Respiratory syncytial virus rna vaccination

Abstract

The present disclosure provides methods for eliciting an immune response against respiratory syncytial virus (RSV) in a subject. The disclosure also provides methods for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 422,621, filed Nov. 4, 2022, and U.S. Provisional Patent Application Serial No. 63 / 523,543, filed Jun. 27, 2023, the disclosures of which are hereby incorporated by reference in their entireties. Background of the Invention

[0002] Respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease in infants and a major cause of respiratory disease in the elderly. Despite decades of research, the need for an RSV vaccine remains unmet. Recently, clinical programs using RSV F antigen in its post - fusion conformation have failed to elicit sufficient efficacy in adults. See, Faloon et al. (2017) JID [Journal of Infectious Diseases] 216:1362 - 1370. However, the RSV F antigen stabilized in the pre - fusion conformation elicits a much higher neutralizing response compared to the post - fusion antigen, potentially conferring a high level of protective efficacy against RSV disease in the elderly.

[0003] RNA - based vaccines (e.g., mRNA vaccines) have recently emerged as an effective vaccine type against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS - CoV - 2). The Coronavirus Disease 2019 (COVID - 19) mRNA vaccines have demonstrated a rapid, safe, and cost - effective production process. Usually in combination with a delivery vehicle such as lipid nanoparticles (LNPs), COVID - 19 mRNA vaccines can achieve high efficacy. In the absence of an available effective RSV vaccine, there is a need for an RNA - based RSV vaccine that elicits a strong immune response against the RSV pre - fusion F protein for effective neutralization of RSV infection. Summary of the Invention

[0004] In some aspects, provided is a method of eliciting an immune response against Respiratory Syncytial Virus (RSV) in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consisting of the amino acid sequence of SEQ ID NO:3.

[0005] In some exemplary embodiments, the RSV F protein antigen is a pre - fusion protein.

[0006] In certain exemplary embodiments, the RSV vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In certain exemplary embodiments, the RSV vaccine is administered intramuscularly. In certain exemplary embodiments, the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

[0007] In certain exemplary embodiments, the subject is 18 to 50 years old. In certain exemplary embodiments, the subject is at least 60 years old.

[0008] In certain exemplary embodiments, the RSV vaccine does not contain an adjuvant.

[0009] In certain exemplary embodiments, the mRNA is formulated in lipid nanoparticles (LNPs). In certain exemplary embodiments, the LNP contains at least one cationic lipid. In certain exemplary embodiments, the at least one cationic lipid is biodegradable or non - biodegradable. In certain exemplary embodiments, the at least one cationic lipid is cleavable or non - cleavable. In certain exemplary embodiments, the at least one cationic lipid is selected from the group consisting of: OF - 02, cKK - E10, GL - HEPES - E3 - E10 - DS - 3 - E18 - 1, GL - HEPES - E3 - E12 - DS - 4 - E10, GL - HEPES - E3 - E12 - DS - 3 - E14, cKK - E10 or GL - HEPES - E3 - E12 - DS - 4 - E10 and IM - 001.

[0010] In certain exemplary embodiments, an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject. In certain exemplary embodiments, each of the one or more booster doses is administered to the subject at least 11 months after a previous dose, at least 12 months after a previous dose, about 12 months after a previous dose, or about 10 months to about 14 months after a previous dose.

[0011] In certain exemplary embodiments, an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject.

[0012] In certain exemplary embodiments, the booster dose is administered to the subject at least 11 months after the initial dose, at least 12 months after the initial dose, about 12 months after the initial dose, or about 10 months to about 14 months after the initial dose.

[0013] In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 15 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 10 micrograms.

[0014] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 20 micrograms to about 40 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 30 micrograms.

[0015] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 65 micrograms to about 95 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 75 micrograms.

[0016] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 100 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 110 micrograms.

[0017] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14.

[0018] In certain exemplary embodiments, the RSV vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In certain exemplary embodiments, the RSV vaccine is administered intramuscularly. In certain exemplary embodiments, the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

[0019] In certain exemplary embodiments, the subject is 18 to 50 years old. In certain exemplary embodiments, the subject is at least 60 years old.

[0020] In certain exemplary embodiments, the RSV vaccine does not contain an adjuvant.

[0021] In certain exemplary embodiments, the mRNA is formulated in a lipid nanoparticle (LNP). In certain exemplary embodiments, the LNP comprises at least one cationic lipid. In certain exemplary embodiments, the at least one cationic lipid is biodegradable or non - biodegradable. In certain exemplary embodiments, the at least one cationic lipid is cleavable or non - cleavable. In certain exemplary embodiments, the at least one cationic lipid is selected from the group consisting of: OF - 02, cKK - E10, GL - HEPES - E3 - E10 - DS - 3 - E18 - 1, GL - HEPES - E3 - E12 - DS - 4 - E10, GL - HEPES - E3 - E12 - DS - 3 - E14, cKK - E10, GL - HEPES - E3 - E12 - DS - 4 - E10, and IM - 001.

[0022] In certain exemplary embodiments, an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject. In certain exemplary embodiments, each of the one or more booster doses is administered to the subject at least 11 months after a previous dose, at least 12 months after a previous dose, about 12 months after a previous dose, or about 10 months to about 14 months after a previous dose.

[0023] In certain exemplary embodiments, an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject.

[0024] In certain exemplary embodiments, the booster dose is administered to the subject at least 11 months after the initial dose, at least 12 months after the initial dose, about 12 months after the initial dose, or about 10 months to about 14 months after the initial dose.

[0025] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 5 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 5 micrograms to about 15 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 10 micrograms.

[0026] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 20 micrograms to about 40 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 30 micrograms.

[0027] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 65 micrograms to about 95 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 75 micrograms.

[0028] In other aspects, a method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject is provided, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:3.

[0029] In certain exemplary embodiments, the RSV F protein antigen is a prefusion protein.

[0030] In certain exemplary embodiments, the vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In certain exemplary embodiments, the RSV vaccine is administered intramuscularly. In certain exemplary embodiments, the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

[0031] In certain exemplary embodiments, the subject is 18 to 50 years old. In certain exemplary embodiments, the subject is at least 60 years old.

[0032] In certain exemplary embodiments, the RSV vaccine does not contain an adjuvant.

[0033] In certain exemplary embodiments, the mRNA is formulated in lipid nanoparticles (LNPs). In certain exemplary embodiments, the LNP contains at least one cationic lipid. In certain exemplary embodiments, the at least one cationic lipid is biodegradable or not biodegradable. In certain exemplary embodiments, the at least one cationic lipid is cleavable or not cleavable. In certain exemplary embodiments, the at least one cationic lipid is selected from the group consisting of: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, cKK-E10, GL-HEPES-E3-E12-DS-4-E10, and IM-001.

[0034] In certain exemplary embodiments, an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject. In certain exemplary embodiments, each of the one or more booster doses is administered to the subject at least 11 months after a previous dose, at least 12 months after a previous dose, about 12 months after a previous dose, or about 10 months to about 14 months after a previous dose.

[0035] In certain exemplary embodiments, an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject. In certain exemplary embodiments, the booster dose is administered to the subject at least 11 months after the initial dose, at least 12 months after the initial dose, about 12 months after the initial dose, or about 10 months to about 14 months after the initial dose.

[0036] In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 15 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 10 micrograms.

[0037] In certain exemplary embodiments, the vaccine is administered in a dose of from about 20 micrograms to about 40 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 30 micrograms.

[0038] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 65 micrograms to about 95 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 75 micrograms.

[0039] In certain exemplary embodiments, the RSV vaccine is administered in a dose of from about 100 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered in a dose of about 110 micrograms.

[0040] In certain exemplary embodiments, one or more symptoms of RSV infection are selected from the group consisting of: acute respiratory disease (ARD), medically attended acute respiratory disease (MAARD), severe ARD, lower respiratory tract disease (LRTD) not requiring medical attention, LRTD requiring medical attention, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

[0041] In other aspects, provided is a method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consisting of the nucleic acid sequence of SEQ ID NO:14.

[0042] In certain exemplary embodiments, the vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In certain exemplary embodiments, the RSV vaccine is administered intramuscularly. In certain exemplary embodiments, the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

[0043] In certain exemplary embodiments, the subject is between 18 and 50 years old. In certain exemplary embodiments, the subject is at least 60 years old.

[0044] In certain exemplary embodiments, the RSV vaccine does not contain an adjuvant.

[0045] In certain exemplary embodiments, the mRNA is formulated in lipid nanoparticles (LNPs). In certain exemplary embodiments, the LNP comprises at least one cationic lipid. In certain exemplary embodiments, the at least one cationic lipid is biodegradable or non - biodegradable. In certain exemplary embodiments, the at least one cationic lipid is cleavable or non - cleavable. In certain exemplary embodiments, the at least one cationic lipid is selected from the group consisting of: OF - 02, cKK - E10, GL - HEPES - E3 - E10 - DS - 3 - E18 - 1, GL - HEPES - E3 - E12 - DS - 4 - E10, GL - HEPES - E3 - E12 - DS - 3 - E14, cKK - E10, GL - HEPES - E3 - E12 - DS - 4 - E10, and IM - 001.

[0046] In certain exemplary embodiments, an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject. In certain exemplary embodiments, each of the one or more booster doses is administered to the subject at least 11 months after a previous dose, at least 12 months after a previous dose, about 12 months after a previous dose, or about 10 months to about 14 months after a previous dose.

[0047] In certain exemplary embodiments, an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject. In certain exemplary embodiments, the booster dose is administered to the subject at least 11 months after the initial dose, at least 12 months after the initial dose, about 12 months after the initial dose, or about 10 months to about 14 months after the initial dose.

[0048] In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 120 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 5 micrograms to about 15 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 10 micrograms.

[0049] In certain exemplary embodiments, the vaccine is administered at a dose of about 20 micrograms to about 40 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 30 micrograms.

[0050] In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 65 micrograms to about 95 micrograms. In certain exemplary embodiments, the RSV vaccine is administered at a dose of about 75 micrograms.

[0051] In certain exemplary embodiments, the one or more symptoms of RSV infection are selected from the group consisting of: acute respiratory disease (ARD), medically attended acute respiratory disease (MAARD), severe ARD, lower respiratory tract disease (LRTD) not requiring medical attention, LRTD requiring medical attention, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

[0052] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is 18 to 50 years old or at least 60 years old, and administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consisting of the amino acid sequence of SEQ ID NO:3.

[0053] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is 18 to 50 years old or at least 60 years old, and administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consisting of the nucleic acid sequence of SEQ ID NO:14.

[0054] In other aspects, provided is a method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising selecting a subject who is 18 to 50 years old or at least 60 years old, and administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consisting of the amino acid sequence of SEQ ID NO:3.

[0055] In other aspects, provided is a method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising selecting a subject who is 18 to 50 years old or at least 60 years old, and administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consisting of the nucleic acid sequence of SEQ ID NO:14.

[0056] In other aspects, there is provided a respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:3, and wherein the RSV F protein antigen is a pre-fusion protein.

[0057] In other aspects, there is provided a respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:3.

[0058] In other aspects, there is provided a respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14.

[0059] In other aspects, there is provided a respiratory syncytial virus (RSV) vaccine for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:3.

[0060] In other aspects, there is provided a respiratory syncytial virus (RSV) vaccine for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14.

[0061] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is at least 60 years old, and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 110 micrograms.

[0062] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is at least 60 years old, and administering an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

[0063] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is at least 60 years old, and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

[0064] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is at least 60 years old, and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

[0065] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising selecting a subject who is at least 60 years old, and administering to the subject an RSV vaccine comprising messenger ribonucleic acid (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

[0066] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger ribonucleic acid (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 110 micrograms.

[0067] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger ribonucleic acid (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

[0068] In other aspects, provided is a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger ribonucleic acid (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

[0069] In other aspects, a method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject is provided, the method comprising: selecting a subject at least 60 years of age; and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consisting of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 110 micrograms. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings.

[0071] Figure 1 An overview of the study design of Study A cohort (sentinel cohort, 18 to 50 years of age) is depicted graphically. AE: adverse event; AESI: adverse event of special interest; BL: blood sample; MAAE: medically attended adverse event; RSV: respiratory syncytial virus; SAE: serious adverse event; SCR: screening. Note: D01 (V01) blood sampling is completed before vaccination.

[0072] Figure 2 An overview of the study design of Study B cohort (main cohort, 60 years and older) is depicted graphically. AE: adverse event; AESI: adverse event of special interest; BL: blood sample for immunogenicity; MAAE: medically attended adverse event; RSV: respiratory syncytial virus; SAE: serious adverse event; SCR: screening; VAC: vaccine; WB: blood sample for CMI; *D04 (V02) is not applicable to the main cohort; D01 (V01) blood sampling is completed before vaccination.

[0073] Figure 3Graphically depicts an overview of the study design for the study C cohort (boost cohort, participants aged 60 and above). AE: Adverse event; AESI: Adverse event of special interest; BL: Blood sample for immunogenicity; MAAE: Medically attended adverse event; RSV: Respiratory syncytial virus; SAE: Serious adverse event; SCR: Screening. Note 1: Approximately 200 participants (100 participants from the selected formulation group and 100 participants from the placebo group) will be randomly assigned at a 1:1 ratio at M12 to receive a booster vaccination with the selected RSV mRNA vaccine formulation (i.e., the dose level and LNP formulation are selected based on the primary cohort safety and immunogenicity results) or placebo. Note 2: BL0005 obtained at V07 of the primary cohort will be used as the pre-vaccination sample in the boost cohort.

[0074] Figure 4 Depicts a table showing the activity schedule for the study A cohort (sentinel cohort, participants aged 18 to 50 years). AE = Adverse event; AESI = Adverse event of special interest; BL = Blood sampling for immunogenicity; BS = Blood sampling for safety assessment; CRF = Case report form; D or d = Day; DC = Diary card; M = Month; MA = Memory aid; MAAE = Medically attended adverse event; NS = Nasal swab; PRN = As needed; SAE = Serious adverse event; TC = Telephone call; UN = Blood sampling for disease visit; V = Visit; vac = Vaccination. An asterisk (*) indicates off-site visit contact by telephone at the planned time points of the study. The dagger Indicates a brief physical examination for all in-person visits after visit 01. The double dagger The electrocardiogram (ECG) to be performed at screening is used as the baseline, and the researchers review the ECGs to identify features of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the conduct of the study, additional ECGs will be performed as soon as possible (i.e., during unscheduled visits, if necessary). The section sign (§) indicates that body temperature is measured using a standard digital thermometer via the oral route (preferred) or the axillary route and recorded in the source document. Two asterisks (**) indicate that safety laboratory assessments will include serum chemistry, hematology, and clotting times. At screening and V03, serum volume samples will be collected for troponin I level testing as part of the safety laboratory assessments; some of the samples collected at V04, V05, V06, and V07 will be stored for possible future troponin I testing if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis (blood samples at screening will be used as the baseline). In the case of abnormal safety laboratory results, unscheduled visits may occur at the discretion of the researchers. The blood volume for safety laboratories may be adjusted according to local regulations. Two daggers Indicates that nasal swab samples for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants during disease visits (including unscheduled visits during the study). If a participant visits any other non-study doctor / hospital for a serious adverse event at any time during the study, once the subject is discharged, nasal swab samples will be obtained at the study site if the researchers deem it appropriate. All nasal swab samples will be collected in recommended viral transport medium tubes and stored at -60°C to -80°C until ready for shipment. The need for a home or site disease visit will first be evaluated by video call (preferred) or (if a video call is not possible) a conventional telephone call in order to be able to remotely evaluate the severity and remotely manage mild (grade 1) disease, as appropriate to the researchers. Two double daggers Indicates that any spontaneously reported systemic adverse event occurring within 30 minutes after vaccine administration will be recorded in the case report form as a spontaneously reported immediate systemic adverse event. Two section signs (§§) indicate that participants will record information in the diary card regarding solicited reactions, spontaneously reported adverse events (AEs), and AEs requiring medical attention from day 0 (D0) to day 28 (D28) after vaccine administration, as well as AESIs and SAEs throughout the study. Three asterisks (***) indicate that only medications that may have an impact on the immune response or may have an impact on both safety and the immune response will be collected. Three daggers Indicates that in the event that a participant terminates at a particular visit, the entire visit will be completed. Three double daggers Or "X" indicates that the nasopharyngeal swab number for each site is unique. Nasopharyngeal swabs will be collected for central laboratory testing. In addition to the UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may also be collected. If the collection date is more than 14 days after symptom resolution, swabs will not be collected during the disease visit.

[0075] Figure 5 Depicts a table showing the activity schedule for Study B cohort (primary cohort, participants aged 60 and above). AE = adverse event; AESI = adverse event of special interest; BL = blood sampling for immunogenicity; BS = blood sampling for safety assessment; CMI = cell-mediated immunity; CRF = case report form; D or d = day; DC = diary card; M = month; MA = memory aid; MAAE = adverse event requiring medical attention; NS = nasal swab; PRN = as needed; SAE = serious adverse event; TC = telephone call; UN = blood sample for disease visit; V = visit; vac: vaccination; WB = blood sample for TruCulture. An asterisk (*) indicates that visit 04 (V02) is not applicable to the primary cohort. Dagger Indicates off-site visit contact by telephone at the planned time points of the study. Double dagger Indicates a brief physical examination for all in-person visits after visit 01. The section sign (§) indicates that an electrocardiogram is performed at screening as a baseline and reviewed by the investigator for characteristics of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the conduct of the study, an additional ECG will be performed as soon as possible (i.e., in an unscheduled visit, if necessary). Double asterisk (**) indicates the body temperature measured and recorded in the source document by the oral route (preferred) or axillary route using a standard digital thermometer. Two daggers Indicates samples collected from a subgroup of 140 participants for CMI determination evaluated by TruCulture. Two double daggers It is indicated that the safety laboratory assessments will include serum chemistry, hematology, and clotting times. At screening and V03, serum volume samples will be collected for troponin I levels as part of the safety laboratory assessments; a portion of the samples collected at V01, V04, V05, V06, and V07 will be stored for possible future troponin I testing if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis (blood samples at screening will be used as baseline). In the case of abnormal safety laboratory results, unscheduled visits may occur at the discretion of the investigator. Two section signs (§§) indicate that nasal swab samples for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from the participant during disease visits (including medically attended visits during the study). If the participant visits any other non-study doctor / hospital for an SAE at any time during the study, once the subject is discharged, nasal swab samples will be obtained at the study site if the investigator deems it appropriate. All nasal swab samples will be collected in recommended viral transport medium tubes and will be stored at -60°C to -80°C until ready for shipment. The requirement for a home or site disease visit will be evaluated first by video call (preferred) or (if video call is not possible) a regular phone call in order to be able to remotely evaluate the severity and remotely manage mild (Grade 1) disease as appropriate by the investigator of the study. Three asterisks (***) indicate that any actively reported systemic AE occurring within 30 minutes after vaccine administration will be recorded in the case report form as an actively reported immediate systemic AE. Three daggers It is indicated that the participant will record information on solicited reactions, actively reported AEs and MAAEs from Day 0 to Day 28 after vaccine administration, and AESIs and SAEs throughout the study in the diary card. Three double daggers It is indicated that only drugs that may have an impact on the immune response or may have an impact on both safety and the immune response will be collected. Three section signs (§§§) indicate that if the participant terminates at a particular visit, the entire visit will be completed. Four asterisks (****) or "X" indicate that the nasopharyngeal swab number is unique for each site. Nasopharyngeal swabs will be collected for central laboratory testing. In addition to the UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may also be collected. If the collection date is more than 14 days after symptom resolution, swabs will not be collected during the disease visit. Four double daggers It is indicated that participants who will not proceed into the booster cohort.

