pre-fusion form of an immunogenic RSV F glycoprotein, wherein the modified polynucleotide comprises: (a) a first polynucleotide encoding a transmembrane (TM) domain of the RSV F glycoprotein; and (b) a second polynucleotide encoding a cytoplasmic tail (CT) region of the RSV F glycoprotein. The modified polynucleotide of claim 1 further includes a third polynucleotide encoding an extracellular (EC) domain. The modified polynucleotide of claim 1 or 2 further includes a fourth polynucleotide encoding the trimeric domain of T4 cellulose. The modified polynucleotide of claim 3, wherein the T4 cellulose is located between the EC domain and the TM domain. The modified polynucleotide of any of claims 1 to 4, wherein the RSV F glycoprotein lacks the p27-fusion protein (p27-FP) domain; or wherein the RSV F glycoprotein replaces the p27-FP domain with a linker (e.g., a GS linker). The modified polynucleotide of any of claims 1 to 5, wherein, relative to SEQ ID NO:1, the RSV F glycoprotein comprises one or more amino acid substitutions selected from the group consisting of: S46X (e.g., S46G), E92X (e.g., E92D), P102X (e.g., P102A), S215X (e.g., S215P), I379X (e.g., I379V), L373X (e.g., L373R), M447X (e.g., M447V), and K465X (e.g., K465Q), wherein X is any amino acid other than the original amino acid. The modified polynucleotide of any one of claims 1 to 6, wherein, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of A149C and Y458C. The modified polynucleotide of any of claims 1 to 7, wherein, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of S155C and S290C. The modified polynucleotide of any of claims 1 to 8, wherein, relative to SEQ ID NO:1, the RSV F glycoprotein includes substitutions of one or more amino acids selected from the group consisting of: S190X (e.g., S190F) and V207X (e.g., V207L). The modified polynucleotide of any one of claims 1 to 9, wherein, relative to SEQ ID NO:1, the RSV F glycoprotein includes an amino acid substitution F572A. The modified polynucleotide of any of claims 1 to 10, wherein the RSV F glycoprotein includes a truncated CT (e.g., composed essentially of a peptide sequence of KAR/a truncated CT composed of it). The modified polynucleotide of claim 11, wherein the truncated CT includes amino acid positions 4-24 of SEQ ID NO:16. The modified polynucleotide of any one of claims 1 to 12, wherein the modified polynucleotide is an RNA comprising a multi(A) tail, the multi(A) tail being 50 to 150 nucleotides in length, as appropriate. The modified polynucleotide of any of claims 1 to 13 comprises a nucleic acid sequence that is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6% or 99.8% identical to SEQ ID NO: 8, 9, 10, 11, 12, 13, 29, 32, 33 or 34. The modified polynucleotide of claim 14 includes the nucleic acid sequence of SEQ ID NO:45. The modified polynucleotide of any one of claims 1 to 15, wherein the RSV F glycoprotein is at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6% or 99.8% identical to SEQ ID NO:17, 18, 19, 20, 21, 22 or fragments thereof (e.g. SEQ ID NO:17, 20, 21 or 22). The modified polynucleotide of claim 16, wherein the RSV F glycoprotein includes the amino acid sequence of SEQ ID NO:46. The modified polynucleotide of any one of claims 1 to 17, wherein the RSV F glycoprotein is at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6% or 99.8% identical to SEQ ID NO:1 or a fragment thereof. The modified polynucleotide of any of claims 1 to 18 further includes a 5' untranslated region (UTR) containing the nucleic acid sequence of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42 or 43. The modified polynucleotide of any of claims 1 to 19 further includes a 3’ UTR containing the nucleic acid sequence of SEQ ID NO:5 or 44. The modified polynucleotide of any of claims 1 to 20 further includes a 5' cap (e.g., cap-0 or ^m7 G(5')ppp(5')N1 _m pNp cap). The modified polynucleotide of any of claims 1 to 21 further includes chemical modification. Such as the modified polynucleotide of claim 22, wherein the chemical modification is the substitution of uridine in the polynucleotide by N1-methylpseudouridine; where appropriate, all or substantially all of the uridine in the polynucleotide is substituted by N1-methylpseudouridine. A polypeptide encoded by a modified polynucleotide as described in any of claims 1 to 23. A pharmaceutical composition comprising a modified polynucleotide as claimed in any of claims 1 to 24. The pharmaceutical composition of claim 25 includes the modified polynucleotide formulated in lipid nanoparticles (LNPs). The pharmaceutical composition of claim 26, wherein the LNP comprises a mixture of lipids containing the following lipids: (1) about 20%-60%, about 30%-50% or about 40% (moles percentage) of ionizable cationic lipids; (2) about 30%-70% (e.g., about 40%-60% or about 50%) (moles percentage) of sterol lipids; (3) about 5%-30% (e.g., about 5%-15% or about 10%) (moles percentage) of phospholipids; or (4) about 0%-5% (e.g., about 1%-3% or about 2%) (moles percentage) of occult lipids or PEG-modified lipids. The pharmaceutical composition of claim 27, wherein the lipid mixture comprises an ionizable