Respiratory syncytial virus RNA vaccination

Abstract

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

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 422,621, filed November 4, 2022, and U.S. Provisional Patent Application No. 63 / 523,543, filed June 27, 2023, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease in infants and a significant cause of respiratory disease in the elderly. Despite decades of research, there remains an unmet need for a vaccine against RSV. Recent clinical programs using the RSV F antigen in its postfusion conformation have failed to induce sufficient efficacy in adults. See Faloon et al. (2017) JID 216:1362-1370. However, RSV F antigen stabilized in the prefusion conformation induces a much more neutralizing response than that of the postfusion antigen, potentially conferring a high level of protective efficacy against RSV disease in the elderly. Summary of the Invention [Problem to be solved by the invention]

[0003] Recently, RNA-based vaccines (e.g., mRNA vaccines) have emerged as an effective vaccine type against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) mRNA vaccines have demonstrated a rapid, safe, and cost-effective production process. Often combined with a delivery vehicle such as lipid nanoparticles (LNPs), COVID-19 mRNA vaccines can achieve high efficacy. Due to the lack of available effective RSV vaccines, there is a need for an RNA-based RSV vaccine that induces a strong immune response against the RSV pre-fusion F protein for potent neutralization of RSV infection. [Means for solving the problem]

[0004] In certain embodiments, a method for inducing an immune response to respiratory syncytial virus (RSV) in a subject is provided, comprising administering to the subject a prophylactically effective amount of a RSV vaccine comprising messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a 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 certain exemplary embodiments, the RSV F protein antigen is a prefusion 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 subject's upper arm.

[0007] In certain exemplary embodiments, the subject is between the ages of 18 and 50. In certain exemplary embodiments, the subject is at least 60 years old.

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

[0009] 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, or GL-HEPES-E3-E12-DS-4-E10, and IM-001.

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

[0011] In certain exemplary embodiments, the subject receives a primary dose of the RSV vaccine and a booster dose of the RSV vaccine.

[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 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.

[0015] 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.

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

[0017] In another aspect, a method of inducing an immune response to respiratory syncytial virus (RSV) in a subject is provided, comprising administering to the subject a prophylactically effective amount of a RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to, or consists 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 subject's upper arm.

[0019] In certain exemplary embodiments, the subject is between the ages of 18 and 50. In certain exemplary embodiments, the subject is at least 60 years old.

[0020] In certain exemplary embodiments, the RSV vaccine does not include 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, a subject is administered an initial dose of a RSV vaccine and one or more booster doses of a RSV vaccine, wherein each of the one or more booster doses is administered to the subject at least 11 months after the previous dose, at least 12 months after the previous dose, about 12 months after the previous dose, or about 10 to about 14 months after the previous dose.

[0023] In certain exemplary embodiments, the subject receives a primary dose of the RSV vaccine and a booster dose of the RSV vaccine.

[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 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.

[0026] In certain exemplary embodiments, the RSV 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.

[0027] 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.

[0028] In another aspect, a method for preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject is provided, comprising administering to the subject a prophylactically effective amount of a RSV vaccine comprising messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a 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.

[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 subject's upper arm.

[0031] In certain exemplary embodiments, the subject is between the ages of 18 and 50. In certain exemplary embodiments, the subject is at least 60 years old.

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

[0033] 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.

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

[0035] In certain exemplary embodiments, a subject is administered an initial dose of a RSV vaccine and a booster dose of a RSV vaccine, hi 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 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.

[0038] 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.

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

[0040] In certain exemplary embodiments, the one or more symptoms of RSV infection are selected from the group consisting of acute respiratory disease (ARD), medically-induced acute respiratory disease (MAARD), severe ARD, non-medical-induced lower respiratory tract disease (LRTD), medically-induced LRTD, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

[0041] In another aspect, a method is provided for preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, 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, or consists 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 subject's upper arm.

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

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

[0045] 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.

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

[0047] In certain exemplary embodiments, a subject is administered an initial dose of a RSV vaccine and a booster dose of a RSV vaccine, hi 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-induced acute respiratory disease (MAARD), severe ARD, non-medical-induced lower respiratory tract disease (LRTD), medically-induced LRTD, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

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

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

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

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

[0056] In another aspect, a respiratory syncytial virus (RSV) vaccine for use in eliciting an immune response to RSV in a subject is provided, comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to or consisting of the amino acid sequence of SEQ ID NO: 3, wherein the RSV F protein antigen is a prefusion protein.

[0057] In another aspect, a respiratory syncytial virus (RSV) vaccine for use in eliciting an immune response to RSV in a subject is provided, comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a 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.

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

[0059] In another aspect, a respiratory syncytial virus (RSV) vaccine for use in preventing or reducing one or more symptoms of a RSV infection in a subject is provided, comprising a messenger RNA (mRNA) containing an open reading frame (ORF) encoding a 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.

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

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

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

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

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

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

[0066] In another aspect, a method of inducing an immune response to respiratory syncytial virus (RSV) in a subject is provided, comprising selecting a subject who is at least 60 years of age and administering to the subject a RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to or consists of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in lipid nanoparticles (LNPs) comprising cKK-E10, and wherein the RSV vaccine is administered at a dose of about 110 micrograms.

[0067] In another aspect, a method of inducing an immune response to respiratory syncytial virus (RSV) in a subject is provided, comprising selecting a subject who is at least 60 years of age and administering to the subject a RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to or consists of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in lipid nanoparticles (LNPs) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 30 micrograms.

[0068] In another aspect, a method of inducing an immune response to respiratory syncytial virus (RSV) in a subject is provided, comprising selecting a subject who is at least 60 years of age and administering to the subject a RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to or consists of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in lipid nanoparticles (LNPs) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 75 micrograms.