[0076] Figure 6Depicts a table showing the activity schedule for the study C cohort (boost cohort, participants aged 60 and above). AE = adverse event; AESI = adverse event of special interest; BL = blood sampling for immunogenicity; BS = blood sampling for safety assessment; CRF = case report form; D or d = day; DC = diary card; M = month; MA = memory aid; MAAE = adverse event requiring medical attention; NS = nasal swab; PRN = as needed; SAE = serious adverse event; TC = telephone call; UN = blood sample for disease visit; V = visit; vac = vaccination; Vac2 = booster vaccination. An asterisk (*) indicates off-site visit contact by telephone at the planned time points of the study. Dagger Indicates a brief physical examination for all in-person visits after visit 08. Body temperature is measured by the oral route (preferred) or axillary route using a standard digital thermometer and recorded in the source document. Double dagger Indicates that an electrocardiogram (ECG) is performed at screening as a baseline and the ECG is reviewed by the investigator for characteristics of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the conduct of the study, an additional ECG will be performed as soon as possible (i.e., in an unscheduled visit, if necessary). The section sign (§) indicates that body temperature is measured by the oral route (preferred) or axillary route using a standard digital thermometer and recorded in the source document. Two asterisks (**) mark blood sample 5, called BL0005, obtained at V07 and used as a pre-vaccination sample in the boost cohort. Double dagger Indicates that safety laboratory assessments will include serum chemistry, hematology, and clotting times. At screening and V09, serum volume samples will be collected for troponin I level testing as part of the safety laboratory assessment; some samples collected at V10, V11, V12, and V13 will be stored for possible future troponin I testing if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis (blood samples at screening will be used as a baseline). In the case of abnormal safety laboratory results, unscheduled visits may occur at the discretion of the investigator. Two double daggers Indicates that nasal swab samples will be collected from participants during disease visits (including medical visits during the study period) for the detection of RSV and respiratory pathogens (including COVID-19). If a participant visits any other non-study doctor / hospital due to an SAE at any time during the study, once the subject is discharged, if the study investigator deems it appropriate, nasal swab samples will be obtained at the study site. If a participant is unable to attend an in-person disease visit and / or unable to receive a home visit, self-collection of the sample should be performed. All nasal swab samples will be collected in recommended viral transport medium tubes and will be stored at -60°C to -80°C until ready for shipment. The requirements for in-home or in-person disease visits will first be evaluated via video call (preferred) or (if video call is not possible) a conventional phone call in order to be able to remotely evaluate the severity and remotely manage mild (level 1) disease, as appropriate by the study investigator. Two double-section symbols (§§) indicate administration 12 months after the first injection. Three asterisks (***) indicate that any unsolicited systemic AEs occurring within 30 minutes after vaccine administration will be recorded in the case report form as unsolicited immediate systemic AEs. Three daggers Indicates that participants will record information regarding solicited reactions, unsolicited AEs and MAAEs from day 0 to day 28 (D0 to D28) after vaccine administration and AESIs and SAEs throughout the study in a diary card. Three double daggers Indicates that only drugs that may have an impact on the immune response or may have an impact on both safety and the immune response will be collected. Three section symbols (§§§) or "X" indicate that the nasopharyngeal swab number is unique for each site. Nasopharyngeal swabs will be collected for central laboratory testing. In addition to the UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may also be collected. If the collection date is more than 14 days after symptom resolution, swabs will not be collected during the disease visit.

[0077] Figure 7 Is a table depicting the demographic characteristics of the primary cohort.

[0078] Figure 8Graphically depicts the summary of the geometric mean titer (GMT) of RSV-A neutralizing antibody (NAb) titers and the geometric mean titer ratio (GMTR) of neutralizing antibodies at D29 and D01 after primary vaccination. X-axis: 1 = cKK-E10, 10 μg, n = 93; 2 = cKK-E10, 30 μg, n = 95; 3 = cKK-E10, 75 μg, n = 97; 4 = GL-HEPES-E3-E12-DS-4-E10, 10 μg, n = 99; 5 = GL-HEPES-E3-E12-DS-4-E10, 30 μg, n = 96; and 6 = GL-HEPES-E3-E12-DS-4-E10, 75 μg, n = 92.

[0079] Figure 9 Graphically depicts participants in the entire primary cohort (aged 60 and above) with a ≥4-fold and <4-fold increase in RSV-A neutralizing antibody titers after primary vaccination.

[0080] Figure 10 Graphically depicts a subset of the primary cohort (aged 60 and above), summarizing the geometric mean of IgG antibody titers and the geometric mean titer ratio of IgG antibodies at D29 and D01 after primary vaccination. X-axis: 1 = cKK-E10, 10 μg, n = 38; 2 = cKK-E10, 30 μg, n = 35; 3 = cKK-E10, 75 μg, n = 44; 4 = GL-HEPES-E3-E12-DS-4-E10, 10 μg, n = 45; 5 = GL-HEPES-E3-E12-DS-4-E10, 30 μg, n = 41; and 6 = GL-HEPES-E3-E12-DS-4-E10, 75 μg, n = 38.

[0081] Figure 11 Graphically depicts the summary of the geometric mean titer (GMT) of RSV-A neutralizing antibody (NAb) titers and the geometric mean titer ratio (GMTR) of neutralizing antibodies at D29 and D01 after primary vaccination in the sentinel cohort (aged 18 - 50). X-axis: 1 = cKK-E10, 10 μg, n = 9; 2 = cKK-E10, 30 μg, n = 8; 3 = cKK-E10, 75 μg, n = 10; 4 = GL-HEPES-E3-E12-DS-4-E10, 10 μg, n = 7; 5 = GL-HEPES-E3-E12-DS-4-E10, 30 μg, n = 10; and 6 = GL-HEPES-E3-E12-DS-4-E10, 75 μg, n = 10.

[0082] Figure 12Graphically depicts participants in the sentinel cohort (aged 18 - 50 years) with ≥4-fold and <4-fold increases in RSV-A neutralizing antibody titers after primary vaccination.

[0083] Figure 13A - Figure 13B Graphically summarizes the fold increase in RSV-A neutralizing antibody titers in the sentinel cohort after primary vaccination. (A) cKK-E10. (B) GL-HEPES-E3-E12-DS-4-E10.

[0084] Figure 14 Graphically summarizes the responses to requests as percentages within 7 days after primary vaccination. X-axis: A = cKK-E10; B = GL-HEPES-E3-E12-DS-4-E10; C = placebo.

[0085] Figure 15 Graphically summarizes the injection site reactions to requests as percentages within 7 days after primary vaccination. X-axis: A = cKK-E10; B = GL-HEPES-E3-E12-DS-4-E10; C = placebo.

[0086] Figure 16 Graphically summarizes the systemic reactions to requests as percentages within 7 days after primary vaccination. X-axis: A = cKK-E10; B = GL-HEPES-E3-E12-DS-4-E10; C = placebo.

[0087] Figure 17 Is a table (main cohort) summarizing the safety overview after primary vaccination.

[0088] Figure 18 Is a table (main cohort) summarizing the responses to requests within 7 days after primary vaccination.

[0089] Figure 19 Is a table summarizing the spontaneously reported adverse events. Since no dose response was observed, pooled data are provided. ￥ After the cutoff date for biostatistical output, the investigators downgraded AESIs to non-AESIs (diagnosis changed from myocarditis to asymptomatic myocardial injury). *There were no meaningful disorders, only observed numerical disorders caused by musculoskeletal and connective tissue disorders (such as arthralgia, myalgia, muscle spasm) and gastrointestinal disorders (such as abdominal pain, diarrhea, nausea). δSAEs were evenly distributed across the mRNA groups (two in LNP cKK-E10 and three in LNP GL-HEPES-E3-E12-DS-4-E10). Only one SAE (asymptomatic myocardial injury) was evaluated as related to the IMP (LNP cKK-E10 low dose). The events were: hypotension with dehydration and syncope in LNP cKK-E10 (low or high dose); asymptomatic myocardial injury in LNP cKK-E10 (low dose); constipation in LNP GL-HEPES-E3-E12-DS-4-E10 (low dose); hydronephrosis with pyelonephritis and RSV infection in LNP GL-HEPES-E3-E12-DS-4-E10 (medium dose); hypertensive crisis in LNP GL-HEPES-E3-E12-DS-4-E10 (high dose).

[0090] Figure 20 is a table summarizing the actively provided adverse events caused by musculoskeletal disorders, connective tissue disorders, and gastrointestinal disorders compared to placebo. Detailed Description

[0091] The present disclosure particularly relates to RNA (e.g., mRNA) vaccine compositions encoding the RSV F protein and methods of vaccination therewith. In addition, the present disclosure relates to vaccine compositions comprising mRNA encoding the prefusion F protein of RSV formulated in lipid nanoparticles (LNPs) and methods of vaccination therewith. I. Definitions

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are described below. In case of conflict, the present specification, including definitions, shall control. In general, the nomenclature and the techniques described herein in connection with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry, protein and nucleic acid chemistry and hybridization are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications as commonly accomplished in the art or as described herein. Further, unless the context requires otherwise, singular terms shall include plural and plural terms shall include singular. Throughout this specification and the examples, the words "have" and "comprise" or variations thereof (such as "has / having", "comprises / comprising") shall be understood to mean including the stated integer or group of integers but not excluding any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not mean that any of these documents constitutes a part of the common general knowledge in the art.

[0093] It should be noted that the term "a" or "an" entity refers to one or more of the said entities; for example, "a nucleotide sequence" should be understood to represent one or more nucleotide sequences. Thus, the terms "a or an", "one or more" and "at least one" can be used interchangeably herein.

[0094] In addition, as used herein, "and / or" is regarded as a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0095] It should be understood that, regardless of how many aspects are described herein in language using the word "comprising", other similar aspects are also provided that are described in terms of "consisting of" and / or "consisting essentially of".

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary Of Cell And Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press can provide a general dictionary of many of the terms used in this disclosure for those skilled in the art.

[0097] Units, prefixes, and symbols are expressed in their internationally accepted SI form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations on the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification (in its entirety).

[0098] The term "about" or "approximately" is used herein to mean roughly, generally, or around. When the term "about" is used in connection with a numerical range, it modifies that range by extending the boundaries above and below the numerical values. Generally speaking, the term "about" can modify a numerical value to be higher and lower (greater or lesser) than the stated value by a variance (e.g., 10%, up or down). In some embodiments, the term represents a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±10% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±5% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±4% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±3% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±2% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±1% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.9% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.8% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.7% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.6% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.5% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.4% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.3% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.1% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.05% from the indicated numerical value. In some embodiments, "about" represents a deviation of ±0.01% from the indicated numerical value.

[0099] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA can contain one or more coding regions and non-coding regions. The coding region may alternatively be referred to as an open reading frame (ORF). The non-coding regions in an mRNA include a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail. An mRNA can be purified from a natural source or produced using a recombinant expression system (e.g., in vitro transcription). In various embodiments, the mRNA can be purified or chemically synthesized.

[0100] As used herein, the term "F protein" or "RSV F protein" refers to the RSV protein that is responsible for driving the fusion of the viral envelope with the host cell membrane during virus entry.

[0101] As used herein, the term "RSV F polypeptide" or "F polypeptide" refers to a polypeptide that includes at least one epitope of the F protein.

[0102] As used herein, the term "post-fusion" with respect to RSV F refers to the stable conformation of RSV F that emerges after viral and cell membrane fusion.

[0103] As used herein, the term "pre-fusion" with respect to RSV F refers to the conformation adopted by RSV F prior to virus-cell interaction.

[0104] As used herein, the term "protomer" refers to the structural unit of an oligomeric protein. In the case of RSV F, the individual unit of the RSV F trimer is a protomer.

[0105] As used herein, the term "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. Thus, an N-glycan is formed by the process of N-glycosylation. This glycan can be a polysaccharide.

[0106] As used herein, the term "glycosylation" refers to the addition of sugar units to a protein.

[0107] As used herein, the term "immune response" refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells, to a stimulus such as an antigen or a vaccine. The immune response can include any cells in the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. The immune response includes, but is not limited to, innate and / or adaptive immune responses.

[0108] As used herein, an "antibody response" is an immune response that produces antibodies.

[0109] As used herein, "antigen" refers to an agent that elicits an immune response; and / or an agent that is bound by a T cell receptor when exposed or administered to an organism (e.g., when presented by an MHC molecule) or that binds to an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, the antigen elicits a cellular response in an organism (e.g., T cells whose receptors interact specifically with the antigen). A particular antigen may elicit an immune response in one or a few members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the members of the target organism species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not induce a specific physiological response in an organism. In some embodiments, for example, the antigen may bind to an antibody and / or a T cell receptor in vitro, whether or not such interaction occurs in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity. Antigens include RSV polypeptides encoded by mRNA as described herein.

[0110] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which the antigen is adsorbed; oil-in-water or water-in-oil emulsions in which an antigen solution is emulsified in a mineral oil or in water (e.g., Freund's incomplete adjuvant). Killed mycobacteria (e.g., Freund's complete adjuvant) are sometimes included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (e.g., see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can also include biomolecules such as toll-like receptor (TLR) agonists and costimulatory molecules.

[0111] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, the "subject" refers to a human. In some embodiments, the "subject" refers to a non-human animal. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, the subject can be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments, the subject is an adult, adolescent, or infant. In some embodiments, the terms "subject" or "patient" are used and are intended to be interchangeable with "subject". In certain exemplary embodiments, the subject is a premature neonate (e.g., less than 37 weeks gestational age), a neonate (e.g., 0 - 27 days old), an infant or toddler (e.g., 28 days to 23 months old), a child (e.g., 2 to 11 years old), an adolescent (e.g., 12 to 17 years old), an adult (e.g., 18 to 50 years old or 18 to 64 years old), or an elderly person (e.g., 65 years old or older). In an exemplary embodiment, the subject is an elderly person (e.g., an adult 60 years old or older).

[0112] As used herein, the terms "vaccination" or "vaccinate" refer to the administration of a composition intended to, for example, elicit an immune response against a pathogenic agent. Vaccination can be administered before, during, and / or after exposure to the pathogenic agent and / or the occurrence of one or more symptoms, and in some embodiments, shortly before, during, and / or after exposure to the pathogenic agent. In some embodiments, vaccination includes administering the vaccination composition multiple times at appropriate intervals (e.g., approximately 12 months after a previous dose).

[0113] This disclosure describes nucleic acid sequences (e.g., DNA sequences and RNA sequences) and amino acid sequences that have a certain degree of identity to a given nucleic acid sequence or amino acid sequence (reference sequence), respectively.

[0114] The terms "percent identical", "identity percent", or similar terms are intended to refer, in particular, to the percentage of identical nucleotides or amino acids in the most preferably aligned sequences to be compared. The percentage is purely statistical, and the differences between two sequences can, but do not necessarily, randomly distribute over the entire length of the sequences to be compared. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. The comparison of two sequences is generally carried out by comparing the sequences with respect to a segment or "comparison window" after the most preferably alignment to identify local regions of the corresponding sequences. The best alignment for comparison can be carried out manually or by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. [Advances in Applied Mathematics] 2, 482, by means of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48, 443, by means of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 88, 2444, or by means of a computer program using the said algorithms (e.g., GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group, 575 Science Drive, Madison, Wis., USA).

[0115] The percentage of identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0116] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some embodiments, consecutive nucleotides) of the reference nucleic acid sequence. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0117] Nucleic acid sequences or amino acid sequences that have a specific degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional property of the given sequence and, for example and in some cases, are functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a specific degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0118] As used herein, the term "kit" refers to a packaged set of related components, such as one or more compounds or compositions and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants. II. RSV mRNA Vaccine

[0119] Respiratory syncytial virus (RSV) is an antisense single-stranded RNA virus belonging to the family Pneumoviridae. RSV can cause respiratory tract infections. RSV is an enveloped virus. The surface of the RSV virion contains three proteins: the attachment glycoprotein (G), the fusion protein (F), and the small hydrophobic (SH) protein.

[0120] The RSV F protein is responsible for the fusion of the virus and the host cell membrane and has at least three conformations (pre-fusion, intermediate, and post-fusion conformations). When in the pre-fusion conformation (pre-fusion, Pre-F), the F protein exists as a trimer, in which the major antigenic sites are exposed. The site serves as the major target for neutralizing antibodies produced by RSV-infected subjects (see, Coultas et al., Thorax. 74:986-993. 2019; McLellan et al., Science. 340(6136):1113-7. 2013). After binding to its target on the host cell surface, Pre-F undergoes a conformational change during which the site is no longer exposed. Pre-F transitions to a transient intermediate conformation, enabling the F protein to insert into the host cell membrane, resulting in the fusion of the virus and the host cell membrane. The final conformational change results in a more stable and elongated protein form (post-fusion, Post-F). Sites II and IV of the F protein are unique to Post-F, while site I is present in both the Pre-F and Post-F conformations (McLellan et al., J. Virol. 85(15):7788-7796. 2011).

[0121] Respiratory syncytial virus (RSV) is a major viral agent causing severe respiratory diseases in the elderly globally. Currently, there is no vaccine available for the prevention of RSV in the elderly, and there is no effective antiviral treatment. The economic and clinical burden imposed on the healthcare system during the RSV epidemic season will remain at a high level until preventive treatment options become available.

[0122] As provided herein, mRNA-based vaccines have been developed based on three different RSV proteins. The F protein designated as FD1 corresponds to the wild-type RSV F protein. The F protein designated as FD2 corresponds to the soluble RSV F protein, which lacks the transmembrane domain and cytoplasmic tail and contains a C-terminal fibritin trimerization domain (also known as the T4 foldon). The F protein designated as FD3 corresponds to the prefusion RSV F protein.

[0123] As used herein, the term "antigenic site" " or "site" "epitope" refers to the site located at the vertex of the prefusion RSV F trimer, which contains amino acid residues 62-69 and 196-209 of wild-type RSV F (i.e., FD1 or SEQ ID NO:1): The F protein designated as FD1 corresponds to the WTRSV F protein. The epitope is the binding site of antibodies specific for pre-fusion RSV F (such as D25 and AM14), and binding of the antibody to the epitope blocks the cell surface attachment of RSV (see, e.g., McLellan et al., Science 340(6136): 1113-1117, 2013). The recombinant human anti-RSV antibody D25 (Creative Catalog No.: PABL-322) and the recombinant human anti-RSV antibody AM14 (Creative Catalog No.: PABL-321) are each commercially available. Catalog No.: PABL-322) and the recombinant human anti-RSV antibody AM14 (Creative Catalog No.: PABL-321) are each commercially available.

[0124] FD2 or SEQ ID NO:2 is as follows: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0125] FD3 or SEQ ID NO:3 is as follows: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNPETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGWYCDNAGNVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNELINQSLAFINQSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN。

[0126] The mRNA described herein may include an open reading frame (ORF) encoding an RSV F protein antigen, at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and at least one polyadenylation (poly(A)) sequence. The mRNA may further comprise a 5' cap having the following structure:

[0127] The nucleic acid sequences of each mRNA open reading frame (ORF) encoding RSV FD1, FD2, and FD3 proteins are listed separately below.

[0128] FD1 mRNA ORF:

[0129] FD2 mRNA ORF:

[0130] FD3 mRNA ORF:

[0131] The nucleic acid sequences of each DNA template encoding the RSV FD1, FD2, and FD3 proteins are listed separately below.

[0132] FD1DNA

[0133] FD2 DNA

[0134] FD3 DNA

[0135] The nucleic acid sequences of the 5’UTR and 3’UTR are listed below.

[0136] 5’UTR GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO:10).

[0137] 3’UTR CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:11).

[0138] The nucleic acid sequences of each full-length mRNA encoding the RSV FD1, FD2, and FD3 proteins are listed below.

[0139] FD1 mRNA

[0140] FD2 mRNA

[0141] FD3 mRNA

[0142] One aspect of the present disclosure relates to methods of eliciting or stimulating an immune response against RSV in a subject. Another aspect of the present disclosure relates to methods of preventing RSV infection or reducing one or more symptoms of RSV infection in a subject. These methods can include administering or providing to the subject an RNA (e.g., mRNA) RSV vaccine. The RNA RSV vaccine can include mRNA, wherein the mRNA includes an open reading frame (ORF) encoding an RSV F protein antigen or a portion of the RSV F protein antigen.

[0143] In certain embodiments, a prophylactically effective amount of the RNA RSV vaccine can be administered (e.g., by a healthcare practitioner) to the subject. In certain embodiments, the RSV F protein antigen can include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO:3. In other embodiments, the RSV F protein antigen is encoded by an RNA sequence (e.g., an mRNA sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO:14.

[0144] Another aspect of the present disclosure relates to methods of eliciting or stimulating an immune response against RSV in a subject, wherein the methods can include selecting a subject who is at least 60 years old and administering to the subject a prophylactically effective amount of the RNA RSV vaccine. Another aspect of the present disclosure relates to methods of preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, wherein the methods can include selecting a subject who is at least 60 years old and administering to the subject a prophylactically effective amount of the RNA RSV vaccine. The step of selecting a subject who is 60 years of age or older can be performed by a healthcare worker (e.g., any one or more of a physician, physician assistant, nurse, pharmacist, pharmacy technician, medical technician, etc.), or can be performed by the subject himself or herself (i.e., self-selection). Then a prophylactically effective amount of the RNA RSV vaccine of the present disclosure can be administered to the selected subject.