cationic lipid, cholesterol, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), and 1,2-dimyristyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), wherein the ionizable cationic lipid is selected from the group consisting of lipids 2, 4, 5, and 8 having the following structures: Lipid 2 Lipid No. 4 Lipid No. 5 and lipid No. 8 . For example, in the pharmaceutical composition of claim 26, the molar ratio of ionizable cationic lipids, cholesterol, DSPC and DMG-PEG2000 is about 40: about 48: about 10: about 2. For example, the pharmaceutical composition of any of claims 26 to 29, wherein the average diameter of the LNP is less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm or less than 60 nm; (e.g., about 50-70 nm or about 60 nm). The pharmaceutical composition of any of claims 26 to 30, wherein the polydispersity index (PDI) of the LNP is from about 0.01 to about 0.15 or from about 0.05 to about 0.10. The pharmaceutical composition of any of claims 26 to 31, wherein the zeta potential of the LNP is about 1.00 mV to about 5.00 mV or about 1.50 mV to about 4.00 mV. The pharmaceutical composition of any of claims 26 to 32, wherein the modified polynucleotide (e.g., mRNA) is formulated into the lipid nanoparticle (LNP) to achieve a final mRNA concentration of about 50 μg/mL by mixing the modified polynucleotide (e.g., mRNA) with the LNP in a container, wherein the mixing includes inverting the container for about 30 seconds to mix thoroughly. The pharmaceutical composition of claim 33, wherein the modified polynucleotide (e.g., mRNA) is equilibrated to room temperature for about 30 minutes before being mixed with the LNP, after being stored, for example, in a solution or suspension at about 1 µg/µL or in a freeze-dried powder at -60°C to -80°C; and wherein the LNP is stored at 4°C and protected from light. For example, in the pharmaceutical composition of claim 33 or 34, the mixture derived from the modified polynucleotide (e.g., mRNA) and the LNP is cultured at room temperature for about 10 minutes prior to use for immunization. A method of treating an individual with RSV infection, comprising administering to the individual a therapeutically effective amount of a modified polynucleotide as described in any of claims 1 to 23 or a pharmaceutical composition as described in any of claims 25 to 35. As in the method of claim 36, the individual is a human being 5 years of age or younger or 60 years of age or older. As in the method of request item 36 or 37, in which the individual has an impaired immune system or suffers from lung disease. The method of any of claims 36 to 38, wherein the RSV glycoprotein folds into a stable pre-fusion conformation in vivo. The method of claim 39, wherein administration of the pharmaceutical composition causes an individual’s Th1/Th2 balance. The method of any of the requests 36 to 40, wherein the RSV is hRSV. The method of any of claims 36 to 41 includes administering at least two doses of the modified polynucleotide or the pharmaceutical composition to the individual. A modified polynucleotide comprising a 5' untranslated region (UTR) comprising a nucleic acid sequence having a secondary structure substantially identical to any of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 and fragments thereof, and wherein, if applicable, being at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6% or 99.8% identical to any of SEQ ID NO:4, 35, 36, 37, 38, 39, 40, 41, 42, 43 and fragments thereof. The modified polynucleotide of claim 43, wherein the 5’UTR comprises a nucleic acid sequence that is identical to SEQ ID NO:4 by at least about 90%, 92%, 95%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6% or 99.8%. The modified polynucleotide of claim 43 or 44, wherein the secondary structure includes one or more (e.g., all) of the following characteristics: 1) having a length of about 50 nucleotides; 2) having binding sites for the translation initiation factor and ribosome; 3) having a GC-rich hairpin region that stabilizes the secondary structure; 4) having a short AT-rich region for rapid ribosome passage; 5) having a GC-rich sequence adjacent to the Kozak sequence for efficient binding of the ribosome to the AUG start codon; and/or, 6) lacking a repressive domain for translation (e.g., lacking a non-canonical start codon). The modified polynucleotide of any of claims 43 to 45 further includes a 3'UTR. The modified polynucleotide of claim 46, wherein the 3’ UTR includes the nucleic acid sequence of SEQ ID NO:5 or 44. The modified polynucleotide of any of claims 43 to 47 further includes a nucleic acid sequence encoding a polypeptide. Such as the modified polynucleotide in claim 48, wherein the polypeptide is an antigen. Such as the modified polynucleotide in claim 49, wherein the antigen is a viral antigen. Such as the modified polynucleotide in claim 50, wherein the viral antigen is the hRSV antigen. The modified polynucleotide of any of claims 49 to 51, wherein the antigen induces a higher antibody titer when expressed in a host mammal (e.g., human) compared to the same antigen encoded by a control polynucleotide lacking the 5'UTR.