[0069] In another aspect, a method is provided for inducing an immune response to respiratory syncytial virus (RSV) in a subject, the method comprising: selecting a subject who is at least 60 years of age; and administering to the subject a RSV vaccine comprising messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid sequence having at least 98% identity to or consists of the nucleic acid sequence of SEQ ID NO: 14, wherein the mRNA is formulated in lipid nanoparticles (LNPs) comprising IM-001, and wherein the RSV vaccine is administered at a dose of about 110 micrograms.

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

[0071] [Figure 1] A graphic overview of the study design for Study A cohort (sentinel cohort, 18-50 years of age) is shown. AE: adverse event; AESI: adverse event of special interest; BL: blood sample; MAAE: adverse event requiring medical attention; RSV: respiratory syncytial virus; SAE: serious adverse event; SCR: screening. Note: Complete blood draw on D01 (V01) prior to vaccination. [Figure 2] A diagrammatic overview of the study design for Study B cohort (primary cohort, ≥60 years of age) is shown. AE: adverse event; AESI: adverse event of special interest; BL: blood sample for immunogenicity; MAAE: adverse event requiring medical consultation; RSV: respiratory syncytial virus; SAE: serious adverse event; SCR: screening; VAC: vaccine; WB: blood sample for CMI; *D04 (V02) not applicable to primary cohort; †D01 (V01) blood sample must be completed prior to vaccination. [Figure 3]The study design for Study C cohort (booster cohort, age 60 years or older) is outlined in the figure. AE: adverse event; AESI: adverse event of special interest; BL: blood sample for immunogenicity; MAAE: adverse event requiring medical consultation; 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 randomized in a 1:1 ratio in M12 to receive a booster vaccination with the selected formulation of the RSV mRNA vaccine (i.e., the dose level and LNP formulation selected based on the safety and immunogenicity results of the primary cohort) or placebo. Note 2: BL0005 obtained in V07 of the primary cohort will be used as the pre-vaccination sample for the booster cohort. [Figure 4]A table showing the activity schedule for Study A cohort (sentinel cohort, participants aged 18-50 years) is shown. AE = adverse event; AESI = adverse event of special interest; BL = blood draw for immunogenicity; BS = blood draw for safety assessment; CRF = case report form; D or d = day; DC = diary card; M = month; MA = memory aid; MAAE = adverse event requiring medical consultation; NS = nasal swab; PRN = as needed; SAE = serious adverse event; TC = telephone contact; UN = blood draw for illness visit; V = visit; vac = vaccination. A single asterisk (*) indicates that non-visit contact will be conducted by phone at the scheduled time point in the study. A dagger (†) indicates an abbreviated physical examination at all in-person visits after Visit 01. A double dagger (‡) indicates an electrocardiogram (ECG) performed at screening as a baseline and reviewed by the investigator for features of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the study, an additional ECG will be performed as soon as possible (i.e., at an unscheduled visit, if necessary). A section mark (§) indicates that body temperature will be measured by oral (preferred) or axillary route using a standard digital thermometer and recorded on source documentation. Two asterisks (**) indicate that safety laboratory assessments will include serum chemistry, hematology, and clotting time. At screening and V03, serum volume samples will be collected for troponin I level testing as part of the safety laboratory assessments. Portions of samples collected at V04, V05, V06, and V07 will be stored for potential future testing of troponin I (using the screening blood sample as a baseline) if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis. If safety laboratory results are abnormal, an unscheduled visit may be conducted at the investigator's discretion. Blood volume for safety testing may be adjusted based on local regulations. Two daggers (††) indicate that nasal swab specimens for detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants during illness visits, including visits requiring consultation during the study.If a participant visits any other off-study physician / hospital for a serious adverse event at any time during the study, a nasal swab specimen will be obtained at the study site immediately upon discharge, if deemed appropriate by the investigator. All nasal swab specimens will be collected in the recommended viral transport media tube and stored at -60°C to -80°C until ready for shipment. If deemed appropriate by the investigator, the requirement for a sick visit or sick visit will be initially assessed by video call (preferred) or regular telephone if video call is not possible, to allow for remote assessment of severity and remote management of mild (Grade 1) illness. Two double daggers (‡‡) represent any spontaneous systemic adverse event occurring within 30 minutes of vaccine administration, which will be recorded on the case report form as an immediate spontaneous systemic adverse event. Two section marks (§§) indicate that participants will record information on diary cards regarding induced reactions, spontaneous 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 affect the immune response or both safety and immune response will be collected. Three daggers (†††) indicate that the entire visit will be completed if the participant discontinues at the visit. Three double daggers (‡‡‡) 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 UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may be collected. Swabs will not be collected during the illness visit if the collection date is more than 14 days after symptom resolution. [Figure 4-1] Same as above. [Figure 4-2] Same as above. [Figure 5]A table showing the activity schedule for Study B Cohort (primary cohort, participants aged 60 years and older) is shown. AE = adverse event; AESI = adverse event of special interest; BL = blood draw for immunogenicity; BS = blood draw 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 contact; UN = blood sample for illness visit; V = visit; vac: vaccination; WB = blood sample for TruCulture. A single asterisk (*) indicates that visit 04 (V02) is not applicable to the primary cohort. A dagger (†) indicates that non-visit contacts will be made by phone at scheduled times during the study. A double dagger (‡) indicates an abbreviated physical examination at all in-person visits after Visit 01. A section mark (§) indicates that an electrocardiogram will be performed at screening as a baseline and reviewed by the investigator for features of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during study conduct, an additional ECG will be performed as soon as possible (i.e., at an unscheduled visit, if necessary). A double asterisk (**) indicates that temperature will be measured by oral (preferred) or axillary route using a standard digital thermometer and recorded on source documentation. A double dagger (††) indicates that samples collected from a subset of 140 participants for CMI assay will be evaluated by TruCulture. A double dagger (‡‡) indicates that safety laboratory assessments will include serum chemistry, hematology, and clotting time. At screening and V03, serum volume samples will be collected for troponin I levels as part of safety laboratory assessments. A portion of the samples collected in V01, V04, V05, V06, and V07 will be stored for potential future testing of Troponin I (using the screening blood sample as a baseline) if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis. If safety test results are abnormal, unscheduled visits may be conducted based on the investigator's discretion.Two section marks (§§) indicate that nasal swab specimens for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants during illness visits, including visits requiring a study consultation. If a participant visits any other off-study physician / hospital for an SAE at any time during the study, a nasal swab sample will be obtained at the study site immediately upon discharge, if deemed appropriate by the investigator. All nasal swab specimens will be collected in recommended viral transport media tubes and stored at -60°C to -80°C until ready for shipment. If deemed appropriate by the investigator, requirements for illness visits or illness visits will be initially assessed by video call (preferred) or regular telephone if video call is not possible, to allow for remote assessment of severity and remote management of mild (Grade 1) illness. Three asterisks (***) indicate any spontaneous systemic AE occurring within 30 minutes of vaccine administration, which will be recorded in the case report form as an immediate spontaneous systemic AE. Three daggers (†††) indicate that participants will record information on a diary card regarding induced reactions from day 0 to day 28 after vaccine administration, as well as spontaneous AEs and MAAEs, and AESIs and SAEs throughout the study. Three double daggers (‡‡‡) indicate that only medications that may affect the immune response or that may affect both safety and immune response will be collected. Three section marks (§§§) indicate that the entire visit will be completed if the participant discontinues at the visit. 