[0145] Another aspect of the present disclosure relates to an RNA (e.g., mRNA) RSV vaccine for eliciting or stimulating an immune response against RSV in a subject. Another aspect of the present disclosure relates to an RNA (e.g., mRNA) RSV vaccine for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject. In certain embodiments, the symptoms of RSV infection include, but are not limited to, acute respiratory disease (ARD), medically attended acute respiratory disease (MAARD), severe ARD, lower respiratory tract disease (LRTD) without medical attendance, LRTD with medical attendance, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis. RSV infection can be confirmed by laboratory tests (e.g., by RT-PCR, ELISA, etc.).

[0146] As used herein, "ARD" refers to RSV infection that includes any respiratory symptoms, which include nasal congestion, sore throat, hoarseness, new or worsening cough, sputum production, and dyspnea with or without fever.

[0147] As used herein, "severe ARD" refers to RSV infection that includes acute respiratory disease with a history of fever or a measured fever of ≥38°C, and a cough that requires hospitalization and has onset within the past 10 days.

[0148] As used herein, "LRTD" refers to RSV infection that includes ARD and has one or more symptoms of lower respiratory tract disease, which includes, but is not limited to, involvement of the lower respiratory tract: trachea, bronchi, and lungs, which can be combined (e.g., bronchopneumonia and / or tracheobronchitis), and the onset of ARD symptoms is 10 days.

[0149] As used herein, "LRTD with medical attendance" refers to RSV infection that includes ARD and has one or more symptoms of lower respiratory tract disease, which includes, but is not limited to, involvement of the lower respiratory tract: trachea, bronchi, and lungs, which can be combined (e.g., bronchopneumonia and / or tracheobronchitis), and the onset of ARD symptoms is 10 days, and seeks medical care (e.g., emergency room visit, hospitalization, or outpatient visit).

[0150] As used herein, "reducing one or more symptoms of RSV infection" means that one or more symptoms and / or viral load are reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% in subjects vaccinated with the RSV vaccine disclosed herein compared to unvaccinated subjects.

[0151] In certain embodiments, the RSV vaccine described herein can be administered to a subject by an administration method that includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratracheal, epidural, and oral administration routes. In certain embodiments, it is administered intramuscularly in the arm muscle or leg muscle of the subject. In certain embodiments, it is administered in the deltoid muscle of the upper arm of the subject. The RSV vaccine described herein can be delivered intramuscularly with a standard needle and syringe or by any other suitable injection device.

[0152] In certain embodiments, the RSV vaccine described herein can be administered to a subject by an administration method that includes skin injection, e.g., injection into the epidermis, dermis, or subcutaneous tissue of the skin. In some embodiments, the RSV vaccine described herein is provided in a device suitable for skin injection (such as a needle (e.g., an epidermal needle, a dermal needle, or a subcutaneous tissue needle), a needleless device, a microneedle device, or a microprojection array device). Examples of microneedle or microprojection array devices suitable for skin injection according to the present invention are described in US20230270842 A1, US20220339416 A1, US 20210085598A1, US20200246450 A1, US20220143376 A1, US20180264244A1, US20180263641 A1, and US20110245776 A1.

[0153] In certain embodiments, an RSV RNA vaccine composition is formulated to exhibit a reduced amount of ionizable lipid-mRNA adduct impurities (e.g., aldehyde-mRNA adduct impurities), which can form due to covalent modification of mRNA by reactive species (e.g., secondary amines or reactive aldehydes) generated by degradation of the ionizable lipid component of the LNP (Packer et al., “A Novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.” Nature Communications, (2021) 12:6777). In some embodiments, the vaccine composition comprises less than about 10% (e.g., less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, less than about 0.05%, less than about 0.01%, or less than 0.001%) of the mRNA in the form of adduct impurities, as measured by reversed-phase ion-pair high-performance liquid chromatography (RP-IP HPLC). In some embodiments, when stored at a temperature of about 25° C. or lower, the amount of adduct impurities in the LNP composition increases at an average rate of less than 2%, less than 1%, less than 0.5%, or less than 0.2% per day. In some embodiments, when stored at a temperature of about 25° C. or lower, the amount of the adduct does not substantially increase (e.g., does not increase by more than 0.05%, more than 0.01%, more than 0.005%, or more than 0.001%).

[0154] In some embodiments, the buffer or pH of the RSV RNA vaccine composition can be adjusted to reduce the amount of adduct impurities formed in the LNP composition (e.g., to inhibit the decomposition of the ionizable lipid). For example, some embodiments may comprise a composition having a TRIS (tris(hydroxymethyl)aminomethane) buffer at a concentration of about 10 mM or higher, such as a TRIS buffer at a concentration of about 20 mM, about 30 mM, about 50 mM, about 60 mM, about 75 mM, about 100 mM, about 120 mM, or about 150 mM. In some embodiments, the composition comprises from about 10 mM to about 150 mM of TRIS, such as from about 15 mM to about 120 mM of TRIS or from about 20 mM to about 100 mM of TRIS. In some embodiments, the composition does not contain a PBS buffer. In some embodiments, the pH of the composition is from about 6.5 to about 9.0, such as from about 7-8, from about 7-7.5, about 7.4, or about 7.5.

[0155] In some embodiments, the RSV RNA vaccines (e.g., mRNA vaccine compositions) described herein comprise a unit dose volume of about 0.3 mL, about 0.35 mL, about 0.4 mL, about 0.45 mL, about 0.5 mL, about 0.55 mL, about 0.6 mL, about 0.65 mL, or about 0.7 mL and one or more pharmaceutically acceptable carriers, one or more diluents, and / or one or more excipients. In some embodiments, the RSV RNA vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject in a volume of about 0.3 mL, about 0.35 mL, about 0.4 mL, about 0.45 mL, about 0.5 mL, about 0.55 mL, about 0.6 mL, about 0.65 mL, or about 0.7 mL.

[0156] In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject in a dose of about 5 μg to about 400 μg, about 5 μg to about 300 μg, about 5 μg to about 200 μg, about 5 μg to about 100 μg, or about 5 μg to about 15 μg to vaccinate the subject, wherein the μg consists of the amount of mRNA formulated in lipid nanoparticles (LNP) and does not include any diluents, etc. In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject in a dose of about 5 μg to about 160 μg, about 5 μg to about 120 μg, about 10 μg to about 80 μg, about 10 μg to about 60 μg, or about 20 μg to about 40 μg to vaccinate the subject, wherein the μg consists of the amount of mRNA formulated in LNP and does not include any diluents, etc. In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject in a dose of about 45 μg to about 130 μg, about 50 μg to about 120 μg, about 55 μg to about 110 μg, about 60 μg to about 100 μg, or about 65 μg to about 95 μg to vaccinate the subject, wherein the μg consists of the amount of mRNA formulated in LNP and does not include any diluents, etc.

[0157] In some embodiments, the RSV vaccine described herein (e.g., an mRNA vaccine composition) is administered to a subject at a dose of about 25 μg, about 50 μg, about 100 μg, about 110 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, about 700 μg, about 750 μg, about 800 μg, about 850 μg, about 900 μg, about 950 μg, or about 1000 μg, where μg consists of the amount of mRNA formulated in the LNP and does not include any diluents, etc. In some embodiments, the RSV vaccine described herein (e.g., an mRNA vaccine composition) is administered to a subject at a dose of about 10 μg, about 30 μg, about 75 μg, or about 110 μg, where μg consists of the amount of mRNA formulated in the LNP and does not include any diluents, etc.

[0158] In some embodiments, a 10-μg RSV RNA vaccine composition is administered to a subject at a 0.5-mL dose. In some embodiments, a 30-μg RSV RNA vaccine composition is administered to a subject at a 0.5-mL dose. In some embodiments, a 75-μg RSV RNA vaccine composition is administered to a subject at a 0.5-mL dose. μg consists of the amount of mRNA formulated in the LNP and does not include any diluents, etc.

[0159] In some embodiments, the RSV RNA vaccine composition (e.g., an mRNA vaccine composition) used in a method of vaccinating a subject is administered to the subject as a single dose. In some embodiments, the RSV RNA vaccine composition (e.g., an mRNA vaccine composition) used in a method of vaccinating a subject is administered to the subject as two doses (e.g., an initial dose and a booster dose). In some embodiments, the RSV RNA vaccine composition (e.g., an mRNA vaccine composition) used in a method of vaccinating a subject is administered to the subject as three or more doses (e.g., an initial dose and two or more booster doses (e.g., a first booster dose, a second booster dose, etc.)).

[0160] In some embodiments, the RSV RNA vaccine composition (e.g., an mRNA vaccine composition) used in a method of vaccinating a subject is administered as an initial dose of the RSV vaccine and a booster dose of the RSV vaccine, where the initial dose and the booster dose are temporally spaced apart.

[0161] In certain embodiments, a booster dose is administered to a subject from about 1 month to about 24 months, about 2 months to about 23 months, about 3 months to about 22 months, about 4 months to about 21 months, about 5 months to about 20 months, about 6 months to about 19 months, about 7 months to about 18 months, about 8 months to about 17 months, about 9 months to about 16 months, about 10 months to about 15 months, about 10 months to about 14 months, about 11 months to about 14 months, or about 11 months to about 13 months after an initial dose.

[0162] In certain embodiments, a booster dose is administered to a subject at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months after an initial dose.

[0163] In some embodiments, an RSV RNA vaccine composition (e.g., an mRNA vaccine composition) for use in a method of vaccinating a subject is administered to the subject as an initial dose of an RSV vaccine and one or more booster doses of an RSV vaccine.

[0164] In certain embodiments, a booster dose is administered to a subject from about 1 month to about 24 months, about 2 months to about 23 months, about 3 months to about 22 months, about 4 months to about 21 months, about 5 months to about 20 months, about 6 months to about 19 months, about 7 months to about 18 months, about 8 months to about 17 months, about 9 months to about 16 months, about 10 months to about 15 months, about 10 months to about 14 months, about 11 months to about 14 months, or about 11 months to about 13 months after a previous initial dose or a previous booster dose.

[0165] In certain embodiments, a booster dose is administered to a subject at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months after a previous initial dose or a previous booster dose. III. Pharmaceutical Compositions

[0166] RNA purified according to the present disclosure can be used as a component in a pharmaceutical composition, for example, as a vaccine (e.g., an RSV RNA vaccine). These compositions will typically contain RNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure may also contain a delivery system for the RNA, such as liposomes, oil-in-water emulsions, or microparticles. In some embodiments, the pharmaceutical composition contains lipid nanoparticles (LNPs). In certain embodiments, the composition contains a nucleic acid molecule encoding an antigen encapsulated within the LNP.

[0167] In some embodiments, the RSV RNA vaccine composition contains a pharmaceutically acceptable carrier, diluent, excipient, and / or LNP, and does not contain an adjuvant. In some embodiments, the vaccine composition contains a pharmaceutically acceptable carrier, diluent, excipient, and / or LNP, and includes one or more adjuvants. In some embodiments, the carrier, diluent, excipient, and / or LNP includes buffered saline. In some embodiments, the carrier, diluent, excipient, and / or LNP contains octylphenol ethoxylate (Triton X-100). In some embodiments, the carrier, diluent, excipient, and / or LNP includes buffered saline and octylphenol ethoxylate (Triton X-100). In some embodiments, the carrier, diluent, excipient, and / or LNP includes sodium chloride. In some embodiments, the carrier, diluent, excipient, and / or LNP includes sodium phosphate. In some embodiments, the carrier, diluent, excipient, and / or LNP includes disodium hydrogen phosphate. In some embodiments, the carrier, diluent, excipient, and / or LNP includes water. In some embodiments, the carrier, diluent, excipient, and / or LNP includes formaldehyde. In some embodiments, the carrier, diluent, excipient, and / or LNP includes ovalbumin. In some embodiments, the carrier, diluent, excipient, and / or LNP includes sodium chloride, sodium phosphate (sodium dihydrogen phosphate, disodium hydrogen phosphate, or both), and water. In some embodiments, the carrier, diluent, excipient, and / or LNP includes sodium chloride, sodium phosphate (sodium dihydrogen phosphate, disodium hydrogen phosphate, or both), water, formaldehyde, ovalbumin, and Triton X-100.

[0168] In certain embodiments, the composition will be in an aqueous form upon administration, but may be stored in a non-liquid form and resuspended prior to administration.

[0169] In some embodiments, the RSV RNA vaccine composition contains a preservative (e.g., thiomersal or 2-phenoxyethanol). However, in some embodiments, the RSV RNA vaccine composition is substantially free of mercury materials, e.g., free of thiomersal. In some embodiments, the RSV RNA vaccine composition does not contain a preservative.

[0170] In some embodiments, the RSV RNA vaccine composition comprises a physiological salt, such as a sodium salt. In some embodiments, the RSV RNA vaccine composition comprises sodium chloride (NaCl). In some embodiments, the RSV RNA vaccine composition comprises from about 1 to 20 mg / ml of NaCl. In some embodiments, the RSV RNA vaccine composition comprises from about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L of NaCl. Other salts that may be present include sodium phosphate, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, disodium hydrogen phosphate dehydrate, magnesium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, or other salts known to those skilled in the art. In some embodiments, the RSV RNA vaccine composition comprises from about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L of sodium dihydrogen phosphate. In some embodiments, the RSV RNA vaccine composition comprises from about 1, 2, 3, 4, or 5 g / L of disodium hydrogen phosphate. In some embodiments, the RSV RNA vaccine composition comprises from about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L of sodium dihydrogen phosphate and from about 1, 2, 3, 4, or 5 g / L of disodium hydrogen phosphate. When the adjuvant is in a separate container from the antigen, the salt (e.g., sodium chloride) may be present in both containers. In some embodiments, the RSV RNA vaccine composition comprises from about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L of NaCl, from about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L of sodium dihydrogen phosphate, and from about 1, 2, 3, 4, or 5 g / L of disodium hydrogen phosphate. When the adjuvant is in a separate container from the antigen, the salt (e.g., sodium chloride) may be present in both containers.

[0171] In certain embodiments, the acceptable materials included in the RSV RNA vaccine composition are non-toxic to the recipient at the doses and concentrations employed. In certain embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, sucrose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring agents, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters, polysorbates (such as polysorbate-20, polysorbate-80), triton, tromethamine, lecithin, cholesterol, or tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (sodium chloride or potassium chloride) or mannitol sorbitol); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants.

[0172] Suitable vehicles or carriers can be water for injection, saline solution, or artificial cerebrospinal fluid. In some embodiments, the vehicle or carrier may be supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles.

[0173] In some embodiments, the RSV RNA vaccine composition comprises one or more buffers. Exemplary buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (e.g., with aluminum hydroxide adjuvant); or citrate buffer. The buffer is typically included in the range of 5 - 20 mM. The pH of the RSV RNA vaccine composition is typically from 5.0 to 8.1, more typically from 6.0 to 8.0, such as from 6.5 to 7.5 or from 7.0 to 7.8.

[0174] In some embodiments, the buffer can be included in the RSV RNA vaccine composition at a concentration of at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM. In some embodiments, the buffer can be included at a concentration up to 50 mM, up to 60 mM, up to 70 mM, up to 80 mM, up to 90 mM, up to 100 mM, up to 110 mM, up to 120 mM, up to 130 mM, up to 140 mM, up to 150 mM, or up to 160 mM. In an exemplary embodiment, the buffer comprises 30 mM L-histidine / L-histidine hydrochloride.

[0175] In some embodiments, the RSV RNA vaccine composition can comprise a humectant (including, for example, sorbitol or a suitable substitute thereof).

[0176] The components of the RSV RNA vaccine composition may be present at a concentration acceptable for the site of administration. In certain embodiments, a buffer may be used to maintain the composition at physiological pH or slightly below physiological pH. In some embodiments, the pH of the composition may be at least 5, at least 5.1, at least 5.2, at least 5.3, at least 5.4, at least 5.5, at least 5.6, at least 5.7, at least 5.8, at least 5.9, at least 6.0, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, at least 6.6, at least 6.7, at least 6.8, at least 6.9, at least 7.0, at least 7.1, at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, or at least 7.9. In some embodiments, the pH of the composition may be up to 5.1, up to 5.2, up to 5.3, up to 5.4, up to 5.5, up to 5.6, up to 5.7, up to 5.8, up to 5.9, up to 6.0, up to 6.1, up to 6.2, up to 6.3, up to 6.4, up to 6.5, up to 6.6, up to 6.7, up to 6.8, up to 6.9, up to 7.0, up to 7.1, up to 7.2, up to 7.3, up to 7.4, up to 7.5, up to 7.6, up to 7.7, up to 7.8, up to 7.9, or up to 8.0. In an exemplary embodiment, the pH of the composition may be in the range of 5 to 8. In an exemplary embodiment, the pH of the composition may be in the range of 5.5 to 6.5. In an exemplary embodiment, the pH of the composition may be 6.0.

[0177] In some embodiments, the RSV RNA vaccine composition may comprise an ionic excipient. An ionic excipient may be included in the antibody formulation to alter the charge state of the antibody in the formulation, to alter the distribution of the antibody in the formulation, and / or to colloidal-stabilize the antibody in the formulation. In some embodiments, the ionic excipient may include charged amino acids (including, for example, lysine and / or arginine). In some embodiments, the ionic excipient may include salts (including, for example, arginine hydrochloride (arginine-HCl), lysine hydrochloride (lysine-HCl), or sodium chloride (NaCl)). In some embodiments, the amino acid or amino acid salt may include an amino acid of biological activity (e.g., L-form). In some embodiments, the ionic excipient may be included at a concentration of at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM. In some embodiments, the ionic excipient may be included at a concentration up to 50 mM, up to 60 mM, up to 70 mM, up to 80 mM, up to 90 mM, up to 100 mM, up to 110 mM, up to 120 mM, up to 130 mM, up to 140 mM, up to 150 mM, or up to 160 mM. In an exemplary embodiment, the ionic excipient may be present at a concentration in the range of 50 mM to 150 mM. In an exemplary embodiment, the ionic excipient may be present at a concentration in the range of 75 mM to 100 mM. In an exemplary embodiment, the ionic excipient may include L-arginine hydrochloride present at a concentration of 75 mM or 80 mM.

[0178] In some embodiments, the RSV RNA vaccine composition may further comprise a sugar (including, for example, sucrose). In some embodiments, the composition may comprise up to 0.5% (w / v) sucrose, up to 1% (w / v) sucrose, up to 5% (w / v) sucrose, up to 10% (w / v) sucrose, or up to 15% (w / v) sucrose. In some embodiments, the sugar may be included at a concentration of at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM. In some embodiments, the sugar may be included at a concentration up to 60 mM, up to 70 mM, up to 80 mM, up to 90 mM, up to 100 mM, up to 110 mM, up to 120 mM, up to 130 mM, up to 140 mM, up to 150 mM, or up to 160 mM. In an exemplary embodiment, the sugar includes sucrose at a concentration in the range of 100 mM to 140 mM. For example, the composition may include sucrose at a concentration of 120 mM.

[0179] In some embodiments, the RSV RNA vaccine composition may further comprise a surfactant (including, for example, polysorbate). Polysorbate may include, for example, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In some embodiments, the surfactant may be included at a concentration of 0.0001% (w / v), at least 0.001% (w / v), at least 0.002% (w / v), at least 0.01% (w / v), at least 0.02% (w / v), at least 0.03% (w / v), at least 0.04% (w / v), at least 0.05% (w / v), at least 0.06% (w / v), at least 0.07% (w / v), at least 0.08% (w / v), at least 0.09% (w / v), or at least 0.1% (w / v). In some embodiments, the surfactant may be included at a concentration of up to 0.0001% (w / v), up to 0.0005% (w / v), up to 0.001% (w / v), up to 0.002% (w / v), up to 0.01% (w / v), up to 0.02% (w / v), up to 0.03% (w / v), up to 0.04% (w / v), up to 0.05% (w / v), up to 0.06% (w / v), up to 0.07% (w / v), up to 0.08% (w / v), up to 0.09% (w / v), or up to 0.1% (w / v). For example, in an exemplary embodiment, the surfactant may be included at a concentration in the range of 0.001% (w / v) to 0.5% (w / v), in the range of 0.002% (w / v) to 0.1% (w / v), or in the range of 0.01% (w / v) to 0.05% (w / v). In an exemplary embodiment, polysorbate 80 is included in the range of 0.01% (w / v) to 0.05% (w / v). In another exemplary embodiment, the composition comprises 0.02% (w / v) polysorbate 80. In another exemplary embodiment, the composition comprises 0.04% (w / v) polysorbate 80.