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 UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may be collected. Swabs will not be collected during the disease visit if the collection date is more than 14 days after symptom resolution. Four double daggers (††††) indicate participants who did not continue in the booster cohort. [Figure 5-1] Same as above. [Figure 5-2] Same as above. [Figure 6]A table showing the activity schedule for Study C cohort (booster cohort, participants aged 60 years and older) is shown. AE = adverse event; AESI = adverse event of special interest; BL = blood draw for immunogenicity; BS = blood draw 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 contact; UN = blood sample for illness visit; V = visit; vac = vaccination; Vac2 = booster vaccination. An asterisk (*) indicates that non-visit contact will be made by phone at the scheduled time point in the study. A dagger (†) denotes an abbreviated physical examination at all in-person visits after Visit 08. Body temperature will be measured by oral (preferred) or axillary route using a standard digital thermometer and recorded in source documentation. The double dagger (‡) indicates that an electrocardiogram (ECG) will be performed at screening as a baseline and reviewed by the investigator for features of prior myocarditis, pericarditis, and / or myopericarditis. For any participant who develops symptoms of myocarditis, pericarditis, and / or myopericarditis during study conduct, an additional ECG will be performed as soon as possible (i.e., at an unscheduled visit, if necessary). The section mark (§) indicates that body temperature will be measured by oral (preferred) or axillary route using a standard digital thermometer and recorded on source documentation. The two asterisks (**) annotate blood sample 5, designated BL0005, obtained in V07 for use as a pre-vaccination sample in the booster cohort. The double dagger (††) indicates that safety laboratory assessments will include serum chemistry, hematology, and clotting time. At screening and V09, serum volume samples will be collected for troponin I level testing as part of safety laboratory assessments. Portions of the samples collected at V10, V11, V12, and V13 will be stored for potential future testing of Troponin I (using the screening blood sample as a baseline) if the participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis. If safety test results are abnormal, unscheduled visits may be conducted at the investigator's discretion.Two double daggers (‡‡) indicate that nasal swab specimens for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants during illness visits, including visits requiring a medical consultation during the study. If a participant visits any other non-study physician / hospital for an SAE at any time during the study, a nasal swab specimen will be obtained at the study site immediately upon discharge, if deemed appropriate by the investigator. If a participant is unable to attend an illness visit and / or cannot make a house call, they will be required to self-collect the specimen. All nasal swab specimens will be collected in recommended viral transport media tubes and stored at -60°C to -80°C until ready for shipment. If deemed appropriate by the investigator, requirements for illness visits or illness visits will be initially assessed by video call (preferred) or regular telephone if video call is not possible, to allow for remote assessment of severity and remote management of mild (Grade 1) illness. The two section marks (§§) indicate that this will be administered 12 months after the first injection. The three asterisks (***) represent any spontaneous systemic AE occurring within 30 minutes of vaccine administration, which will be recorded as an immediate spontaneous systemic AE in the case report form. The three daggers (†††) indicate that participants will record information on their diary cards regarding induced reactions, spontaneous AEs, and MAAEs from day 0 to day 28 (D0-D28) after vaccine administration, as well as AESIs and SAEs throughout the study. The three double daggers (‡‡‡) indicate that only medications that may affect the immune response or both safety and immune response will be collected. The three section marks (§§§) 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 UN swabs for central laboratory testing, samples for local laboratory testing (clinical care) may be collected. If the collection date is more than 14 days after symptom resolution, the swab will not be taken during the illness visit. [Figure 6-1] Same as above. [Figure 6-2] Same as above. [Figure 7] 1 is a table showing the demographic characteristics of the main cohort. [Figure 8]Graphical summary of RSV-A neutralizing antibody (NAb) geometric mean titers (GMTs) and neutralizing antibody geometric mean titer ratios (GMTRs) 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. [Figure 9] A graph shows participants with a 4-fold or greater and less than 4-fold increase in RSV-A neutralizing antibody titers after primary vaccination for the entire primary cohort (ages 60 years and older). [Figure 10] Graphical representation of the partial core cohort (ages 60 years and older) summarizing the geometric means of IgG antibody titers after primary vaccination and the IgG antibody geometric mean titer ratios at D29 and D01. 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. [Figure 11] Graphical summary of RSV-A neutralizing antibody (NAb) geometric mean titers (GMTs) and neutralizing antibody geometric mean titer ratios (GMTRs) at D29 and D01 after primary vaccination for the sentinel cohort (ages 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. [Figure 12]The graph shows participants in the sentinel cohort (aged 18-50 years) who had a 4-fold or greater increase in RSV-A neutralizing antibody titers and participants who had a less than 4-fold increase after primary vaccination. [Figure 13A] Graphical summary of the fold increase in RSV-A neutralizing antibody titers after primary vaccination in the sentinel cohort: (A) cKK-E10. (B) GL-HEPES-E3-E12-DS-4-E10. [Figure 13B] Graphical summary of the fold increase in RSV-A neutralizing antibody titers after primary vaccination in the sentinel cohort: (A) cKK-E10. (B) GL-HEPES-E3-E12-DS-4-E10. [Figure 14] The induced reactions within 7 days after primary vaccination are summarized in percentage in the graph. X-axis: A=cKK-E10; B=GL-HEPES-E3-E12-DS-4-E10; C=placebo. [Figure 15] The graph summarizes induced injection site reactions in percentage within 7 days after primary vaccination. X-axis: A=cKK-E10; B=GL-HEPES-E3-E12-DS-4-E10; C=placebo. [Figure 16] The graph summarizes induced systemic reactions in percentage within 7 days after primary vaccination. X-axis: A=cKK-E10; B=GL-HEPES-E3-E12-DS-4-E10; C=placebo. [Figure 17] Table summarizing safety profile after primary vaccination (primary cohort). [Figure 18] Table summarizing induced reactions within 7 days after primary vaccination (primary cohort). [Figure 18-1] Same as above. [Figure 19]This table summarizes spontaneous adverse events. Pooled data are presented because no dose response was observed. An AESI was downgraded to a non-AESI by the investigator after the biostatistical output cutoff date (the diagnosis changed from myocarditis to silent myocardial injury). *There were no significant imbalances; the only observed imbalances were those caused by musculoskeletal and connective tissue disorders (e.g., arthralgia, myalgia, muscle cramps) and gastrointestinal disorders (e.g., abdominal pain, diarrhea, nausea). δSAEs were evenly distributed across mRNA groups (two in LNP cKK-E10 and three in LNP GL-HEPES-E3-E12-DS-4-E10). Only one SAE (silent myocardial injury) was assessed as related to IMP (LNP cKK-E10 low dose). The events were as follows: 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); and hypertensive crisis in LNP GL-HEPES-E3-E12-DS-4-E10 (high dose). [Figure 20] 1 is a table summarizing spontaneous adverse events due to musculoskeletal, connective tissue, and gastrointestinal disorders compared to placebo. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure relates, inter alia, to RNA (e.g., mRNA) vaccine compositions encoding the RSV F protein and vaccination methods using the same. Further, the present disclosure relates to vaccine compositions comprising mRNA encoding the RSV pre-fusion F protein formulated in lipid nanoparticles (LNPs) and vaccination methods using the same.