[0180] The RSV RNA vaccine compositions described herein may comprise a detergent, such as a polyoxyethylene sorbitan surfactant (referred to as "Tween"), octylphenol polyether (such as octylphenol polyether-9 (Triton X-100) or tert-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide (CTAB), or sodium deoxycholate, for example, for lysing antigen vaccines or surface antigen vaccines. The detergent may be present only in trace amounts. In some embodiments, the RSV RNA vaccine compositions used in the methods disclosed herein comprise trace amounts of other residual components, such as antibiotics (e.g., neomycin, kanamycin, or polymyxin B). When the adjuvant is in a separate container from the RNA encoding the antigen, this detergent is typically present in the RNA-containing container.

[0181] In an exemplary embodiment, the RSV RNA vaccine composition is sterile. The RSV RNA vaccine composition is typically non-pyrogenic, for example containing less than 0.25 or 0.5 EU (endotoxin units, a standard measure) per dose. For example, the RSV RNA vaccine composition may contain <0.1 EU per dose. The RSV RNA vaccine composition is typically gluten-free.

[0182] In some embodiments, the RSV RNA vaccine composition can be stored at -20 °C to -70 °C.

[0183] In some embodiments, the RSV RNA vaccine composition can be stored at 2 °C to 8 °C. In some embodiments, the RSV RNA vaccine compositions described herein are stable upon long-term storage at room temperature or at a temperature in the range of 2 °C to 8 °C (including, for example, 5 °C). As used herein, room temperature is typically a temperature in the range of 22 °C to 25 °C. Suitably, the RSV RNA vaccine composition is stable after storage at a temperature in the range of 2 °C to 8 °C (including, for example, 5 °C) for at least one month, at least three months, or at least six months. As used herein, the term "stable" over a storage period (or "stability") is used to indicate that the formulation resists aggregation, degradation, half-antibody formation, and / or fragmentation.

[0184] When considering parenteral administration, the RSV RNA vaccine composition can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution that contains the RSV RNA vaccine in a pharmaceutically acceptable vehicle. In some embodiments, a suitable vehicle for parenteral injection is sterile distilled water, in which the antibody is formulated as a sterile isotonic solution that is appropriately preserved. Additionally or alternatively, a formulation suitable for parenteral administration can include a sterile aqueous preparation of the RSV RNA vaccine composition, or a dispersion of a sterile powder of the RSV RNA vaccine composition, which can be isotonic with the recipient's blood. Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride. A solution of the anti-RSV antibody or an antigen-binding fragment thereof can be prepared in water. In certain embodiments, a solution of the anti-RSV antibody or an antigen-binding fragment thereof prepared in water can be mixed with a non-toxic surfactant.

[0185] A dispersion of the RSV RNA vaccine can be prepared in water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), vegetable oils, glycerol esters, and mixtures thereof. In some embodiments, the final dosage form can be sterile, fluid, and stable under manufacturing and storage conditions. The necessary fluidity can be achieved, for example, by using liposomes, in the case of a dispersion, by using an appropriate particle size, or by using surfactants. Sterilization of the liquid preparation can be achieved by any convenient method that maintains the biological activity of the anti-RSV antibody or an antigen-binding fragment thereof (e.g., by filtration sterilization). Methods for preparing the powder include vacuum drying and lyophilization of the sterile injectable solution. Various antimicrobial agents (e.g., antibacterial agents, antiviral agents, and antifungal agents, including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.) can be used to prevent subsequent microbial contamination. Absorption of the anti-RSV antibody or an antigen-binding fragment thereof over an extended period of time can be achieved by including agents for delaying absorption (e.g., aluminum monostearate and gelatin).

[0186] In an exemplary embodiment, the RSV RNA vaccine composition is provided as a liquid solution in a vial. The vaccine can be kept frozen until use. In some embodiments, the RSV RNA vaccine is provided in a 0.5 mL dose containing 10 micrograms, 30 micrograms, 75 micrograms, or 110 micrograms of mRNA. In certain embodiments, the RSV RNA vaccine is diluted with 2.2X PBS (2°C to 8°C). Adjuvant

[0187] In some embodiments, the RSV RNA vaccine composition does not contain an adjuvant. In some embodiments, the RSV RNA vaccine composition contains one or more adjuvants, which can be used to enhance the immune response (humoral and / or cellular response) elicited in a subject receiving the composition. In some embodiments, the RSV RNA vaccine composition contains an oil-in-water emulsion adjuvant. In some embodiments, the RSV RNA vaccine composition contains squalene.

[0188] In some embodiments, the RSV RNA vaccine composition contains an oil-in-water emulsion and at least one surfactant.

[0189] In some embodiments, the RSV RNA vaccine composition contains one or more tocopherols.

[0190] In some embodiments, the RSV RNA vaccine composition contains tocopherol and squalene. In some embodiments, the oil content ranges from 2% to 20% (by volume).

[0191] In some embodiments, the RSV RNA vaccine composition contains an adjuvant that comprises a mineral-containing composition (including calcium salts and aluminum salts (or mixtures thereof)). The calcium salts include calcium phosphate. The aluminum salts include hydroxides, phosphates, sulfates, etc., where the salts take any suitable form (e.g., gel, crystalline, amorphous, etc.). The mineral-containing composition can also be formulated as particles of the metal salt.

[0192] In some embodiments, the RSV RNA vaccine composition contains an adjuvant that comprises one or more saponins, which are a heterogeneous group of sterol glycosides and triterpene glycosides found in the bark, leaves, stems, roots, and flowers of a wide range of plant species. Saponins from the bark of Quillaja saponaria Molina have been widely studied as adjuvants. Saponins from Smilax ornata (Mexican sarsaparilla), Gypsophilla paniculata (“brides veil”), and Saponaria officianalis are also commercially available.

[0193] Saponin adjuvant formulations include purified formulations (such as QS21), as well as lipid formulations (such as ISCOM). QS21 is sold as a product. Combinations of saponins and cholesterol can be used to form unique particles called immunostimulating complexes (ISCOM). In some embodiments, ISCOM includes phospholipids (such as phosphatidylethanolamine or phosphatidylcholine). Any known saponin can be used in ISCOM. In an exemplary embodiment, ISCOM includes one or more of QuilA, QHA, and QHC.

[0194] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant containing a lipid adjuvant.

[0195] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant that comprises a bacterial ADP-ribosylating toxin (e.g., Escherichia coli (E. coli) heat-labile enterotoxin "LT", cholera toxin "CT", or pertussis toxin "PT") and its detoxified derivatives (such as mutant toxins designated LT-K63 and LT-R72).

[0196] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant that comprises a bioadhesive and a mucoadhesive (such as esterified hyaluronic acid microspheres or chitosan and its derivatives).

[0197] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant containing a cytokine inducer.

[0198] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant containing liposomes.

[0199] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant that comprises a polyoxyethylene ether and / or a polyoxyethylene ester. Such formulations further comprise a combination of a polyoxyethylene sorbitan surfactant and an octylphenol polyether and a combination of a polyoxyethylene alkyl ether or ester surfactant and at least one additional nonionic surfactant (such as an octylphenol polyether). Exemplary polyoxyethylene ethers are selected from the group consisting of polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

[0200] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant that comprises muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxyl propionamide (DTP-DPP or THERAMIDE TM ), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0201] The RSV RNA vaccine composition may comprise one or more adjuvants, e.g., 2, 3, 4 or more adjuvants. For example, the RSV RNA vaccine composition may comprise an oil-in-water emulsion and a cytokine inducer. IV. Vaccination Methods

[0202] The RSV vaccines disclosed herein can be administered to a subject to induce an immune response against the RSV F protein, wherein the anti-antigen antibody titer in the subject after vaccination is increased relative to the anti-antigen antibody titer in a subject not vaccinated with the RSV vaccines disclosed herein or relative to an alternative vaccine against RSV. "Anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0203] In one aspect, the present disclosure provides a method of priming an immune response against RSV or protecting a subject from RSV infection, the method comprising administering to the subject an RSV vaccine as described herein. The present disclosure also provides an RSV vaccine as described herein for priming an immune response against RSV or protecting a subject from RSV infection. The present disclosure also provides an RSV mRNA as described herein for manufacturing a vaccine for priming an immune response against RSV or for protecting a subject from RSV infection.

[0204] In certain embodiments, after administration of the RSV vaccine, the subject has a higher serum concentration of anti-RSV neutralizing antibodies relative to a subject administered an RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:1.

[0205] In certain embodiments, after administration of the RSV vaccine, the subject has a comparable serum concentration of anti-RSV neutralizing antibodies relative to a subject administered an RSV protein vaccine co-administered with an adjuvant.

[0206] In certain embodiments, the RSV vaccine increases the serum concentration of antibodies that have binding specificity for the site of the RSV F protein.

[0207] In certain embodiments, after administration of the RSV vaccine, the subject has a lower serum concentration of antibodies that have binding specificity for site I or site II of the RSV F protein relative to a subject administered an RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:2.

[0208] In certain embodiments, the RSV vaccine increases the serum concentration of neutralizing antibodies in subjects with pre-existing RSV immunity. V. RSV F Protein

[0209] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine that comprises messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen.

[0210] In some embodiments, the ORF is codon-optimized. Codon optimization can refer to introducing certain codons (to exchange corresponding wild-type codons encoding the same amino acid), which may be more beneficial for RNA stability and / or the codon usage of the subject.

[0211] In some embodiments, the epitopes shared between Pre-F and Post-F of the RSV F protein are blocked. When RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject, blocking the epitopes reduces or eliminates the production of antibodies against the epitopes. This can increase the proportion of antibodies targeting epitopes specific to a particular conformation of F, such as the pre-fusion conformation (e.g., antibodies to the target site antibodies). Since F has a pre-fusion conformation in the virus that has not yet entered the cell, an increased proportion of antibodies targeting Pre-F can provide greater neutralization (e.g., expressed as a neutralization-to-binding ratio as described herein). Blocking can be achieved by engineering bulky moieties (such as N-glycans) near the shared epitope. For example, N-glycosylation sites that are not present in wild-type F can be added, for example, by mutating appropriate residues to asparagine. In some embodiments, the blocked epitope is an epitope of antigenic site I of RSV F. In some embodiments, two or more epitopes shared between pre-F and post-F are blocked. In some embodiments, two or more epitopes of antigenic site I of RSV F are blocked. In some embodiments, one or more or all epitopes that are topologically overlapping with the blocked epitope are also blocked. The blocked epitope can be an epitope of antigenic site I of RSV F.

[0212] In some embodiments, the RSV F polypeptide comprises an asparagine substitution (i.e., E328N, S348N, or R507N) at one or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO:1. In some embodiments, the RSV F polypeptide comprises asparagine substitutions (i.e., E328N, S348N, or R507N) at two or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO:1. In some embodiments, the RSV F polypeptide comprises asparagine substitutions (i.e., E328N, S348N, and R507N) at positions 328, 348, and 507 of SEQ ID NO:1.

[0213] Asparagine can serve as a glycosylation site (see WO 2019 / 195291, incorporated herein by reference). Additionally, without wishing to be bound by any particular theory, when RNA encoding an antigenic RSV F polypeptide (e.g., mRNA) is administered to a subject, glycans at these sites can inhibit the production of antibodies against nearby epitopes, including epitopes shared by pre-fusion and post-fusion RSV F proteins. In some embodiments, glycosylation of asparagines corresponding to positions 328, 348, or 507 of SEQ ID NO:1 blocks at least one epitope shared between pre-fusion RSV F and post-fusion RSV F, such as the epitope of antigenic site 1. Inhibiting the production of antibodies against epitopes shared by pre-fusion and post-fusion RSV F proteins can be beneficial because it can direct the production of antibodies against epitopes unique to the pre-fusion RSV F protein (such as the site epitope), which may have more effective neutralizing activity than antibodies against other RSV F epitopes. The site epitope involves amino acid residues 62 - 69 and 196 - 209 of SEQ ID NO:1. Thus, in some embodiments, the RSV F polypeptide comprises amino acid residues 62 - 69 and 196 - 209 of SEQ ID NO:1.

[0214] The RSV F polypeptides described herein can have deletions or substitutions of varying lengths relative to wild-type RSV F. For example, in the RSV F polypeptide of SEQ ID NO:1, positions 98 - 144 of the wild-type sequence (SEQ ID NO:1) are replaced with GSGNVGL (SEQ ID NO:15), resulting in a net removal of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO:1 correspond to positions 288, 308, and 467 of SEQ ID NO:3. Alternatively, in the RSV F polypeptide of SEQ ID NO:3, positions 98 - 146 of the wild-type sequence (SEQ ID NO:1) are replaced with GSGNVGLGG (SEQ ID NO:16, positions 98 - 106 of SEQ ID NO:3), resulting in a net removal of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO:1 correspond to positions 290, 310, and 469 of SEQ ID NO:3.

[0215] Generally, positions in the constructs described herein can be mapped to the wild-type sequence of SEQ ID NO:1 by pairwise alignment, for example, using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, gap penalty 10, gap extension penalty 0.5). See also the discussion of structural alignment provided herein as an alternative method for identifying corresponding positions.

[0216] In some embodiments, the RSV F polypeptide comprises a mutation that is the addition of a glycan to block epitopes on the pre-fusion antigen that are structurally similar to those on the post-fusion RSV F surface. In some embodiments, the glycan is added to specifically block epitopes that may be present in the post-fusion conformation of RSV F. In some embodiments, a glycan is added that blocks epitopes that may be present in the post-fusion conformation of RSV F but does not affect one or more epitopes (such as the site epitopes) present on the pre-fusion conformation of RSV F.

[0217] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO:1.

[0218] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO:2.

[0219] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO:3.

[0220] In some embodiments, the RSV F polypeptide comprises DS-CAV1 amino acid substitutions (such as those described in McLellan et al., Science, 342(6158):592-598, 2013), wherein further modifications are made, including at least one, two or three of the above asparagines. Relative to SEQ ID NO:1, the CAV1 mutations are S190F and V207L. Relative to SEQ ID NO:1, the DS mutations are S155C and S290C.

[0221] In some embodiments, the amino acid substitution or amino acid substitution pair is one or more interprotomer stabilizing substitutions. Exemplary substitutions that can be interprotomer stabilizing are V207L; N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L (using the position numbering of SEQ ID NO:1).

[0222] In some embodiments, the amino acid substitution or amino acid substitution pair is protomer-internal stabilizing. Exemplary substitutions that can be protomer-internal stabilizing are V220I; and A74L and Q81L (using the position numbering of SEQ ID NO:1).

[0223] In some embodiments, the amino acid substitution is helix stabilizing, i.e., predicted to stabilize the helical domain of RSV F. Stabilization of the helical domain can generally contribute to the stability of the site epitope and the pre-fusion conformation of RSV F. Exemplary substitutions that can be helix stabilizing are N216P or I217P (using the position numbering of SEQ ID NO:1). Position 217 in SEQ ID NO:1 corresponds to position 177 in SEQ ID NO:3.

[0224] In some embodiments, the amino acid substitution is helix capping. In some embodiments, the amino acid substitution is helix PRO capping. Helix capping is based on the following biophysical observation: while proline residue mutations placed within an α-helix may disrupt helix formation, a proline at the N-terminus of a helical region can help induce helix formation by stabilizing the PHI / PSI bond angles. Exemplary substitutions that can be helix capping are N216P or I217P (using the position numbering of SEQ ID NO:1).

[0225] In some embodiments, the amino acid substitution replaces a disulfide mutation of DS-CAV1. In some embodiments, the engineered disulfide of DS-CAV1 is reverted to the wild type of DS-CAV1 (C69S and / or C212S mutations) (using the position numbering of SEQ ID NO:1). In some embodiments, one or more C residues of DS-CAV1 are replaced with S residues to eliminate disulfide bonds. In some embodiments, the C69S or C212S substitution using the position numbering of SEQ ID NO:1 eliminates the disulfide bond. In some embodiments, the RSV F polypeptide comprises both C69S and C212S using the position numbering of SEQ ID NO:1. In some embodiments, replacing such cysteines and thereby eliminating the disulfide bond prevents reduction of the RSV F polypeptide (i.e., acceptance of electrons from a reducing agent). In some embodiments, the I217P substitution using the position numbering of SEQ ID NO:1 is included in the antigen instead of the substitution at C69 and / or C212.

[0226] In some embodiments, the amino acid substitution prevents proteolysis by trypsin or a trypsin-like protease. In some embodiments, the amino acid substitution that blocks such proteolysis is located in the region of the heptad repeat region B (HRB) of RSV F.

[0227] The appearance of fragments consistent with proteolysis of the RSV F polypeptide containing the wild-type HRB region indicates that lysine or arginine in this region is the target of proteolysis. Amino acid substitutions used to remove K or R residues can be referred to as knockouts (KO). In some embodiments, K or R is substituted with L or Q. In some embodiments, K is substituted with L or Q. In some embodiments, the RSV F polypeptide contains K498L and / or K508Q using the position numbering of SEQ ID NO:1. The corresponding positions in SEQ ID NO:3 are 458 and 468, respectively. In some embodiments, the RSV F polypeptide contains both K498L and K508Q.

[0228] In some embodiments, the amino acid substitution adds a glycan. In some embodiments, the amino acid substitution increases glycosylation by adding a glycan to the RSV F polypeptide. The substitution used to add a glycan can also be referred to as engineered glycosylation compared to native glycosylation (without additional glycans).

[0229] In some embodiments, the amino acid substitution used to add a glycan is substitution with N. In some embodiments, substitution with N allows N-linked glycosylation. In some embodiments, substitution with N is accompanied by substitution with T or S at the second amino acid position at the C-terminus of N, thereby forming an NxT / S glycosylation motif. In some embodiments, N is surface-exposed.

[0230] Each of the above-listed substitutions and mutations in the RSV F polypeptide is described in more detail in WO 2019 / 195291, which is incorporated herein by reference.

[0231] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises one or more of the following substitutions relative to the amino acid sequence shown in SEQ ID NO:1:

[0232] 1) Amino acid positions 98 - 146 of SEQ ID NO:1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO:16);

[0233] 2) Amino acid substitutions S190F and V207L;

[0234] 3) Amino acid substitution I217P;

[0235] 4) Amino acid substitutions E328N, S348N, and R507N;

[0236] 5) Amino acid substitution L373R;

[0237] 6) Amino acid substitution K498L; and

[0238] 7) Amino acid substitution K508Q.

[0239] In another aspect, the present disclosure provides an RSV vaccine comprising an mRNA containing an ORF encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises each of the following substitutions relative to the amino acid sequence shown in SEQ ID NO: 1:

[0240] 1) Amino acid positions 98 - 146 of SEQ ID NO: 1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO: 16);

[0241] 2) Amino acid substitutions S190F and V207L;

[0242] 3) Amino acid substitution I217P;

[0243] 4) Amino acid substitutions E328N, S348N, and R507N;

[0244] 5) Amino acid substitution L373R;

[0245] 6) Amino acid substitution K498L; and

[0246] 7) Amino acid substitution K508Q.

[0247] In certain embodiments, the RSV F protein antigen comprises a transmembrane domain and a cytoplasmic tail amino acid sequence IMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 17).

[0248] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence shown in any one of SEQ ID NOs: 4 - 6. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence shown in any one of SEQ ID NOs: 12 - 14. VI. RSV RNA

[0249] The RSV vaccines of the present disclosure may comprise at least one ribonucleic acid (RNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen.

[0250] In certain embodiments, the RNA is an mRNA comprising an ORF encoding an RSV F protein antigen. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5' UTR, 3' UTR, poly(A) tail, and / or 5' cap.

[0251] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO:12.

[0252] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO:13.

[0253] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO:14. II.A. 5' Cap

[0254] The mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also referred to as "m 7 G" or "cap-0") contains guanosine linked to the first transcribed nucleotide by a 5'-5'-triphosphate bond.

[0255] Typically, the 5' cap is added as follows: First, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyl transferase, generating a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Publication No. US2016 / 0032356 and U.S. Publication No. US2018 / 0125989, which are incorporated herein by reference.

[0256] 5'-capping of polynucleotides can be accomplished concomitantly during in vitro transcription reactions using the following chemical RNA cap analogs according to the manufacturer's protocol to generate 5'-guanosine cap structures: 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; m7G(5')ppp(5')(2'-OMeA)pG; m7G(5')ppp(5')(2'-OMeA)pU; m7G(5')ppp(5')(2'-OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNA can be accomplished post-transcriptionally using vaccinia virus capping enzyme to generate a cap 0 structure: m7G(5')ppp(5')G. A cap 1 structure can be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. A cap 2 structure can be generated from the cap 1 structure and then the 5' penultimate nucleotide is 2'-O-methylated using 2'-O methyltransferase. A cap 3 structure can be generated from the cap 2 structure and then the 5' antepenultimate nucleotide is 2'-O-methylated using 2'-O methyltransferase.

[0257] In certain embodiments, the mRNA of the present disclosure comprises a 5' cap selected from the group consisting of: 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, m7G(5')ppp(5')(2'-OMeA)pG, m7G(5')ppp(5')(2'-OMeA)pU, and m7G(5')ppp(5')(2'-OMeG)pG.

[0258] In certain embodiments, the mRNA of the present disclosure comprises a 5' cap: II.B. Untranslated Regions (UTRs)

[0259] In some embodiments, the mRNA of the present disclosure includes 5' and / or 3' untranslated regions (UTRs). In an mRNA, the 5' UTR starts at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR starts immediately after the stop codon and continues until the transcription termination signal.

[0260] In some embodiments, the mRNA disclosed herein can include a 5'UTR that contains one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5'UTR can have a length of about 10 to 5,000 nucleotides. In some embodiments, the 5'UTR can have a length of about 50 to 500 nucleotides. In some embodiments, the 5'UTR has a length of at least about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0261] In some embodiments, the mRNA disclosed herein can comprise a 3' UTR that includes one or more of the following: a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in a cell, or one or more binding sites for miRNAs. In some embodiments, the 3' UTR can have a length of 50 to 5,000 nucleotides or longer. In some embodiments, the 3' UTR can have a length of 50 to 1,000 nucleotides or longer. In some embodiments, the 3' UTR has a length of at least about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0262] In some embodiments, the mRNA disclosed herein can comprise a 5' or 3' UTR that is derived from a gene different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0263] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, the 5' UTR sequence can include a partial sequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve the nuclease resistance of the mRNA and / or improve the half-life of the mRNA. Inclusion of the sequence encoding human growth hormone (hGH) or a fragment thereof into the 3' end or untranslated region of the mRNA is also contemplated. Generally speaking, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts and include, for example, modifications made to improve such resistance of the mRNA to nuclease digestion in vivo.

[0264] Exemplary 5’UTRs include sequences derived from the CMV immediate early 1 (IE1) gene (U.S. Publication Nos. 2014 / 0206753 and 2015 / 0157565, each incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO:18) (U.S. Publication No. 2016 / 0151409, incorporated herein by reference).

[0265] In various embodiments, the 5’UTR can be derived from the 5’UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5’-terminal oligopyrimidine (TOP) tract. In addition, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5’UTR derived from the 5’UTR of a TOP gene lacks a 5’TOP motif (oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each incorporated herein by reference).

[0266] In certain embodiments, the 5’UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, ibid).

[0267] In certain embodiments, the 5’UTR is derived from the 5’UTR of the hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, ibid).

[0268] In certain embodiments, the 5’UTR is derived from the 5’UTR of the ATP5A1 gene (U.S. Publication No. 2016 / 0166710, ibid).

[0269] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5’UTR.

[0270] In some embodiments, the 5’UTR comprises the nucleic acid sequence set forth in SEQ ID NO:10. In some embodiments, the 3’UTR comprises the nucleic acid sequence set forth in SEQ ID NO:11. The 5’UTR and 3’UTR are further described in detail in WO 2012 / 075040 (incorporated herein by reference).

[0271] II.C. Polyadenylation Tail

[0272] As used herein, the terms "poly(A) sequence", "poly(A) tail", and "poly(A) region" refer to an adenosine nucleotide sequence at the 3' end of an mRNA molecule. The poly(A) tail can confer stability to the mRNA and protect it from exonucleolytic degradation. The poly(A) tail can enhance translation. In some embodiments, the poly(A) tail is substantially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides can substantially have a length of 100 nucleotides. In certain embodiments, the poly(A) tail can be interrupted by at least one nucleotide different from adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides can have a length of more than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides different from adenosine nucleotide). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:19).

[0273] As used herein, "poly(A) tail" typically refers to RNA. However, in the context of the present disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., "poly(T) sequence").

[0274] The poly(A) tail can contain from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0275] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by conventional chemical synthesis methods without transcription from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocol, or alternatively, by using immobilized poly(A) polymerase, e.g., using the methods and means described in WO 2016 / 174271.

[0276] The nucleic acid can comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein most of the nucleic acid molecules comprise from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0277] In some embodiments, the nucleic acid can include a poly(A) tail derived from template DNA and can additionally include at least one additional poly(A) tail produced by enzymatic polyadenylation, such as described in WO 2016 / 091391. In certain embodiments, the nucleic acid comprises at least one polyadenylation signal. In various embodiments, the nucleic acid can comprise at least one poly(C) sequence. As used herein, the term "poly(C) sequence" is intended to be a cytosine nucleotide sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises from about 10 to about 200 cytosine nucleotides, from about 10 to about 100 cytosine nucleotides, from about 20 to about 70 cytosine nucleotides, from about 20 to about 60 cytosine nucleotides, or from about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides. II.D. Chemical Modifications

[0278] The mRNA disclosed herein can be modified or unmodified. In some embodiments, the mRNA can include at least one chemical modification. In some embodiments, the mRNA disclosed herein can contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouridine (5-uridine), dihydro-uridine, 2-thio-uridine, 4-thio-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-(carboxyhydroxymethyl)-uridine, 5-fluoro-uridine, 5-bromo-uridine, 5-carboxymethylaminomethyl-uridine, 5-methyl-2-thio-uridine, 5-methyl-uridine, N-uridine-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine, 5-methoxyaminomethyl-2-thio-uridine, 5'-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid (v), 1-methyl-pseudouridine, wybutosine, β-D-mannosyl-wybutosine, phosphoramidate, thiophosphate, peptide nucleic acid, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0279] In some embodiments, the disclosed mRNA can contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0280] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0281] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0282] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleotides in the mRNA are chemically modified.

[0283] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleotides in the ORF are chemically modified.

[0284] The preparation of such analogs is described, for example, in U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. II.E. mRNA Synthesis

[0285] The mRNA disclosed herein can be synthesized according to any one of a variety of methods. For example, the mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically carried out with: a linear or circular DNA template containing a promoter, a ribonucleoside triphosphate pool, a buffer system that may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or an RNase inhibitor. The exact conditions can vary according to the particular application. The presence of these reagents is generally not desired in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide a contaminant-free and / or homogeneous mRNA suitable for therapeutic use. Although in some embodiments mRNA provided from in vitro transcription reactions may be required, other sources of mRNA can be used according to the present disclosure, including wild-type mRNA produced by bacteria, fungi, plants, and / or animals.

[0286] In certain embodiments, the mRNA comprises the following structural elements: (i) A 5' cap having the following structure: (ii) A 5' untranslated region (5’UTR) having the nucleic acid sequence SEQ ID NO:10; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO:6; (iv) A 3' untranslated region (3’UTR) having the nucleic acid sequence SEQ ID NO:11; and (v) A poly(A) tail.

[0287] In certain embodiments, the length of the poly(A) tail is from about 10 to about 500 adenosine nucleotides. VII. Lipid Nanoparticles (LNP)

[0288] The LNP of the present disclosure can comprise four classes of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids (e.g., cholesterol); and (iv) helper lipids. A. Cationic Lipids

[0289] Ionizable lipids promote mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance. Exemplary cationic lipids are shown in Table 1 below.

[0290] Table 1 - Ionizable Lipids

[0291] The cationic lipid can be selected from the group consisting of: [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butyrate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); bis((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 8-{ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid 9-heptadecyl ester (SM-102); [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[bis(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl] N-[2-(dimethylamino)ethyl]carbamate (DC-Chol); tetra(8-methylnonyl) 3,3′,3″,3″′-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10); (2-(dioctylammonio)ethyl) decyl phosphate (9A1P9); 5,5-bis((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylic acid ethyl ester (A2-Iso5-2DC18); bis(2-(dodecyldithio)ethyl) 3,3’-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B); 1,1’-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexakis(octan-3-yl) 9,9′,9″,9″′,9″″,9″′″-((((benzene-1,3,5-tricarbonyl)tris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetrakis(octadec-9,12-dienoate) (OF-Deg-Lin); TT3; N; 1 ,N 3 ,N 5 -Tris(3-(dodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-bis[oleoyloxy]-benzamide (MVL5); 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octadecan-9-yl ester (Lipid 5); IM-001; and combinations thereof.

[0292] In certain embodiments, the cationic lipid is biodegradable.

[0293] In various embodiments, the cationic lipid is not biodegradable.

[0294] In some embodiments, the cationic lipid is cleavable.

[0295] In certain embodiments, the cationic lipid is not cleavable.

[0296] The cationic lipid is further described in detail in Dong et al. (PNAS. [Proceedings of the National Academy of Sciences of the United States of America] 111(11):3955 - 60. 2014); Fenton et al. (Adv Mater. [Advanced Materials] 28:2939. 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference. B. PEGylated Lipids

[0297] PEGylated lipid components can provide control over the particle size and stability of nanoparticles. The addition of such components can prevent aggregation of the complexes and provide a means for increasing the circulation lifetime of lipid-nucleic acid pharmaceutical compositions and increasing their delivery to target tissues (Klibanov et al., FEBS Letters 268(1):235-7, 1990). These components can be selected to rapidly exchange the pharmaceutical composition in vivo (see, e.g., U.S. Patent No. 5,885,613).

[0298] PEGylated lipids contemplated include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length covalently linked to a lipid having one or more alkyl chains of C 6 -C 20 (e.g., C 8 、C 10 、C 12 、C 14 、C 16 or C 18 ) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG); PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropyl carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0299] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipids herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000. C. Cholesterol-Based Lipids

[0300] The cholesterol component can provide stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent 5,744,335), imidazole cholesterol ester (“ICE”; WO2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmast-5,22-dien-3-ol), ergosterol; cholestanol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadiene-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); zymosterol (5α-cholest-8,24-dien-3β-ol); lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecolesterol (5,24(28)-cholestadiene-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol. D. Co-lipids

[0301] Helper lipids can enhance the structural stability of LNPs and assist LNPs in endosomal escape. Helper lipids can improve the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is an amphipathic lipid that has fusogenic properties for enhancing the uptake and release of the drug payload. Examples of helper lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoyl phosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoyl phosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0302] Other exemplary helper lipids are dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0303] In various embodiments, the LNP of the present invention comprises (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-002; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE. E. Molar ratio of lipid components

[0304] The molar ratios of the above components can play a role in the effective delivery of mRNA by LNP. The molar ratio of the cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid is A:B:C:D, where A + B + C + D = 100%. In some embodiments, the molar ratio of the cationic lipid relative to the total lipids in the LNP (i.e., A) is 35% - 55%, such as 35% - 50% (e.g., 38% - 42%, such as 40% or 45% - 50%). In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is 0.25% - 2.75% (e.g., 1% - 2%, such as 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is 20% - 50% (e.g., 27% - 30%, such as 28.5% or 38% - 43%). In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is 5% - 35% (e.g., 28% - 32%, such as 30%, or 8% - 12%, such as 10%). In some embodiments, the PEGylated lipid + cholesterol component has the same molar amount as the helper lipid. In some embodiments, the molar ratio of the cationic lipid to the helper lipid contained in the LNP is greater than 1.

[0305] In certain embodiments, the LNP of the present disclosure comprises:

[0306] a cationic lipid in a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid in a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%);

[0307] a polyethylene glycol (PEG)-conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid in a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%);

[0308] a cholesterol-based lipid in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., a cholesterol-based lipid in a molar ratio of 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%, or 50%); and

[0309] An auxiliary lipid in a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., an auxiliary lipid in a molar ratio of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%),

[0310] wherein all molar ratios are relative to the total lipid content of the LNP.

[0311] In certain embodiments, the LNP comprises: a cationic lipid in a molar ratio of 40%; a PEGylated lipid in a molar ratio of 1.5%; a cholesterol-based lipid in a molar ratio of 28.5%; and an auxiliary lipid in a molar ratio of 30%.

[0312] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0313] In various embodiments, the cholesterol-based lipid is cholesterol.

[0314] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0315] In certain embodiments, the LNP comprises: OF-02 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0316] In certain embodiments, the LNP comprises: cKK-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0317] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0318] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0319] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0320] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0321] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0322] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid A".

[0323] In certain embodiments, the LNP comprises: cKK-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid B".

[0324] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid C".

[0325] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid D".

[0326] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid E".

[0327] In certain embodiments, the LNP comprises: 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid 9-heptadecyl ester (SM-102) at a molar ratio of 50%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0328] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159) at a molar ratio of 1.6%.

[0329] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159) at a molar ratio of 1.7%.

[0330] In certain embodiments, the LNP comprises: IM-001 at a molar ratio of 35% to 55%; a polyethylene glycol (PEG)-conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75%; a cholesterol-based lipid at a molar ratio of 20% to 45%; and a helper lipid at a molar ratio of 5% to 35%, wherein all molar ratios are relative to the total lipid content of the LNP.

[0331] In certain embodiments, the LNP comprises: IM-001 at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%, wherein all molar ratios are relative to the total lipid content of the LNP.

[0332] In certain embodiments, the LNP comprises: IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%, wherein all molar ratios are relative to the total lipid content of the LNP.

[0333] To calculate the actual amount of each lipid to be placed in the LNP formulation, the molar amount of the cationic lipid can first be determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. Next, the molar amount of each other lipid can be calculated based on the molar amount of the cationic lipid and the selected molar ratios. These molar amounts can then be converted to weights using the molecular weight of each lipid. F. Buffers and Other Components

[0334] To stabilize the nucleic acid and / or the LNP (e.g., to extend the shelf life of the vaccine product), to facilitate the administration of the LNP pharmaceutical composition, and / or to enhance the in vivo expression of the nucleic acid, the nucleic acid and / or the LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing agents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients include, but are not limited to, parabens, thimerosal, sodium thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0335] The LNP compositions of the present disclosure can be provided in a cryogenic liquid form or a lyophilized form. A variety of cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextran, etc. The cryoprotectant can account for 5%-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, 5%-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.

[0336] The LNP composition can be provided to a patient in an aqueous buffer solution: if previously frozen, thawed, or if previously lyophilized, reconstituted at the bedside in an aqueous buffer solution. The buffer solution can be isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate-buffered saline (PBS). VIII. Vectors

[0337] In one aspect, the present disclosure provides vectors comprising the mRNA compositions disclosed herein. RNA sequences encoding a protein of interest (e.g., mRNA encoding the RSV F protein) can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Suitable vectors can include expression vectors, replication vectors, probe-generating vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0338] In certain embodiments, the vector can be used to express mRNA in a host cell. In various embodiments, the vector can be used as a template for IVT. The construction of the most preferably translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin et al. (2014). Nat. Rev. Drug Discov. [Nature Reviews: Drug Discovery] 13, 759-780; Weissman (2015). Expert Rev. Vaccines [Expert Review of Vaccines] 14, 265-281.

[0339] In some embodiments, the vectors disclosed herein can include at least the following from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding the 5'UTR; a polynucleotide sequence encoding the ORF; a polynucleotide sequence encoding the 3'UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein can contain a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0340] Multiple RNA polymerase promoters are known. In some embodiments, the promoter can be a T7 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences for the T7, T3, and SP6 promoters are known.

[0341] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising the vectors or RNA compositions disclosed herein.

[0342] Any of a variety of different methods can be used to introduce polynucleotides into target cells, e.g., commercially available methods including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, CO)), Multiporator (Eppendorf, Hamburg, Germany), cationic lipid-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems such as the "gene gun" (see, e.g., Nishikawa et al. (2001). Hum Gene Ther. 12(8): 861-70) or TransIT-RNA transfection kit (Mirus, Madison, WI)).

[0343] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0344] Regardless of the method used to introduce foreign nucleic acids into host cells or otherwise expose cells to the inhibitors disclosed herein, a variety of measurements can be made to confirm the presence of the mRNA sequence in the host cells. IX. Self-Replicating RNA and Trans-Replicating RNA Self - replicating RNA

[0345] In one aspect, disclosed herein is self-replicating RNA encoding the RSV F protein.

[0346] Self-replicating RNAs can be generated by using replication elements derived from, for example, alphaviruses and replacing the structural viral proteins with nucleotide sequences encoding a protein of interest (e.g., the RSV F protein). Self-replicating RNAs are typically positive-strand molecules that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA enables the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can be themselves translated to provide in situ expression of the encoded antigen (i.e., the RSV F protein antigen), or can be transcribed to provide further transcripts having the same sense as the delivered RNA, which are translated to provide in situ expression of the antigen. The overall result of this transcriptional sequence is a substantial amplification in the number of introduced replicon RNAs, and thus the encoded antigen becomes the major polypeptide product of the cell.

[0347] A suitable system for achieving self-replication in this way is to use an alphavirus-based replicon. These replicons are positive-strand (sense-strand) RNAs that, upon delivery to a cell, result in the translation of a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that autocleaves to provide a replication complex that produces genomic strand copies of the positive-strand delivered RNA. These negative (-) strand transcripts can themselves be transcribed to produce further copies of the positive-strand parental RNA, and also produce subgenomic transcripts encoding the antigen. Thus, translation of the subgenomic transcripts enables in situ expression of the antigen in the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki forest virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: WO 2005 / 113782, which is incorporated herein by reference.

[0348] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule, and (ii) an RSV F protein antigen. The polymerase can be an alphavirus replicase, for example, comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Although in addition to the non-structural replicase polyprotein, the native alphavirus genome also encodes structural virion proteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, the self-replicating RNA may result in the production of copies of its own genomic RNA in cells, but not in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot perpetuate itself in an infectious form. The alphavirus structural proteins necessary for perpetuation in wild-type viruses are absent from the self-replicating RNAs of the present disclosure, and their positions are replaced by one or more genes encoding the desired immunogen such that the subgenomic transcript encodes the immunogen rather than the alphavirus structural virion proteins. The self-replicating RNA is further described in WO 2011005799, which is incorporated herein by reference. Trans - replicating RNA

[0349] In one aspect, the present disclosure provides a trans-replicating RNA encoding an RSV F protein.

[0350] The trans-replicating RNA has elements similar to the self-replicating RNA described above. However, for the trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the RNA replicase described above (e.g., an alphavirus replicase), and the second RNA molecule encodes the protein of interest (e.g., the RSV F protein antigen). The RNA replicase can replicate one or both of the first RNA molecule and the second RNA molecule, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. The trans-replicating RNA is further described in WO 2017162265, which is incorporated herein by reference. X. Methods for Manufacturing LNP Vaccines

[0351] The LNPs of the present invention can be prepared by a variety of techniques. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by depositing the selected lipids on the inner wall of a suitable container or vessel (by dissolving the lipids in a suitable solvent and then evaporating the solvent to leave a film inside the vessel) or by spray drying. Then, an aqueous phase can be added to the vessel with vortexing, which causes the formation of MLVs. The MLVs can then be formed into unilamellar vesicles (ULVs) by homogenization, sonication, or extrusion. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0352] Multiple methods are described in US2011 / 0244026, US2016 / 0038432, US 2018 / 0153822, US2018 / 0125989, and US2021 / 0046192 and can be used to manufacture LNP vaccines. An exemplary method involves encapsulating mRNA by mixing it with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles, as described in US2016 / 0038432. Another exemplary method involves encapsulating mRNA by mixing pre-formed LNP with mRNA, as described in US2018 / 0153822.