[0073] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In the case of conflict, the present specification, including definitions, shall control. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited in this specification, such citation does not constitute an admission that any of those documents form part of the common general knowledge in the art.

[0074] It should be noted that the term "a" or "an" component refers to one or more of that component; for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0075] Furthermore, as used herein, "and / or" should be understood as specifically disclosing each of the two specified features or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" when used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: 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).

[0076] Whenever an embodiment is described herein with the word "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0077] 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 ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0078] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of 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 of the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0079] The terms "approximately" or "about" are used herein to mean approximately, in the vicinity of, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above and below the stated value by, for example, a variance of 10% above or below (higher or lower). In some embodiments, the term indicates 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 stated numerical value. In some embodiments, "about" indicates a deviation of ±10% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the stated numerical value. In some embodiments, "about" indicates a ±3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value.

[0080] 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. mRNA can contain one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding region of an mRNA includes the 5' cap, 5' untranslated region (UTR), 3' UTR, and poly(A) tail. mRNA can be purified from natural sources or produced using recombinant expression systems (e.g., in vitro transcription). In various embodiments, mRNA can be purified or chemically synthesized.

[0081] As used herein, the term "F protein" or "RSV F protein" refers to a protein of RSV that is responsible for promoting the fusion of the viral envelope with the host cell membrane during viral entry.

[0082] As used herein, the term "RSV F polypeptide" or "F polypeptide" refers to a polypeptide containing at least one epitope of the F protein.

[0083] As used herein, the term "post-fusion" with respect to RSV F refers to the stable conformation of RSV F that occurs after the merging of the viral and cellular membranes.

[0084] As used herein, the term "pre-fusion" with respect to RSV F refers to the conformation of RSV F adopted prior to virus-cell interaction.

[0085] As used herein, the term "protomer" refers to a structural unit of an oligomeric protein. In the case of RSV F, the individual units of the RSV F trimer are protomers.

[0086] 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. The glycan may be a polysaccharide.

[0087] As used herein, the term "glycosylation" refers to the addition of sugar units to a protein.

[0088] As used herein, the term "immune response" refers to the response of a cell of the immune system, e.g., a B cell, a T cell, a dendritic cell, a macrophage, or a polymorphonuclear cell, to a stimulus, e.g., an antigen or a vaccine. An immune response can include any cell of the body that participates in a host defense reaction, including, e.g., epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, an innate immune response and / or an adaptive immune response.

[0089] As used herein, an "antibody response" is an immune response in which antibodies are produced.

[0090] As used herein, "antigen" refers to an agent that, when exposed to or administered to an organism, elicits an immune response and / or binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including the production of antigen-specific antibodies). Alternatively, or additionally, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism's species. In some embodiments, the antigen induces 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 members of the target 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, regardless of whether 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 the mRNAs described herein.

[0091] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions in which an antigen solution is emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria are sometimes included to further enhance antigenicity (e.g., Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, 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 biological molecules, such as Toll-like receptor (TLR) agonists and costimulatory molecules.

[0092] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "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 may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments, the subject is an adult, an adolescent, or an infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject." In certain exemplary embodiments, the subject is a premature infant (e.g., less than 37 weeks gestational age), a newborn (e.g., 0-27 days old), an infant or toddler (e.g., 28 days to 23 months old), a child (e.g., 2-11 years old), an adolescent (e.g., 12-17 years old), an adult (e.g., 18-50 years old or 18-64 years old), or a geriatric (e.g., 65 years old or older). In exemplary embodiments, the subject is a geriatric (e.g., an adult 60 years old or older).

[0093] As used herein, the terms "vaccination" or "vaccinating" refer to the administration of a composition intended to generate an immune response, for example, against a disease-causing agent. Vaccination can occur before, during, and / or after exposure to a disease-causing agent and / or before, during, and / or after the onset of one or more symptoms, in some embodiments before, during, and / or immediately after exposure to a disease-causing agent. In some embodiments, vaccination involves multiple administrations of a vaccinating composition spaced appropriately apart, for example, about 12 months after a previous dose.

[0094] The present disclosure describes nucleic acid sequences (eg, DNA and RNA sequences) and amino acid sequences that have a degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).

[0095] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the compared sequences. Said percentage is purely statistical; the differences between two sequences can be, but are not necessarily, randomly distributed over the entire length of the compared sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "window of comparison" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or using computer programs that employ such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0096] The percentage identity is obtained by determining the number of corresponding identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0097] In some embodiments, the degree of identity is provided over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is provided over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, contiguous nucleotides. In some embodiments, the degree of identity is provided over the entire length of the reference sequence.

[0098] A nucleic acid sequence or amino acid sequence that shares a particular degree of identity with a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional characteristic of the given sequence, e.g., in some instances, is functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that shares a particular degree of identity with a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0099] As used herein, the term "kit" refers to a packaged set of related components, e.g., one or more compounds or compositions and one or more associated materials, e.g., solvents, solutions, buffers, instructions, or desiccants.

[0100] II. RSV mRNA Vaccine Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus belonging to the Pneumoviridae family. RSV can cause infection of the respiratory tract. RSV is an enveloped virus. The surface of the RSV virion contains three proteins: a binding glycoprotein (G), a fusion protein (F), and a small hydrophobic (SH) protein.

[0101] The RSV F protein is involved in the fusion of the viral membrane with the host cell membrane and adopts at least three conformations (pre-fusion, intermediate, and post-fusion conformations). In the pre-fusion conformation (pre-fusion, Pre-F), the F protein exists in a trimer form with the major antigen site Φ exposed. Site Φ serves as the primary 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 site Φ is no longer exposed. Pre-F transitions to a transient intermediate conformation, allowing the F protein to be inserted into the host cell membrane, resulting in fusion of the viral membrane with the host cell membrane. The final conformational shift results in a more stable, extended form of the protein (post-fusion, Post-F). Sites II and IV of the F protein are specific 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).

[0102] Respiratory syncytial virus (RSV) is the leading viral agent causing severe respiratory tract disease in older adults worldwide. Currently, no vaccine is available for the prevention of RSV in older adults, and no effective antiviral treatment is available. The economic and clinical burden imposed on healthcare systems during RSV epidemics will remain high until preventive treatment options become available.

[0103] As provided herein, mRNA-based vaccines based on three different RSV proteins have been developed. The F protein designated FD1 corresponds to the wild-type RSV F protein. The F protein designated FD2 corresponds to a soluble RSV F protein lacking the transmembrane domain and cytoplasmic tail and containing a C-terminal fibritin trimerization domain (also known as the T4 foldon). The F protein designated FD3 corresponds to the pre-fusion RSV F protein.

[0104] As used herein, the term "antigenic site Φ" ​​or "site Φ epitope" refers to the antigenic site Φ of wild-type RSV F, i.e., FD1 or SEQ ID NO: 1. [ka] The Φ epitope is a binding site for antibodies with specificity for pre-fusion RSV F, such as D25 and AM14, and antibody binding to the Φ epitope blocks RSV cell surface attachment (see, e.g., McLellan et al., Science, 340(6136):1113-1117, 2013). Recombinant human anti-RSV antibody D25 (Creative Biolabs®; Catalog No. PABL-322) and recombinant human anti-RSV antibody AM14 (Creative Biolabs®; Catalog No. PABL-321) are commercially available.

[0105] FD2 or SEQ ID NO:2 is written as follows: [ka]

[0106] FD3 or SEQ ID NO: 3 is written as follows: [ka]

[0107] The mRNA described herein may include an open reading frame (ORF) encoding a 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 include a 5' cap having the following structure: [ka]

[0108] The nucleic acid sequences of each of the mRNA open reading frames (ORFs) encoding the RSV FD1, FD2, and FD3 proteins, respectively, are listed below. FD1 mRNA ORF: [ka] FD2 mRNA ORF: [ka] FD3 mRNA ORF: [ka]

[0109] The nucleic acid sequences of each of the DNA templates encoding the RSV FD1, FD2, and FD3 proteins, respectively, are listed below. FD1 DNA: [ka] FD2 DNA: [ka] FD3 DNA: [ka]

[0110] The nucleic acid sequences of the 5'UTR and 3'UTR are listed below. 5'UTR: [ka] 3'UTR: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 11)

[0111] The nucleic acid sequences of each of the full-length mRNAs encoding the RSV FD1, FD2, and FD3 proteins are listed below. FD1 mRNA: [ka] FD2 mRNA: [ka] FD3 mRNA: [ka]

[0112] One aspect of the present disclosure relates to a method for inducing or stimulating an immune response to RSV in a subject. Another aspect of the present disclosure relates to a method for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject. The method can include administering or providing an RNA (e.g., mRNA) RSV vaccine to a subject. The RNA RSV vaccine can include mRNA, where the mRNA includes an ORF encoding a RSV F protein antigen or a portion of the RSV F protein antigen.