[0353] In some embodiments, the method of preparing mRNA-loaded LNP includes the step of heating one or more solutions to a temperature greater than ambient temperature, where the one or more solutions are a solution comprising pre-formed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising LNP-encapsulated mRNA. In some embodiments, the method includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution prior to the mixing step. In some embodiments, the method includes heating one or more of the solution comprising pre-formed LNP, the solution comprising mRNA, and the solution comprising LNP-encapsulated mRNA during the mixing step. In some embodiments, the method includes heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the one or more solutions are heated to the following temperature or greater: about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the one or more solutions are heated to the following temperature ranges: about 25°C - 70°C, about 30°C - 70°C, about 35°C - 70°C, about 40°C - 70°C, about 45°C - 70°C, about 50°C - 70°C, or about 60°C - 70°C. In some embodiments, the temperature is about 65°C.

[0354] A variety of methods can be used to prepare the mRNA solutions suitable for the present disclosure. In some embodiments, the mRNA can be directly dissolved in the buffer solutions described herein. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution prior to mixing with the lipid solution for encapsulation. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution immediately prior to mixing with the lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA at a concentration of or greater than about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml in water or buffer.

[0355] In some embodiments, the mRNA stock solution and the buffer solution are mixed using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than the rate of the mRNA stock solution. For example, the buffer solution can be mixed at a rate of at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100 - 6000 ml / min (e.g., about 100 - 300 ml / min, 300 - 600 ml / min, 600 - 1200 ml / min, 1200 - 2400 ml / min, 2400 - 3600 ml / min, 3600 - 4800 ml / min, 4800 - 6000 ml / min, or 60 - 420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of or greater than about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min.

[0356] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10 - 600 ml / min (e.g., about 5 - 50 ml / min, about 10 - 30 ml / min, about 30 - 60 ml / min, about 60 - 120 ml / min, about 120 - 240 ml / min, about 240 - 360 ml / min, about 360 - 480 ml / min, or about 480 - 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of or greater than about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min.

[0357] The process of incorporating the desired mRNA into lipid nanoparticles is referred to as "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255 - 8. The nucleic acid incorporated into the LNP can be located entirely or partially within the internal space of the lipid nanoparticle, within the bilayer of the lipid nanoparticle membrane, or associated with the outer surface of the lipid nanoparticle membrane. Incorporating mRNA into lipid nanoparticles is also referred to herein as "encapsulation", where the nucleic acid is entirely or substantially included within the internal space of the lipid nanoparticle.

[0358] Suitable LNPs can be prepared in a variety of sizes. In some embodiments, reduced lipid nanoparticle size is associated with more efficient delivery of the mRNA. Selection of an appropriate LNP size can take into account the site of the target cell or tissue and to some extent the application for which the lipid nanoparticles will be used.

[0359] A variety of methods can be used to vary the size of the lipid nanoparticle population. In various embodiments, the methods herein utilize a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of the LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed into the Zetasizer machine. The z - average diameter (nm) or the cumulant average is considered the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles can be alternated with quasi - elastic light scattering (QELS) assessments to guide efficient lipid nanoparticle synthesis.

[0360] In some embodiments, the majority of the purified LNPs (i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs) have a size of about 70 - 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 - 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0361] In certain embodiments, the average diameter of the LNP is 30 - 200 nm.

[0362] In various embodiments, the average diameter of the LNP is 80 - 150 nm.

[0363] In some embodiments, the average size of the LNPs in the compositions of the present invention is less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0364] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs in the compositions of the present invention have a size ranging from about 40 - 90 nm (e.g., about 45 - 85 nm, about 50 - 80 nm, about 55 - 75 nm, or about 60 - 70 nm) or about 50 - 70 nm (e.g., about 55 - 65 nm), which is suitable for pulmonary delivery via nebulization.

[0365] In some embodiments, the dispersity or measure of molecular size heterogeneity (PDI) of the LNPs in the pharmaceutical compositions provided by this disclosure is less than about 0.5. In some embodiments, the PDI of the LNPs is less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured by a Zetasizer machine as described above.

[0366] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate the mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate the mRNA within each individual particle. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is from 50% to 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%. Typically, the encapsulation efficiency of the lipid nanoparticles used herein is at least 90 (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0367] In some embodiments, the N / P ratio of the LNP is from 1 to 10. In some embodiments, the N / P ratio of the lipid nanoparticles is higher than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In certain embodiments, the typical N / P ratio of the LNPs herein is 4.

[0368] In some embodiments, the pharmaceutical composition according to the present disclosure contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, the pharmaceutical composition contains from about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0369] In some embodiments, the mRNA can be prepared by chemical synthesis or by in vitro transcription (IVT) from a DNA template. In this method, during the IVT process, a cDNA template is used to generate the mRNA transcript, and the DNA template is degraded by DNase. The transcript is purified by depth filtration and tangential flow filtration (TFF). The purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.

[0370] mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNP. The amphiphilic solution of the four lipid components used to dissolve the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 - 7.0 (e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5). The buffer can contain other components such as salts (e.g., sodium, potassium, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate and 150 mM NaCl (pH 4.5).

[0371] Exemplary non - limiting methods for preparing the mRNA - LNP composition involve mixing a buffered mRNA solution with an ethanol solution of lipids in a controlled and uniform manner, where the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a cholesterol - based lipid, and a helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed in a "T" - shaped mixer with a near "pulse - free" pump system at a volume ratio of 4:1. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. TFF can be used to concentrate and perform buffer exchange on the resulting nascent LNP immediately after its formation via the T - mixing process. The diafiltration process is a continuous operation where the volume is kept constant by adding an appropriate buffer at the same rate as the permeate stream. XI. Packaging and Use of mRNA - LNP RSV Vaccine

[0372] The mRNA - LNP vaccine can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or for nasopharyngeal (e.g., intranasal) administration. In various embodiments, the mRNA - LNP vaccine can be formulated or packaged for pulmonary administration. In various embodiments, the mRNA - LNP vaccine can be formulated or packaged for intravenous administration. The vaccine composition can be in the form of a ready - to - use formulation where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) just prior to use. The vaccine composition can also be shipped and provided as an aqueous solution or a frozen aqueous solution and can be administered directly to a subject without reconstitution (after thawing if previously frozen).

[0373] Accordingly, the present disclosure provides articles (such as kits) that provide an mRNA-LNP vaccine in a single container, or provide an mRNA-LNP vaccine in one container (e.g., a first container) and a physiological buffer for reconstitution in another container (e.g., a second container). The one or more containers may contain a single-use dose or a multi-use dose. The one or more containers may be pre-treated glass vials or ampoules. The article may also include instructions for use.

[0374] In certain embodiments, an mRNA-LNP vaccine is provided for use in intramuscular (IM) injection. The vaccine may be injected into, for example, the deltoid muscle in the subject's upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use in inhalation and is provided in a pre-filled pump, nebulizer, or inhaler.

[0375] The mRNA-LNP vaccine may be administered to a subject in need thereof in a prophylactically effective amount, which is an amount that provides sufficient immune protection against a target pathogen for a sufficient amount of time (e.g., one year, two years, five years, ten years, or a lifetime). Sufficient immune protection may be, for example, preventing or alleviating symptoms associated with pathogen infection. In some embodiments, multiple doses (e.g., two doses) of the vaccine are administered (e.g., injected) to a subject in need thereof to achieve the desired prophylactic effect. The doses (e.g., a prime dose and a booster dose) may be separated by an interval of at least, for example, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months, five months, six months, one year (i.e., twelve months), two years, five years, or ten years.

[0376] For a better understanding of the present invention, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. Examples

[0377] The foregoing description of specific embodiments will so fully disclose the general nature of the disclosure that others can, by applying knowledge within the scope of the art, readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept of the disclosure. Accordingly, such adaptations and modifications are intended to be included within the meaning and range of equivalents of the disclosed embodiments based on the teaching and guidance presented herein. It is to be understood that the language or terminology herein is for the purpose of description and not of limitation, and thus the terminology or wording of this specification will be interpreted by those skilled in the art in light of the teaching and guidance. Example 1: A Phase I / II, randomized, double-blind, placebo-controlled, multi-group dose-finding study to evaluate the safety and immunogenicity of RSV mRNA vaccine candidates with LNP cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10 in adult participants aged 18 to 50 years in Study A (sentinel cohort) and aged 60 years and older in Studies B and the booster study (primary and booster cohorts). Introduction Background

[0378] Currently, there is no vaccine available for the prevention of RSV in the elderly, and there is no effective antiviral treatment. Therefore, there is an unmet medical need in addressing the prevention of respiratory diseases in the elderly while improving the quality of life of patients.

[0379] This example outlines the parameters for evaluating the efficacy, safety, and immunogenicity of an RSV mRNA LNP vaccine as described herein in adults (aged 18 to 50 years and 60 years and older). Such an RSV mRNA LNP vaccine can prevent LRTD caused by RSV in the elderly. Study rationale

[0380] The clinical trials described herein test the safety and immunogenicity of an RSV mRNA LNP vaccine. The RSV mRNA LNP vaccine contains mRNA encoding the RSV pre-fusion (pre-F) antigen in one of two encapsulated LNP formulations (i.e., LNP containing cKK-E10 (non-biodegradable) or GL-HEPES-E3-E12-DS-4-E10 (biodegradable)), which is administered at three different doses (i.e., low dose (10 μg), medium dose (30 μg), or high dose (75 μg)) to healthy adults aged 18 to 50 years (Study A, i.e., the sentinel cohort) and 60 years and older (Studies B and the booster study (i.e., the primary and booster cohorts)). Study Overview and Study Design Number of participants, intervention group overview, and visit frequency

[0381] The total expected number of participants in the two studies (Study A and Study B) is approximately 790 randomized participants.

[0382] Study A (named "Sentinel Cohort") (initial clinical trial) is a small, randomized, double-blind, dose-escalation safety study (i.e., approximately 90 participants in total, see Table 2 below), followed by a larger Study B (named "Main Cohort"; approximately 700 participants in total; see Table 3 below), to evaluate the safety and immunogenicity of an RSV mRNA vaccine encapsulated in LNPs in healthy adult participants (18 to 50 years old in Study A; 60 years old and above in Study B). The graphical timelines of Study A and Study B are shown respectively in Figure 1 and Figure 2 .

[0383] Study A and Study B include the same six experimental subcohorts and a placebo control group. Three different doses (i.e., low dose (10 μg), medium dose (30 μg), and high dose (75 μg)) of an RSV messenger mRNA vaccine candidate (shown as SEQ ID NO:14) encapsulated in either of two different lipid nanoparticle (LNP)-based formulations (i.e., LNPs containing cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10) are administered to the six experimental subcohorts. Study A and Study B will also have a placebo control group to which 0.9% saline solution will be administered. The vaccine is administered intramuscularly (deltoid muscle in the upper arm) at a dose level of 0.5 ml per dose. The vaccine is stored at -80°C ± 10°C at the study site and diluted with 2.2x PBS.

[0384] After vaccination, the follow-up of participants in Study A and Study B continues for 12 months after vaccination. Study A conducts initial screening participant visits and 7 scheduled site visits at -14 days (D) (-D14), D01, D04, D08, D29, 3 months, 6 months, and 12 months. Study B conducts initial screening participant visits and 6 scheduled site visits at -D14, D01, D08, D29, 3 months, 6 months, and 12 months.

[0385] It is also planned to select 140 participants enrolled in Study B (i.e., 20 participants per group) to be included in the cell-mediated immunity (CMI) subgroup. Participants in the CMI subgroup will be recruited from a limited number of selected sites. Study B also plans to recruit at least 6% Japanese-source participants (i.e., at least approximately 42 participants; 6 participants per group). Table 2. Experimental sample size of Study A (Sentinel Cohort) LNP: Lipid nanoparticle; mRNA: Messenger ribonucleic acid; N / A: Not applicable; RSV: Respiratory syncytial virus; RSV mRNA is shown as SEQ ID NO:14. Table 3. Experimental sample sizes for Study B (main cohort) LNP: Lipid nanoparticle; mRNA: Messenger ribonucleic acid; N / A: Not applicable; RSV: Respiratory syncytial virus; RSV mRNA is as shown in SEQ ID NO:14.

[0386] In addition, these clinical trials evaluated the safety and immunogenicity of a booster vaccination administered 12 months after the primary vaccination in a subgroup of the study population (termed the "booster cohort"; approximately 200 participants from Study B; see Table 4 below). The booster vaccination was administered 12 months after the primary vaccination in a subgroup of the study population from Study B. For the booster vaccination, a single vaccine formulation was used based on the evaluation of the candidate formulation after the first injection. For the subgroup of participants enrolled in the booster cohort, the total duration of participation for each participant was 24 months. Similar to Study B, in addition to the 6 scheduled visits, participants also had a screening visit. Participants received the booster vaccination at the 8th visit, 12 months after the primary vaccination, which could be on the same day as the 12-month Study B follow-up visit (i.e., visit 7). After the booster vaccination, participants returned to the site at D08, D29, 3 months, 6 months, and 12 months. Table 4. Experimental sample sizes for the booster cohort *The dose level and study intervention formulation were determined at the interim analysis. As defined herein, the dropout rate at the time of the booster vaccination was not considered. Participants in the booster cohort were randomized to receive the selected formulation of the booster dose or placebo. Definition of study completion

[0387] If a participant completed the last contact scheduled in the planned activities, they were considered to have completed the study. The planned activities for Study A, Study B, and the booster cohort are as Figure 4 - Figure 6 shown. Study completion was defined as the date of the last contact of the last participant in the study. However, for the periodic safety reports, when the clinical study report was completed, the study was considered to have been completed. Drug administration

[0388] The vaccine is provided as a liquid frozen solution in vials. Each 0.5 mL dose contains: 10 μg, 30 μg, or 75 μg of RSV pre-F mRNA; and an LNP containing cKK-E10 or an LNP containing GL-HEPES-E3-E12-DS-4-E10. The vaccine is formulated as a single-dose mRNA-LNP complex, which is diluted to the desired mRNA dose at the study site using a buffered diluent (diluent = 2.2XPBS (2°C to 8°C)). The sentinel and primary cohorts receive a single intramuscular injection. The booster cohort receives two intramuscular injections, with the second injection administered 12 months after the primary injection. Each dose (vial) of the vaccine is provided in a separate box. Each dose (vial) of the vaccine is stored at -80°C + / - 10°C. Objectives

[0389] Primary objectives. The primary objectives are to evaluate the safety and immunogenicity profiles of the RSV mRNA vaccine described herein encapsulated in an LNP containing cKK-E10 or an LNP containing GL-HEPES-E3-E12-DS-4-E10 at three different dose levels (i.e., low dose (10 μg), medium dose (30 μg), and high dose (75 μg)).

[0390] Secondary objectives. The secondary objectives are to evaluate: (1) the safety profile of a booster vaccination administered 12 months after the primary vaccination in subgroups of participants; (2) the persistence of the immune response at 3, 6, and 12 months after the primary vaccination and before vaccination (D01); and (3) the persistence of the immune response after a booster vaccination at 12 months after the primary vaccination in subgroups of participants.

[0391] The following Table 5 summarizes the primary objectives and the corresponding endpoints. The following Table 6 summarizes the secondary objectives and the corresponding endpoints. The following Table 7 summarizes the exploratory objectives and the corresponding endpoints. Table 5. Primary objectives and corresponding endpoints Table 6. Secondary objectives and corresponding endpoints Table 7. Exploratory objectives and corresponding endpoints Study Population Inclusion and exclusion criteria

[0392] Inclusion criteria. For all studies (Study A, Study B, and the Booster Cohort), participants were eligible to participate in the study only if they met all of the following criteria at screening and at the first visit (Day 01; Visit 01): (I1) The participant must (1) be 18 to 50 years of age on the inclusion date of Study A (i.e., “18 years of age” means from the date of the 18th birthday) or (2) be 60 years of age or older on the inclusion date of Study B and the Booster Cohort (i.e., “60 years of age or older” means from the date of the 60th birthday); (I2) Female participants were eligible if they were not pregnant or lactating and had no reproductive potential (to be considered to have no reproductive potential, a female must have been postmenopausal for at least 1 year or surgically sterile). For the Sentinel Cohort, urine or serum pregnancy tests were performed on women of reproductive potential prior to vaccination; and (I3) The participant must be able to attend all scheduled visits and comply with all study procedures. Additionally, a fourth additional criterion at screening was that the participant signed and dated the informed consent form.

[0393] Exclusion Criteria. For all studies (Study A, Study B, and the Booster Cohort), participants were ineligible if they met any of the following criteria: (E1) Known or suspected congenital or acquired immunodeficiency; or received immunosuppressive therapy (such as anti-cancer chemotherapy or radiotherapy) within the previous 6 months; or long-term systemic corticosteroid therapy (more than 2 consecutive weeks of prednisone or equivalent within the past 3 months); (E2) Known systemic hypersensitivity to any study intervention component (e.g., polyethylene glycol, polysorbate); history of life-threatening reaction to the study intervention used in the study or to products containing any of the same substances, any allergic reaction (e.g., anaphylaxis) after administration of the mRNA COVID-19 vaccine; (E3) History of RSV-related disease diagnosed clinically, serologically, or microbiologically within the past 12 months; (E4) Prior history of myocarditis, pericarditis, and / or myopericarditis; (E5) Thrombocytopenia or bleeding disorder that, in the judgment of the investigator, contraindicates IM injection; (E6) History of bleeding disorder or received anticoagulants within 3 weeks prior to enrollment, contraindicating intramuscular injection; (E7) Chronic disease (e.g., cardiac disorder, renal disorder, autoimmune disorder, diabetes, psychiatric disorder, or chronic infection) that the investigator considered to be in a stage that might interfere with the conduct or completion of the study; (E8) Alcohol, prescription drug, or substance abuse that the investigator considered might interfere with the conduct or completion of the study; (E9) Received any vaccine within 4 weeks prior to any study intervention administration, or planned to receive any vaccine within 4 weeks after any study intervention administration (if a participant enrolls and seeks to receive an authorized influenza or non-mRNA COVID-19 vaccine outside of the study, he / she will be encouraged to discuss this intention proactively with the study investigator and will be permitted to receive the authorized vaccine as early as 28 days after study vaccination and at any time thereafter); (E10) Received any mRNA vaccine within 60 days prior to any study intervention administration, or planned to receive any mRNA vaccine within 60 days after any study intervention administration; (E11) Prior vaccination with an investigational vaccine against RSV; (E12) Received immunoglobulin, blood, or blood-derived products within the past 3 months; (E13) Received oral or injectable antibiotic therapy within 72 hours prior to the first blood draw; (E14) Participated or planned to participate in another clinical study of a study vaccine, drug, medical device, or treatment procedure at the time of study enrollment (or within 4 weeks prior to the first study intervention administration) or during the study; (E15) Loss of freedom due to administrative or court order, or in an emergency situation, or involuntary hospitalization; (E16) Self-reported or documented positive for human immunodeficiency virus (HIV), hepatitis B surface antigen (HbsAg), hepatitis B core antibody (HbcAb), or hepatitis C virus antibody (HCV Ab) detected by any FDA-approved / validated test, or positive SARS-CoV-2 RT-PCR or antigen test;or (E17) is a researcher determined to be directly involved in the proposed study, or an employee of a researcher or research center, or a direct family member (i.e., parent, spouse, natural or adopted child) of a researcher or employee determined to be directly involved in the proposed study.;

[0394] Exclusion criteria E1 - E17 are checked at the participant's initial screening visit. Additionally, at the first visit (Visit 1; Day 1), E1 - E17 plus three additional exclusion criteria E18 - E20 are checked. E18 is that the screening electrocardiogram is consistent with possible myocarditis, pericarditis, and / or myopericarditis, or the researcher believes the screening electrocardiogram demonstrates clinically relevant abnormalities that may affect the safety of the participant or the study results. E19 is having moderate or severe acute illness / infection (as judged by the researcher) or febrile illness (body temperature 38.0°C) on the day of study intervention administration. Participants are not expected to enter the study until the condition has resolved or the febrile episode has cleared. E20 is that for any screening laboratory parameter, the laboratory abnormality is greater than Grade 1 or is considered clinically significant by the researcher.

[0395] If the participant's attending physician is not the researcher, the site should contact this physician with the participant's consent to inform him / her that the participant is participating in the study. Additionally, the site should have this attending physician verify the exclusion criteria related to previous therapies (such as receipt of blood products or previous vaccines). Study Interventions and Concomitant Therapies Method of randomly assigning participants to the intervention groups

[0396] Randomized participants are defined as participants who have been assigned to a randomized intervention, whether or not the treatment has been administered (i.e., participants registered in the IRT). Participants cannot be randomized more than once in the study.