[0113] In certain embodiments, a prophylactically effective amount of an RNA RSV vaccine can be administered to a subject (e.g., by a physician). 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.

[0114] Another aspect of the present disclosure relates to a method for inducing or stimulating an immune response to RSV in a subject, the method may include selecting a subject at least 60 years of age and administering a prophylactically effective amount of an RNA RSV vaccine to the subject. Another aspect of the present disclosure relates to a method for preventing RSV infection or reducing one or more symptoms of RSV infection in a subject, the method may include selecting a subject at least 60 years of age and administering a prophylactically effective amount of an RNA RSV vaccine to the subject. The step of selecting a subject aged 60 years or older can be performed by a medical professional (e.g., one or more of a physician, physician assistant, nurse, pharmacist, pharmacy technician, medical technician, etc.) or can be performed by the subject themselves (i.e., self-selection). The selected subject can then be administered a prophylactically effective amount of the RNA RSV vaccine of the present disclosure.

[0115] Another aspect of the present disclosure relates to an RNA (e.g., mRNA) RSV vaccine for use in inducing or stimulating an immune response to RSV in a subject. Another aspect of the present disclosure relates to an RNA (e.g., mRNA) RSV vaccine for use in preventing RSV infection or reducing one or more symptoms of RSV infection in a subject. In certain embodiments, symptoms of RSV infection include, but are not limited to, acute respiratory disease (ARD), medically required acute respiratory disease (MAARD), severe ARD, non-medical required lower respiratory tract disease (LRTD), medically required LRTD, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis. RSV infection can be confirmed by laboratory tests, such as RT-PCR, ELISA, etc.

[0116] As used herein, "ARD" refers to an RSV infection including any respiratory symptoms including nasal congestion, sore throat, hoarseness, new or worsening cough, sputum production, and difficulty breathing with or without fever.

[0117] As used herein, "severe ARD" refers to an RSV infection that includes acute respiratory illness requiring hospitalization, with a history of fever or a measured fever of 38°C or higher, accompanied by cough that has developed within the past 10 days.

[0118] As used herein, "LRTD" refers to an RSV infection including ARD with one or more symptoms of lower respiratory tract disease, including, but not limited to, lower respiratory tract: tracheal, bronchial, and pulmonary involvement, which may be complicated, e.g., bronchopneumonia and / or tracheobronchitis, with the onset of ARD symptoms for 10 days.

[0119] As used herein, "LRTD requiring medical attention" refers to an RSV infection, including ARD, with one or more symptoms of lower respiratory tract disease, including, but not limited to, lower respiratory tract: tracheal, bronchial, and pulmonary involvement, which may be complicated, e.g., bronchopneumonia and / or tracheobronchitis, accompanied by the onset of ARD symptoms within 10 days, and which seeks medical attention (e.g., emergency room visit, hospitalization, or outpatient clinic visit).

[0120] As used herein, "reducing one or more symptoms of RSV infection" refers to a reduction in one or more symptoms and / or a reduction in viral load of 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 a subject vaccinated with a RSV vaccine of the present disclosure compared to an unvaccinated subject.

[0121] In certain embodiments, the RSV vaccines described herein can be administered to a subject by administration methods including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratracheal, epidural, and oral routes of administration. In certain embodiments, administration is intramuscular in the arm or leg muscles of a subject. In certain embodiments, administration is in the deltoid muscle of the subject's upper arm. The RSV vaccines described herein can also be delivered intramuscularly using a standard needle and syringe, or by any other suitable injection device.

[0122] In certain embodiments, the RSV vaccines described herein can be administered to a subject by a method of administration that includes, for example, cutaneous injection into the epidermis, dermis, or subcutaneous tissue of the skin. In some embodiments, the RSV vaccines described herein are provided in a device suitable for cutaneous injection, such as a needle (e.g., an epidermal, dermal, or hypodermic needle), a needle-free device, a microneedle device, or a microprojection array device. Examples of microneedle or microprojection array devices suitable for dermal injection of the present invention are described in U.S. Patent Application Publication Nos. 20230270842A1, 20220339416A1, 20210085598A1, 20200246450A1, 20220143376A1, 20180264244A1, 20180263641A1, and 20110245776A1.

[0123] In certain embodiments, the RSV RNA vaccine composition is formulated to reduce the amount of ionized lipid-mRNA adduct impurities (e.g., aldehyde-mRNA adduct impurities) that may be formed by covalent modification of mRNA with reactive species (e.g., secondary amines or reactive aldehydes) produced by degradation of the ionized lipid component of the LNP (see 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 contains 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 mRNA in the form of adduct impurities, as measured by reverse-phase ion-pair high-performance liquid chromatography (RP-IP HPLC). In some embodiments, the amount of added 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 upon storage at a temperature of about 25° C. or less. In some embodiments, the amount of added impurities does not increase substantially (e.g., not by more than 0.05%, more than 0.01%, more than 0.005%, or more than 0.001%) upon storage at a temperature of about 25° C. or less.

[0124] In some embodiments, the buffer or pH of the RSV RNA vaccine composition can be adjusted to reduce the amount of additional impurities formed in the LNP composition (e.g., to inhibit degradation of ionized lipids). For example, some embodiments may include a composition having a TRIS (tris(hydroxymethyl)aminomethane) buffer at a concentration of about 10 mM or greater, such as 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 about 10 mM to about 150 mM TRIS, such as about 15 mM to about 120 mM TRIS or about 20 mM to about 100 mM TRIS. In some embodiments, the composition does not contain PBS buffer. In some embodiments, the composition has a pH of about 6.5 to about 9.0, such as about 7 to 8, about 7 to 7.5, about 7.4, or about 7.5.

[0125] In some embodiments, the RSV RNA vaccines (e.g., mRNA vaccine compositions) described herein comprise a unit dose 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 a pharmaceutically acceptable carrier, diluent, and / or excipient. 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.

[0126] In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject at 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, where μg consists of the amount of mRNA formulated in lipid nanoparticles (LNPs) without any diluents, etc. In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject at 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, where μg consists of the amount of mRNA formulated in LNPs without any diluents, etc. In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are administered to a subject at 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, where μg consists of the amount of mRNA formulated in the LNP, without any diluents, etc.

[0127] In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are 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 is the amount of mRNA formulated in the LNP, without any diluents, etc. In some embodiments, the RSV vaccines (e.g., mRNA vaccine compositions) described herein are 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 without any diluents, etc.

[0128] In some embodiments, 10 micrograms of the RSV RNA vaccine composition is administered to a subject in a dose of 0.5 mL. In some embodiments, 30 micrograms of the RSV RNA vaccine composition is administered to a subject in a dose of 0.5 mL. In some embodiments, 75 micrograms of the RSV RNA vaccine composition is administered to a subject in a dose of 0.5 mL. μg refers to the amount of mRNA formulated in LNP, without any diluents, etc.

[0129] In some embodiments, a RSV RNA vaccine composition (e.g., an mRNA vaccine composition) for use in a method for vaccinating a subject is administered to the subject in a single dose. In some embodiments, a RSV RNA vaccine composition (e.g., an mRNA vaccine composition) for use in a method for vaccinating a subject is administered to the subject in two doses, for example, a primary dose and a booster dose. In some embodiments, a RSV RNA vaccine composition (e.g., an mRNA vaccine composition) for use in a method for vaccinating a subject is administered to the subject in three or more doses, for example, a primary dose and two or more booster doses (e.g., a first booster dose, a second booster dose, etc.).

[0130] In some embodiments, the RSV RNA vaccine composition (e.g., mRNA vaccine composition) for use in the method of vaccinating a subject is administered as a primary dose of the RSV vaccine and a booster dose of the RSV vaccine, with the primary dose and the booster being spaced apart in time.

[0131] In certain embodiments, the booster dose is administered to the subject 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 the initial dose.

[0132] In certain embodiments, the booster dose is administered to the 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 the initial dose.

[0133] In some embodiments, the RSV RNA vaccine composition (e.g., mRNA vaccine composition) for use in the method of vaccinating a subject is administered to the subject as a primary dose of the RSV vaccine and one or more booster doses of the RSV vaccine.

[0134] In certain embodiments, the booster dose is administered to the subject 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 the preceding initial dose or the preceding booster dose.

[0135] In certain embodiments, the booster dose is administered to the 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 the preceding initial dose or the preceding booster dose.

[0136] III. Pharmaceutical Compositions The RNA purified according to the present disclosure can be useful, for example, as a component in pharmaceutical compositions for use as vaccines, such as RSV RNA vaccines. These compositions typically contain RNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure can also contain a delivery system for RNA, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle (LNP). In certain embodiments, the composition comprises an antigen-encoding nucleic acid molecule encapsulated in the LNP.

[0137] In some embodiments, the RSV RNA vaccine composition comprises a pharmaceutically acceptable carrier, diluent, excipient, and / or LNP and excludes an adjuvant. In some embodiments, the vaccine composition comprises 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 comprises buffered saline. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises octylphenol ethoxylate (Triton X-100). In some embodiments, the carrier, diluent, excipient, and / or LNP comprises buffered saline and octylphenol ethoxylate (Triton X-100). In some embodiments, the carrier, diluent, excipient, and / or LNP comprises sodium chloride. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises sodium phosphate. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises dibasic sodium phosphate. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises water. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises formaldehyde. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises ovalbumin. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises sodium chloride, sodium phosphate (monobasic, dibasic, or both), and water. In some embodiments, the carrier, diluent, excipient, and / or LNP comprises sodium chloride, sodium phosphate (monobasic, dibasic, or both), water, formaldehyde, ovalbumin, and Triton X-100.

[0138] In certain embodiments, the composition is in aqueous form when administered, but can be stored in a non-liquid form and resuspended prior to administration.

[0139] In some embodiments, the RSV RNA vaccine composition comprises 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 is free of preservatives.

[0140] 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 about 1-20 mg / ml NaCl. In some embodiments, the RSV RNA vaccine composition comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L NaCl. Other salts that may be present include sodium phosphate, potassium chloride, potassium dihydrogen phosphate, disodium phosphate, disodium phosphate dihydrate, magnesium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, or other salts known to those of skill in the art. In some embodiments, the RSV RNA vaccine composition comprises about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L monobasic sodium phosphate. In some embodiments, the RSV RNA vaccine composition comprises about 1, 2, 3, 4, or 5 g / L dibasic sodium phosphate. In some embodiments, the RSV RNA vaccine composition comprises about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L of monobasic sodium phosphate and about 1, 2, 3, 4, or 5 g / L of dibasic sodium phosphate. If the adjuvant is in a separate container from the antigen, a salt, such as sodium chloride, may be present in both containers. In some embodiments, the RSV RNA vaccine composition comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L of NaCl, about 0.1, 0.2, 0.3, 0.4, or 0.5 g / L of monobasic sodium phosphate, and about 1, 2, 3, 4, or 5 g / L of dibasic sodium phosphate. If the adjuvant is in a separate container from the antigen, a salt, such as sodium chloride, may be present in both containers.