[0397] Participants who sign the informed consent form at the screening visit and meet the eligibility criteria at the screening visit and Visit 1 will be randomly assigned to one of the study intervention groups at Visit 1 as follows: · Study A, Cohort 1 (Sentinel Cohort 1): RSV mRNA vaccine with low dose of LNP cKK - E10, RSV mRNA vaccine with low dose of LNP GL - HEPES - E3 - E12 - DS - 4 - E10, or placebo at a 1:1:1 ratio. · Study A, Cohort 2 (Sentinel Cohort 2): RSV mRNA vaccine with medium dose of LNP cKK - E10, RSV mRNA vaccine with medium dose of LNP GL - HEPES - E3 - E12 - DS - 4 - E10, or placebo at a 1:1:1 ratio. · Study A Cohort 3 (Sentinel Cohort 3): High-dose RSV mRNA vaccine with LNP cKK-E10, high-dose RSV mRNA vaccine with LNP GL-HEPES-E3-E12-DS-4-E10, or placebo in a 1:1:1 ratio. · Study B (Main Cohort): Low-dose RSV mRNA vaccine with LNP cKK-E10, low-dose RSV mRNA vaccine with LNP GL-HEPES-E3-E12-DS-4-E10, medium-dose RSV mRNA vaccine with LNP cKK-E10, medium-dose RSV mRNA vaccine with LNP GL-HEPES-E3-E12-DS-4-E10, high-dose RSV mRNA vaccine with LNP cKK-E10, high-dose RSV mRNA vaccine with LNP GL-HEPES-E3-E12-DS-4-E10, or placebo in a 1:1:1:1:1:1:1 ratio.

[0398] At Visit 1, randomization will be stratified by cohort (Sentinel Cohorts 1, 2, 3, or Main Cohort), CMI subgroup (yes or no), and Japanese origin status (yes or no).

[0399] Participants from the placebo group and the selected formulation groups (i.e., both dose level and LNP formulation) in Study B (Main Cohort) will be eligible to receive a booster dose 12 months after the first vaccination.

[0400] At Visit 8, participants meeting the eligibility criteria (including the booster screening visit (Screening Visit 8)) will be randomly assigned in a 1:1 ratio to receive the selected RSV mRNA vaccine formulation or placebo. At Visit 8, randomization will be stratified by the study intervention group randomized at Visit 1.

[0401] Site staff will connect to the Interactive Response Technology (IRT), enter identity and safety information, and confirm the minimum data amount in response to IRT prompts. Then, the IRT will provide the group assignment and have the site staff confirm. The IRT will also state whether the participant has been assigned to the CMI subgroup (i.e., 20 participants per study intervention group in the Main Cohort recruited from a limited number of selected sites). If the participant is not eligible for the study, the information will only be recorded in the participant recruitment log. Participant numbers shall not be reassigned for any reason. The randomization code will be securely stored in the IRT. Concomitant Therapy - Drugs to be Reported

[0402] If any drug received by a participant before the vaccination date, at the time of enrolment, or during the study affects the interpretation of safety data (e.g., antipyretics or analgesics that can reduce the intensity or frequency of adverse events) or may interfere with the development or measurement of the immune response (e.g., use of immunosuppressants, immunomodulators, or some antibiotics that may affect the performance of certain biological assays), the drug must be reported and will be reported by the investigator. Steroid drugs can affect both the safety assessment and the immune response to the vaccine.

[0403] The following is a list of drug classes for which reporting is required: · Drugs that affect or may affect safety assessment (e.g., antipyretics, analgesics, and non-steroidal anti-inflammatory drugs (NSAIDs), systemic steroids / corticosteroids). Note: Topical analgesics should not be applied at the injection site of the study intervention; however, if they are inadvertently applied, they should be recorded. · Drugs that affect or may affect the immune response (e.g., other vaccines, blood products, antibiotics that may interfere with biological assays used in the Sanofi Pasteur laboratory or other test laboratories, systemic steroids / corticosteroids, immunosuppressants, immunomodulators with immunosuppressive properties, antiproliferative drugs such as DNA synthesis inhibitors). · Drugs that affect or may affect both safety and the immune response (e.g., systemic steroids / corticosteroids).

[0404] Medications for which reporting is required will be collected in the case report form (CRF) until the end of the actively provided follow-up period (i.e., 28 days after vaccination). Drugs that may affect the immune response or may affect both safety and the immune response will be collected throughout the study. One or more mRNA vaccines will be collected throughout the study (including 28 days after vaccination).

[0405] Dose and route of administration will not be recorded for homeopathic medications, topical and inhaled steroids, and topical, ophthalmic, and otic treatments (except for topical analgesics applied at the injection site of the study intervention).

[0406] Medications given in response to an adverse event will only be recorded in the "Actions Taken" section of the case report form. Details will not be recorded in the concomitant medications table of the CRF unless one or more of the medications received fall into one of the pre-listed categories. Medications will be coded. Information on previous medications (including previous influenza vaccinations and previous mRNA vaccinations / products received by the participant) will be recorded in the participant's eCRF. Rescue medications

[0407] In the event of an allergic, vasovagal, or other immediate hypersensitivity reaction, appropriate medical equipment and emergency medications (including epinephrine) (1:1000) must be available at the study site. Termination of Study Interventions

[0408] If a participant experiences at least one of the 13 reasons listed below as an explicit contraindication, the participant will permanently discontinue the study intervention, i.e., will not be eligible to receive a booster dose. For safety reasons, additional unscheduled visits may be conducted and information will be reported in the source documents. Three temporary contraindications

[0409] If a participant experiences one of the three situations listed here, the investigator will postpone further vaccination (i.e., administer a booster dose to participants eligible to enroll in the booster cohort) until the situation is no longer present. The postponement must still be within the time frame for vaccination specified in the planned activities. (1) A febrile illness (temperature ≥ 38°C [≥ 100.4°F]) or moderate or severe acute illness / infection on the day of vaccination, in the judgment of the investigator. (2) Receiving any vaccine other than the study mRNA vaccine within 4 weeks before any study vaccine administration intervention, or planning to receive any vaccine other than the mRNA vaccine within 4 weeks after any study intervention. (3) Receiving any mRNA vaccine within 60 days before any study vaccine administration intervention, or planning to receive any mRNA vaccine within 60 days after any study intervention. Thirteen explicit contraindications for withdrawing from the study

[0410] If a participant experiences at least one of the situations listed in this document, the researcher will explicitly terminate the vaccination: (1) an allergic reaction or anaphylaxis to a previous dose of the vaccine; (2) abnormal laboratory parameters of grade 2 or 3, and evaluated by the researcher as related to a previous dose of the vaccine; (3) an SAE evaluated as related to the study vaccine after a previous dose of the vaccine according to the researcher's judgment; (4) myocarditis, pericarditis, and / or myopericarditis; (5) RSV-related diseases diagnosed clinically, serologically, or microbiologically; (6) thrombocytopenia or hemorrhagic disorders; (7) chronic diseases (such as heart diseases, kidney diseases, autoimmune diseases, diabetes, mental disorders, or chronic infections) that the researcher believes are in a stage that may interfere with the conduct or completion of the study; (8) known or suspected congenital or acquired immunodeficiency; or receipt of immunosuppressive therapy (such as anti-cancer chemotherapy or radiotherapy) within the previous 6 months; or long-term systemic corticosteroid therapy (more than 2 consecutive weeks of prednisone or equivalent taken in the past 3 months); (9) receipt of anticoagulants within 3 weeks before the booster injection; (10) receipt of immunoglobulins, blood, or blood-derived products within the past 3 months; (11) receipt of oral or injectable antibiotic therapy within 72 hours before blood sampling (BL0005) at visit 7 for the booster vaccination; (12) participation or planned participation in a clinical study of another study vaccine, drug, medical device, or treatment procedure during the booster period of the study; (13) self-reported or recorded seropositivity for human immunodeficiency virus (HIV) antigen and / or antibody, hepatitis B virus surface antigen (hBsAg), hepatitis B core antibody (hBcAb), or hepatitis C virus antibody (HCV Ab).

[0411] In the case of an influenza vaccine or any other vaccine immunization program carried out locally or nationally as needed, participants who receive the vaccine at any time during the study will not be withdrawn from the study.

[0412] A participant may withdraw from the study at any time at his / her own request, or may be withdrawn from the study at any time at the researcher's discretion for reasons of safety, behavior, or compliance. If a participant withdraws consent, the participant will permanently terminate the study intervention and the study at that time. Withdrawn participants will not be replaced. Study Assessments and Procedures

[0413] Obtain the study assessment data collected at each visit (including routine clinical management (such as blood cell count, electrocardiogram, physical examination)), as depicted in the schedule of planned activities for Study A (sentinel cohort) as shown in Figure 4 , Study B (main cohort) as shown in Figure 5 , and the booster cohort as shown in Figure 6 . Blood samples

[0414] At the time of the visit, blood samples are collected according to the schedule of planned activities for each cohort as Figure 4 - Figure 6 shown and are used to assess safety, immunogenicity, and to test for serology against HIV, hepatitis B, and hepatitis C. The maximum volume of blood collected from each participant during the study (including any additional evaluations that may be required) will not exceed 255 mL. As shown in Tables 8, 9, and 10 below, the volume of blood collected at each visit ranges from 15 mL to 50 mL. Repeat or unscheduled sampling may be performed for safety reasons or technical issues with the samples. Table 8. Blood sampling volume (mL) per visit - Study A (Sentinel cohort (participants aged 18 to 50 years)) BL: Blood sample for immunogenicity; BS: Blood sample for safety; ELISA: Enzyme-linked immunosorbent assay; hBcAb: Hepatitis B core antibody; hBsAg: Hepatitis B surface antigen; HCVAb: Hepatitis C virus antibody; HIV: Human immunodeficiency virus; IgG: Immunoglobulin G; MN: Microneutralization; RSV: Respiratory syncytial virus Table 9. Blood sampling volume (mL) per visit - Study B (Main cohort (participants aged 60 years and older)) Abbreviations: BL: Blood sample for immunogenicity; BS: Blood sample for safety; CMI: Cell-mediated immunity; ELISA: Enzyme-linked immunosorbent assay; hBcAb: Hepatitis B core antibody; hBsAg: Hepatitis B surface antigen; HCVAb: Hepatitis C virus antibody; HIV: Human immunodeficiency virus; IgG: Immunoglobulin G; MN: Microneutralization; RSV: Respiratory syncytial virus; WB: Blood sample for TruCulture. * Visit on Day 3 (V02) is not applicable to the main cohort Applicable only to a subgroup of participants at some selected sites S samples will be collected and stored for potential future safety and immunology studies Table 10. Blood sampling volume (mL) per visit - Study C (Booster cohort (participants aged 60 years and older)) Abbreviations: BL: Blood sample for immunogenicity; BS: Blood sample for safety; ELISA: Enzyme-linked immunosorbent assay; hBcAb: Hepatitis B core antibody; hBsAg: Hepatitis B surface antigen; HCVAb: Hepatitis C virus antibody; HIV: Human immunodeficiency virus; IgG: Immunoglobulin G; MN: Microneutralization; RSV: Respiratory syncytial virus * Samples will be collected and stored for potential future safety and immunology studies Immunogenicity Assessment RSV anti-F IgG ELISA

[0415] Antibodies against RSV-F antigen were measured using RSV anti-F IgG ELISA. Briefly, RSV-F antigen was coated on microtiter plates, and serial two-fold dilutions of human serum samples were added and incubated to allow binding to RSV-F antigen. Then, horseradish peroxidase (HRP)-conjugated anti-human IgG detection antibody was added, followed by a colorimetric substrate. Concentrations of IgG antibodies against RSV-F antigen in serial six-fold dilutions were calculated relative to a qualified in-house reference calibrated against the WHO international standard (1st International Standard of RSV antiserum) with a specified value (international units / mL). RSV neutralizing antibody assessment

[0416] RSV neutralizing antibodies were measured using the microneutralization (MN) assay, Nexelis PRNT A2 assay, or A Long assay. Serial two-fold dilutions of serum samples were heat inactivated and then mixed with a constant concentration of RSV A2 strain (ATCC VR-1540). The mixtures were inoculated into wells of a 96-well microplate with permissive hEp-2 cells (ATCC CCL-23) and incubated for 2 days. Virus infectivity (viral antigen production) was detected by ELISA due to the reduction in neutralization by antibodies present in the serum samples. After washing and fixation, production of RSV antigen in the cells was detected by sequential incubation with mouse anti-RSV specific monoclonal antibody, HRP-anti-mouse IgG conjugate, and a chromogenic substrate. The resulting optical density was measured using a microplate reader. Reduction in RSV infectivity (compared to virus control wells) constituted a positive neutralization reaction, indicating the presence of neutralizing antibodies in the serum samples. Cell-mediated immunity

[0417] T helper cell responses were evaluated by using fresh whole blood (TruCulture). T cell analysis in whole blood collected and cultured using the RBM TruCulture whole blood collection and culture system enables consistent and reliable assessment of T helper cell polarization. Triculture tubes containing selected stimulants or antigens allow almost instantaneous stimulation of cells in the presence of all blood components after the blood is drawn into the tube, thus minimizing variability that may arise due to handling and manipulation of the blood, including handling of peripheral blood mononuclear cells (PBMCs). Preliminary laboratory results and previous literature reports have shown the robustness of this method. Duffy et al. (2017), Clin Immunol [Clinical Immunology], 183:325-335. The assay relies on the fact that T helper cytokines are secreted in a single tube after stimulation and the supernatant is collected at 24 or 48 hours. Measurements are made using a panel of cytokines from the Luminex xMAP technology of RBM TruCulture to determine the state of T helper cell polarization. Safety Assessment

[0418] All planned time points for safety assessments are provided in the planned activity schedule for each cohort as shown in Figure 4 - Figure 6 Before enrollment, participants' past and ongoing pre-existing conditions and diseases are evaluated and such conditions are recorded. Important (clinically relevant) medical history (as per diagnostic reports) is reported in the case report form, including conditions / diseases for which the participant is or has been followed by a physician or conditions / diseases that may recur or result in an SAE or repeated outpatient care during the study. In addition, the history of receiving mRNA-based vaccines will be recorded. Physical examination and vital signs

[0419] At the screening visit, the investigator or designee will perform a comprehensive physical examination. A comprehensive physical examination will also be completed at visit 1. A targeted / simplified physical examination will also be completed at all subsequent face-to-face visits at the time points specified in the planned activity schedule for each cohort as shown in Figure 4 - Figure 6 Oral or axillary temperature will be systematically collected by the investigator before vaccination. Tympanic, skin, and temporal artery thermometers shall not be used. Electrocardiogram

[0420] ECG will be performed at screening visit for use as a baseline and to exclude participants who are likely or possibly suffering from myocarditis, pericarditis, and / or myopericarditis, as well as to identify participants with clinically relevant abnormalities that may affect the safety of the participants or the study results. In the event that a participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the study, an additional one or more ECGs will be performed as soon as possible at an unscheduled visit (if necessary). The ECGs will be recorded and any assessment thereof will be based on standard medical care. Clinical safety laboratory assessments

[0421] Table 11 below lists the clinical laboratory tests. Laboratory tests will be performed during the time points specified in the schedule of planned activities for each cohort as shown Figure 4 - Figure 6 . The investigator reviews the laboratory reports and records any clinically significant changes that occur after the primary or booster vaccination as adverse events.

[0422] After the primary or booster vaccination, all laboratory tests with values considered to be clinically significantly abnormal will be repeated until the value returns to normal or baseline, or is no longer considered clinically significant by the investigator. If a clinically significant / any value does not return to normal / baseline within a reasonable period of time judged by the investigator, the cause should be identified and the sponsor notified. Table 11. Clinical safety laboratory tests Nasopharyngeal swab collection

[0423] Nasopharyngeal swab samples for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants with any episode of respiratory disease. If a participant visits any other non-study doctor / hospital at any time during the study, once the subject is discharged, nasopharyngeal swab samples will be obtained at the study site if the investigator deems it appropriate. All nasopharyngeal swab samples will be collected in recommended viral transport medium tubes and stored at -60°C to -80°C until ready for shipment. Requests for home or in-site disease visits will first be evaluated by video call in order to be able to remotely evaluate the severity and remotely manage mild (grade 1) disease, as appropriate for the investigators of the study. Lower respiratory tract and acute respiratory disease assessments

[0424] In the case of RSV confirmed by RT-PCR, the following RSV disease categories will be used: (1) RSV acute respiratory disease (ARD) is any respiratory symptom including the following: nasal congestion, sore throat, hoarseness, new or worsening cough, sputum production, and dyspnea with or without fever; (2) Severe RSV ARD is an acute respiratory disease confirmed by RT-PCR, with a history of fever or measured fever ≥ 38 °C, and cough onset within the last 10 days, and requires hospitalization; (3) RSV lower respiratory tract disease (LRTD) not requiring medical attention is ARD with one or more symptoms of lower respiratory tract disease (which includes involvement of the lower respiratory tract: trachea, bronchi, and lungs, which can be combined, i.e., bronchopneumonia, tracheobronchitis), ARD symptom onset for 10 days and RSV confirmed by RT-PCR; (4) RSV LRTD requiring medical attention is ARD with one or more symptoms of lower respiratory tract disease (which includes involvement of the lower respiratory tract: trachea, bronchi, and lungs, which can be combined, i.e., bronchopneumonia, tracheobronchitis) seeking medical care, ARD symptom onset for 10 days and confirmed by RT-PCR. Medical care is divided into emergency room, hospitalization, and outpatient visit. Example 2: Interim Analysis 2 - Potency and Safety Data

[0425] The planned sample size (IA2) was 790, of which 90 were in the sentinel cohort and 700 were in the main cohort. The IA2 sample size (partial main cohort) was 667 participants for the analysis of immunogenicity (D0 and D29); and 698 participants for the analysis of safety data (up to Day 29 (D29)). Demographic characteristics of the main cohort are shown in Figure 7 in. Potency

[0426] Results of the geometric mean titer (GMT) of RSV-A neutralizing antibody (nAb) and geometric mean titer ratio (GMTR) D29 / D01 (PPAS-1) after primary vaccination summarized for the entire main cohort (aged 60 and above) are shown in Figure 8 in. Compared with the GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 75 mcg group (GMTR of 5.44), the cKK-E10+RSV mRNA 75 mcg group showed a slightly higher GMT at D29 (GMTR of 5.66). Followed by the GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 30 mcg group (GMTR of 4.74).

[0427] Data on the fold increase of RSV-A neutralizing antibody in the entire main cohort after primary vaccination are shown in Figure 9Among them, the percentage range of participants with at least a ≥4-fold increase according to dose and LNP was 38.4% to 69.6%.

[0428] Results for the geometric mean of IgG antibody titers and IgG antibody GMTR after primary vaccination summarized for a subset of the main cohort (aged 60+) are shown in Figure 10 Among them, the GL-HEPES-E3-E12-DS-4-E10 + 75 mcg group showed a higher IgG GM at D29, with the highest fold increase (10.4), followed by the cKK-E10 + 75 mcg group (10.3) and the GL-HEPES-E3-E12-DS-4-E10 + 30 mcg group (8.29). These data are related to the group showing the best RSV-A GMT response.

[0429] Results for the GMT of RSV A neutralizing antibody titers (PPAS-1) and the geometric mean titer ratio of neutralizing antibodies (GMTR) D29 / D01 after primary vaccination in the sentinel cohort (aged 18 - 50) are summarized in Figure 11 Among the sentinel cohort, the GL-HEPES-E3-E12-DS-4-E10 + 75 mcg group showed a higher GMT at D29 (GMTR of 11.5), followed by the cKK-E10 + 75 mcg group (GMTR of 8.64), and the cKK-E10 and GL-HEPES-E3-E12-DS-4-E10 + 30 mcg groups (GMTRs of 6.43 and 6.06, respectively).

[0430] Data on the fold increase of RSV-A neutralizing antibodies in the sentinel cohort after primary vaccination are shown in Figure 12 Among them, the percentage range of participants with at least a ≥4-fold increase according to dose and LNP was 55.6% to 90.0%.

[0431] A summary of RSV-A neutralizing antibody titers after primary vaccination in the sentinel cohort is shown in Figure 13A - Figure 13B Among them. Conclusions Regarding Immunogenicity

[0432] The GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 75 mcg group showed a higher GMT at D29 (GMTR of 5.22), followed by the cKK-E10 + RSV mRNA 75 mcg group (GMTR of 4.56), and the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 30 mcg group (GMTR of 4.4).

[0433] The IgG results were associated with RSV-A neutralizing antibody responses (higher titers and fold increases in the same groups).

[0434] Higher doses of mRNA (75 mcg) showed higher immunogenicity.

[0435] GL-HEPES-E3-E12-DS-4-E10 was associated with higher immunogenicity (i.e., the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 75 mcg and GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 30 mcg groups). Safety

[0436] Safety data in the elderly showed that all RSV mRNA formulations were generally well tolerated.

[0437] Compared to cKK-E10, the GL-HEPES-E3-E12-DS-4-E10 group showed a better trend in reactogenicity, particularly pain at the injection site and myalgia (the most commonly reported solicited reactions). Solicited reactions were generally mild to moderate, with a small number of grade 3 reactions. A dose response was observed between the mRNA groups.

[0438] Spontaneously reported adverse events (AEs) were generally balanced in the mRNA groups (with a slightly higher trend of reporting in the mRNA groups compared to placebo). No dose response was observed.

[0439] In five participants (with normal baselines) in the mRNA groups, cardiac biomarkers (troponin I levels) were observed to increase relative to baseline on day 8 (this observation was considered not meaningful at this stage). One high troponin level was reported as a related serious adverse event (SAE) (asymptomatic myocardial injury; adjudicated by the Cardiac Adjudication Committee). Vigorous exercise was identified as a potential cause of the troponin elevation.

[0440] A summary of solicited reactions (%) within 7 days after the primary vaccination is shown in Figure 14 . Most solicited reactions were mild to moderate and of short duration. A summary of injection site reactions (%) within 7 days after the primary vaccination is shown in Figure 15 . Most injection site reactions were mild to moderate and of short duration. A summary of solicited systemic reactions (%) within 7 days after the primary vaccination is shown in Figure 16 . Most solicited systemic reactions were mild to moderate and of short duration.

[0441] The safety overview after the primary vaccination is depicted inFigure 17 Among. Overview of Reactogenicity

[0442] Overall, injection site pain was the most frequently reported solicited injection site reaction among the 6 mRNA groups (39.4% to 71.0% in the cKK-E10 group and 28.7% to 53.1% in the GL-HEPES-E3-E12-DS-4-E10 group).

[0443] Among the 6 mRNA groups, myalgia, fatigue, and headache were the most frequently reported solicited systemic reactions. Myalgia (range 17.2% to 41.0% in the cKK-E10 group), fatigue (13.1% to 29.0% in the cKK-E10 group), and headache (16.2% to 23.0% in the cKK-E10 group and 23% at the medium dose) were the most frequently reported solicited systemic reactions in the cKK-E10 group, followed by arthralgia and chills at the highest dose (21.0% and 15.0%, respectively). The same trend was observed in the GL-HEPES-E3-E12-DS-4-E10 group (myalgia range 19.8% to 27.6%; fatigue 15.8% to 26.0% and 26.0% at the medium dose; and headache 14.9% to 25.5%), except for arthralgia and chills, which tended to be reported in the placebo group (even for the highest dose, 8.2% and 9.2%, respectively).

[0444] Most solicited reactions started between D01 - D04 and most were of short duration (lasting 1 - 3 days). Solicited reactions were generally mild to moderate, with few grade 3 reactions (<4.1% in the GL-HEPES-E3-E12-DS-4-E10 group and <7.0% in the cKK-E10 group), and all grade 3 reactions lasted 1 to 3 days at maximum intensity (except for one case where injection site erythema lasted 5 days at maximum intensity).

[0445] A dose response was observed between the mRNA groups, with a better reactogenicity trend in the GL-HEPES-E3-E12-DS-4-E10 group compared to the cKK-E10 group (GL-HEPES-E3-E12-DS-4-E10 high dose ≈ cKK-E10 medium dose).

[0446] No delayed injection site reactions were observed.

[0447] A summary of solicited reactions within 7 days after the primary vaccination is shown in Figure 18 Among. Adverse Events (AEs) Reported Spontaneously

[0448] Two subjects, both in the high mRNA dose group, reported spontaneously volunteered immediate AEs / Adverse Reactions (ARs). Both reported hypertension (SOC: Vascular disorders) assessed as related to the IMP (both Grade 1).

[0449] Compared to placebo, spontaneously volunteered AEs reported in the mRNA groups tended to be slightly more frequent, and within the mRNA groups, an overall balance was observed between the mRNA groups: 25.7% of subjects reported at least 1 AE in the combined cKK-E10, 22.7% in the combined GL-HEPES-E3-E12-DS-4-E10, and 17.2% in placebo. No dose-response was observed. The minor imbalances observed were considered not meaningful and were caused by musculoskeletal and connective tissue disorders (such as arthralgia, myalgia, muscle spasm) and gastrointestinal disorders (such as abdominal pain, diarrhea, nausea). There were a small number of spontaneously volunteered ARs (AEs assessed as related to the IMP): 5% of subjects in the combined cKK-E10; 4% in the combined GL-HEPES-E3-E12-DS-4-E10; and 2% in placebo.

[0450] Twenty-three subjects had Medical Adverse Events (MAE) that required medical attention. MAEs were balanced between the groups.

[0451] Five subjects experienced Serious Adverse Events (SAEs) within 28 days. All were in the mRNA groups and were evenly distributed across the groups (2 in LNP cKK-E10 and 3 in LNP GL-HEPES-E3-E12-DS-4-E10). Only one was assessed as related to the investigational medicinal product IMP (mRNA LNP cKK-E10 low dose) (asymptomatic myocardial injury).

[0452] No Adverse Events of Special Interest (AESIs) (i.e., anaphylactic reactions (including bronchospasm and laryngospasm), myocarditis, pericarditis, and myopericarditis) were observed. After the cut-off date, the investigators downgraded an AESI in the biostatistical tables to a non-AESI (diagnosis changed from myocarditis to asymptomatic myocardial injury). No AEs and deaths leading to study termination were reported.

[0453] The summary of spontaneously volunteered AEs is shown in Figure 19 The summary of spontaneously volunteered AEs caused by gastrointestinal disorders, musculoskeletal disorders, and connective tissue disorders is shown in Figure 20 below. Overall Conclusions

[0454] The GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 75 mcg group showed a higher GMT at D29 (GMTR was 5.22), followed by the cKK-E10+RSV mRNA 75 mcg group (GMTR was 4.56), and the GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 30 mcg group (GMTR was 4.4).

[0455] Higher doses of mRNA (75 mcg) showed higher immunogenicity.

[0456] GL-HEPES-E3-E12-DS-4-E10 was associated with higher immunogenicity (i.e., the GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 75 mcg and GL-HEPES-E3-E12-DS-4-E10+RSV mRNA 30 mcg groups). The GL-HEPES-E3-E12-DS-4-E10 group showed a better trend of reactogenicity compared to cKK-E10 (GL-HEPES-E3-E12-DS-4-E10 high dose ≈ cKK-E10 medium dose).

[0457] GL-HEPES-E3-E12-DS-4-E10+mRNA 30 mcg had the best overall safety regarding grade 3 and grade 2 solicited events and special events. Example 3: Phase IIb / III - Phase II XII. Study rationale

[0458] RSV is a major viral agent causing severe respiratory diseases in the elderly globally. There is a medical need to improve the quality of life of patients by preventing lower respiratory tract diseases (LRTD). The clinical trials described in Study A (i.e., the sentinel cohort) and Study B (i.e., the main cohort) of Example 1 will include a subsequent Phase IIb / III study, which will enroll approximately 13,482 adults over 60 years old to evaluate the efficacy, immunogenicity, and safety of the LNPs selected in Phase 1 (i.e., Phase I / IIa as described in Examples 1 and 2), using a 110 μg dose of RSV mRNA vaccine to prevent LRTD caused by RSV.

[0459] During Phase 1, a 75 μg dose of the RSV mRNA vaccine in combination with the selected LNPs showed the highest immune response and had a favorable safety profile. Therefore, Example 3 will test an 110 μg dose of the RSV mRNA vaccine with the selected LNPs. The IIb / III phase of this study aims to evaluate the safety of an 110 μg dose of the RSV mRNA vaccine with the selected LNPs and primarily demonstrate the clinical efficacy of the selected RSV mRNA vaccine candidate in preventing RSV-LRTD. A. Study Overview and Study Design Number of Participants, Overview of Intervention Groups, and Duration

[0460] Number of participants. A total of 13,482 participants are planned to be randomly assigned, with 50 participants (25 participants per group) in the IIb phase sentinel cohort, 5,190 participants (2,595 participants per group) in the IIb phase main cohort, as shown in Table 12 below. Table 12. IIb / III Phase Experimental Sample Size (Participants Aged 60 and Above)

[0461] For the Phase III cohort, an additional 8,242 participants (4,121 participants per group) are planned to be randomly assigned. Approximately 2,000 participants (1,000 per group) from the IIb phase and 4,000 participants (2,000 per group) from the Phase III cohort will be included in the reactogenicity subgroup for collecting solicited injection site reactions and systemic reactions occurring within 7 days after vaccination. In addition, approximately 500 participants (250 per group) from each of the IIb phase and Phase III cohorts will be included in the immunogenicity subgroup to evaluate the persistence of neutralizing antibody responses. Approximately 100 participants (50 per group) from the Phase III cohort will also be selected to be included in the CMI subgroup. Participants in the CMI subgroup will be recruited from a limited number of selected sites, which are chosen based on previous experience in collecting and processing blood samples for CMI assays.

[0462] Intervention group. The intervention group in Phase 2 will be eligible participants randomized at a 1:1 ratio to receive a single intramuscular (IM) administration of the RSV mRNA vaccine candidate or placebo.

[0463] Expected duration. The expected total duration of the study for participants in the IIb phase sentinel cohort is approximately 6 months, and the expected total duration of the study for participants in the IIb phase main cohort / Phase III cohort is approximately 12 months.

[0464] Composition. Participants in Phase 2 will receive, via IM injection, a dose (0.5 mL) of the liquid solution in a vial containing 110 μg of RSV pre-F mRNA with the selected LNP in PBS 2.2x diluent, or a dose (0.5 mL) of the liquid solution in a vial containing 0.9% saline. B. Objectives

[0465] Primary objective. The primary objective is to evaluate the safety of the 110 μg dose of the RSV mRNA vaccine with the selected LNP and to demonstrate the same clinical efficacy of the mRNA RSV vaccine candidate in preventing RSV-LRTD during the first season that occurs ≥ 14 days after vaccination.

[0466] Secondary objectives. The secondary objectives are to demonstrate the clinical efficacy of the mRNA RSV vaccine candidate in preventing RSV-ARD (RSV - Acute Respiratory Disease) and RSV-MAARD (RSV - Medically Attended Acute Respiratory Disease) during the first season that occur ≥ 14 days after vaccination.

[0467] The following Table 13 summarizes the primary objective and the corresponding endpoints. The following Table 14 summarizes the secondary objectives and the corresponding endpoints. The following Table 15 summarizes the immunogenicity objectives and the corresponding endpoints. The following Table 16 summarizes the safety objectives and the corresponding endpoints. The following Table 17 summarizes the exploratory objectives and the corresponding endpoints. Table 13. Primary Objectives and Corresponding Endpoints Table 14. Secondary Objectives and Corresponding Endpoints a The baseline frailty status was evaluated using a gait speed test. A walking speed < 0.4 m / sec or inability to perform the test indicated a frail status, a walking speed of 0.4 to 0.99 m / sec indicated a pre-frail status, and a walking speed of 1 m / sec or faster indicated a healthy status. Table 15. Immunogenicity Objectives and Corresponding Endpoints Table 16. Safety Objectives and Corresponding Endpoints Table 17. Exploratory Objectives and Corresponding Endpoints

[0468] In view of the disclosure and practice disclosed herein, other embodiments of the present disclosure will be apparent to those skilled in the art. The specification and examples are intended to be considered only as exemplary, and the true scope and spirit of the present disclosure are indicated by the following claims.

[0469] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

2. The method according to claim 1, wherein the RSV F protein antigen is a prefusion protein.

3. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

4. The method according to any one of claims 1-3, wherein the RSV vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously or intradermally.

5. The method according to claim 4, wherein the RSV vaccine is administered intramuscularly.

6. The method according to claim 5, wherein the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

7. The method according to any one of claims 1-6, wherein the subject is at least 60 years old.

8. The method according to any one of claims 1-7, wherein the RSV vaccine does not contain an adjuvant.

9. The method according to any one of claims 1-8, wherein the mRNA is formulated in a lipid nanoparticle (LNP).

10. The method according to claim 9, wherein the LNP comprises at least one cationic lipid.

11. The method according to claim 10, wherein the at least one cationic lipid is biodegradable.

12. The method according to claim 10, wherein the at least one cationic lipid is not biodegradable.

13. The method according to claim 10, wherein the at least one cationic lipid is cleavable.

14. The method according to claim 10, wherein the at least one cationic lipid is not cleavable.

15. The method according to claim 10, wherein the at least one cationic lipid is selected from the group consisting of: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 or IM-001.

16. The method according to claim 15, wherein the at least one cationic lipid is cKK-E10.

17. The method according to claim 15, wherein the at least one cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

18. The method according to claim 15, wherein the at least one cationic lipid is IM-001.

19. The method according to any one of claims 1-18, wherein an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject.

20. The method according to claim 19, wherein each of the one or more booster doses is administered to the subject at least 11 months after a previous dose.

21. The method according to claim 19, wherein each of the one or more booster doses is administered to the subject at least 12 months after a previous dose.

22. The method according to claim 19, wherein each of the one or more booster doses is administered to the subject from about 10 months to about 14 months after a previous dose.

23. The method according to claim 19, wherein each of the one or more booster doses is administered to the subject about 12 months after a previous dose.

24. The method according to any one of claims 1-18, wherein an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject.

25. The method according to claim 24, wherein the booster dose is administered to the subject at least 11 months after the initial dose.

26. The method according to claim 24, wherein the booster dose is administered to the subject at least 12 months after the initial dose.

27. The method according to claim 24, wherein the booster dose is administered to the subject from about 10 months to about 14 months after the initial dose.

28. The method according to claim 24, wherein the booster dose is administered to the subject about 12 months after the initial dose.

29. The method according to any one of claims 1-28, wherein the RSV vaccine is administered at a dose of about 5 micrograms to about 120 micrograms.

30. The method according to claim 29, wherein the RSV vaccine is administered at a dose of about 5 micrograms to about 15 micrograms.

31. The method according to claim 30, wherein the RSV vaccine is administered at a dose of about 10 micrograms.

32. The method according to claim 29, wherein the RSV vaccine is administered at a dose of about 20 micrograms to about 40 micrograms.

33. The method according to claim 32, wherein the RSV vaccine is administered at a dose of about 30 micrograms.

34. The method according to claim 29, wherein the RSV vaccine is administered at a dose of about 65 micrograms to about 95 micrograms.

35. The method according to claim 34, wherein the RSV vaccine is administered at a dose of about 75 micrograms.

36. The method according to claim 29, wherein the RSV vaccine is administered at a dose of about 100 micrograms to about 120 micrograms.

37. The method according to claim 36, wherein the RSV vaccine is administered at a dose of about 110 micrograms.

38. A method for preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

39. The method according to claim 38, wherein the RSV F protein antigen is a prefusion protein.

40. A method for preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising administering to the subject a prophylactically effective amount of an RSV vaccine, the RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

41. The method according to any one of claims 38-40, wherein the vaccine is administered intramuscularly, intranasally, intravenously, subcutaneously or intradermally.

42. The method according to claim 41, wherein the RSV vaccine is administered intramuscularly.

43. The method according to claim 42, wherein the RSV vaccine is administered into the deltoid muscle of the upper arm of the subject.

44. The method according to any one of claims 38-43, wherein the subject is at least 60 years old.

45. The method according to any one of claims 38-44, wherein the RSV vaccine does not contain an adjuvant.

46. The method according to any one of claims 38-45, wherein the mRNA is formulated in lipid nanoparticles (LNPs).

47. The method according to claim 46, wherein the LNP comprises at least one cationic lipid.

48. The method according to claim 47, wherein the at least one cationic lipid is biodegradable.

49. The method according to claim 47, wherein the at least one cationic lipid is not biodegradable.

50. The method according to claim 47, wherein the at least one cationic lipid is cleavable.

51. The method according to claim 47, wherein the at least one cationic lipid is not cleavable.

52. The method according to claim 47, wherein the at least one cationic lipid is selected from the group consisting of: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

53. The method according to claim 52, wherein the at least one cationic lipid is cKK-E10.

54. The method according to claim 52, wherein the at least one cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

55. The method according to claim 52, wherein the at least one cationic lipid is IM-001.

56. The method according to any one of claims 38-55, wherein an initial dose of the RSV vaccine and one or more booster doses of the RSV vaccine are administered to the subject.

57. The method according to claim 56, wherein each of the one or more booster doses is administered to the subject at least 11 months after a previous dose.

58. The method according to claim 56, wherein each of the one or more booster doses is administered to the subject at least 12 months after a previous dose.

59. The method according to claim 56, wherein each of the one or more booster doses is administered to the subject from about 10 months to about 14 months after a previous dose.

60. The method according to claim 56, wherein each of the one or more booster doses is administered to the subject about 12 months after a previous dose.

61. The method according to any one of claims 38-55, wherein an initial dose of the RSV vaccine and a booster dose of the RSV vaccine are administered to the subject.

62. The method according to claim 61, wherein the booster dose is administered to the subject at least 11 months after the initial dose.

63. The method according to claim 61, wherein the booster dose is administered to the subject at least 12 months after the initial dose.

64. The method according to claim 61, wherein the booster dose is administered to the subject from about 10 months to about 14 months after the initial dose.

65. The method according to claim 61, wherein the booster dose is administered to the subject about 12 months after the initial dose.

66. The method according to any one of claims 38-65, wherein the RSV vaccine is administered at a dose of about 5 micrograms to about 120 micrograms.

67. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 5 micrograms to about 15 micrograms.

68. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 10 micrograms.

69. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 20 micrograms to about 40 micrograms.

70. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 30 micrograms.

71. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 65 micrograms to about 95 micrograms.

72. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 75 micrograms.

73. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 100 micrograms to about 120 micrograms.

74. The method according to claim 66, wherein the RSV vaccine is administered at a dose of about 110 micrograms.

75. The method according to any one of claims 38 - 74, wherein the RSV vaccine is administered in a device suitable for subcutaneous injection.

76. The method according to any one of claims 38 - 74, wherein the one or more symptoms of RSV infection are selected from the group consisting of: acute respiratory disease (ARD), medically attended acute respiratory disease (MAARD), severe ARD, lower respiratory tract disease (LRTD) not requiring medical attention, LRTD requiring medical attention, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

77. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

78. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

79. A method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

80. A method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject a prophylactically effective amount of an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

81. A respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity with SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:3, and wherein the RSV F protein antigen is a pre-fusion protein.

82. A respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity with SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

83. A respiratory syncytial virus (RSV) vaccine for eliciting an immune response against RSV in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity with SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

84. A respiratory syncytial virus (RSV) vaccine for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity with SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:

3.

85. A respiratory syncytial virus (RSV) vaccine for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, wherein the RSV vaccine comprises messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity with SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:

14.

86. A method for eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject at least 60 years old; and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity with SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 110 micrograms.

87. A method for eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: Select subjects who are at least 60 years old; and administer an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

88. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: Select subjects who are at least 60 years old; and administer to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising GL-HEPES-E3-E12-DS-4-E10, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

89. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: Select subjects who are at least 60 years old; and administer to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

90. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: Select subjects who are at least 60 years old; and administer to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

91. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: Select subjects who are at least 60 years old; and administer to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14 or consists of the nucleic acid sequence of SEQ ID NO:14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising cKK-E10, and The RSV vaccine is administered at a dose of approximately 110 micrograms.

92. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO: 14 or consisting of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of approximately 30 micrograms.

93. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO: 14 or consisting of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of approximately 75 micrograms.

94. A method of eliciting an immune response against respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years old; and administering to the subject an RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO: 14 or consisting of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in a lipid nanoparticle (LNP) comprising IM-001, and wherein the RSV vaccine is administered at a dose of approximately 110 micrograms.

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