[0141] In certain embodiments, acceptable materials included in RSV RNA vaccine compositions are non-toxic to recipients at the dosages and concentrations used. In certain embodiments, pharmaceutical compositions may include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; 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); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, 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 or potassium chloride, or mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0142] Suitable vehicles or carriers may be water for injection, saline, or artificial cerebrospinal fluid. In some embodiments, the vehicle or carrier may be supplemented with other materials common in compositions for parenteral administration. Further exemplary vehicles are neutral buffered saline or saline mixed with serum albumin.

[0143] In some embodiments, the RSV RNA vaccine composition comprises one or more buffers. Typical buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer (e.g., containing aluminum hydroxide adjuvant), or citrate buffer. Buffers are typically present in the range of 5 to 20 mM. The pH of the RSV RNA vaccine composition is generally 5.0 to 8.1, more typically 6.0 to 8.0, e.g., 6.5 to 7.5 or 7.0 to 7.8.

[0144] In some embodiments, the buffer may 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 may be included at a concentration of 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.

[0145] In some embodiments, the RSV RNA vaccine composition can include a humectant, including, for example, sorbitol or a suitable substitute thereof.

[0146] The RSV RNA vaccine composition components can be present at a concentration acceptable to the site of administration. In certain embodiments, a buffer can be used to maintain the composition at physiological pH or slightly below physiological pH. In some embodiments, the pH of the composition can 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 at most 5.1, at most 5.2, at most 5.3, at most 5.4, at most 5.5, at most 5.6, at most 5.7, at most 5.8, at most 5.9, at most 6.0, at most 6.1, at most 6.2, at most 6.3, at most 6.4, at most 6.5, at most 6.6, at most 6.7, at most 6.8, at most 6.9, at most 7.0, at most 7.1, at most 7.2, at most 7.3, at most 7.4, at most 7.5, at most 7.6, at most 7.7, at most 7.8, at most 7.9, or at most 8.0. In exemplary embodiments, the pH of the composition may be in the range of 5 to 8. In exemplary embodiments, the pH of the composition may be in the range of 5.5 to 6.5. In exemplary embodiments, the pH of the composition may be 6.0.

[0147] In some embodiments, the RSV RNA vaccine composition may include an ionic excipient. An ionic excipient may be included in an antibody formulation to alter the charge state of the antibody in the formulation, alter the distribution of the antibody in the formulation, and / or colloidally stabilize the antibody in the formulation. In some embodiments, the ionic excipient may include, for example, a charged amino acid, including lysine and / or arginine. In some embodiments, the ionic excipient may include, for example, a salt, including arginine hydrochloride (arginine HCl), lysine hydrochloride (lysine HCl), or sodium chloride (NaCl). In some embodiments, the amino acid or amino acid salt may include a biologically active amino acid (e.g., L-form). In some embodiments, the ionic excipient may be present 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 present at a concentration of 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 exemplary embodiments, the ionic excipient may be present at a concentration ranging from 50 mM to 150 mM. In exemplary embodiments, the ionic excipient may be present at a concentration ranging from 75 mM to 100 mM, hi exemplary embodiments, the ionic excipient may include L-arginine hydrochloride present at a concentration of 75 mM or 80 mM.

[0148] 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 present 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, sugars may be included at a concentration of 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 exemplary embodiments, the sugar includes sucrose at a concentration ranging from 100 mM to 140 mM. For example, the composition may include sucrose at a concentration of 120 mM.

[0149] In some embodiments, the RSV RNA vaccine composition can further comprise a surfactant, including, for example, polysorbate, such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In some embodiments, the surfactant can be present 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 exemplary embodiments, the surfactant may be present at a concentration ranging from 0.001% (w / v) to 0.5% (w / v), from 0.002% (w / v) to 0.1% (w / v), or from 0.01% (w / v) to 0.05% (w / v). In exemplary embodiments, polysorbate 80 is present at a concentration ranging from 0.01% (w / v) to 0.05% (w / v). In a further exemplary embodiment, 0.02% (w / v) polysorbate 80 is present in the composition. In another exemplary embodiment, 0.04% (w / v) polysorbate 80 is present in the composition.

[0150] The RSV RNA vaccine compositions described herein may contain surfactants, such as polyoxyethylene sorbitan ester surfactants (known as "Tween"), octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide (CTAB), or sodium deoxycholate, for example, for split or surface antigen vaccines. The surfactant may be present only in trace amounts. In some embodiments, the RSV RNA vaccine compositions for use in the methods disclosed herein contain trace amounts of other residual components, such as antibiotics (e.g., neomycin, kanamycin, or polymyxin B). If the adjuvant is in a container separate from the RNA encoding the antigen, the surfactant is typically present in the container containing the RNA.

[0151] 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 less than 0.1 EU per dose. The RSV RNA vaccine composition is typically gluten-free.

[0152] In some embodiments, the RSV RNA vaccine composition can be stored at -20°C to -70°C.

[0153] In some embodiments, the RSV RNA vaccine compositions can be stored at 2° C. to 8° C. In some embodiments, the RSV RNA vaccine compositions described herein are stable for long-term storage at room temperature or at a temperature ranging from 2° C. to 8° C. (e.g., including 5° C.). As used herein, room temperature generally refers to a temperature ranging from 22° C. to 25° C. Preferably, the RSV RNA vaccine compositions are stable after storage at a temperature ranging from 2° C. to 8° C. (e.g., including 5° C.) for at least 1 month, at least 3 months, or at least 6 months. As used herein, the term "stable" (or "stability") with respect to shelf life is used to indicate that the formulation resists aggregation, degradation, half-antibody formation, and / or fragmentation.

[0154] When parenteral administration is intended, the RSV RNA vaccine composition may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the RSV RNA vaccine in a pharmaceutically acceptable vehicle. In some embodiments, a vehicle suitable for parenteral injection is sterile distilled water, in which the antibody is formulated as a sterile isotonic solution and properly stored. Additionally or alternatively, a formulation suitable for parenteral administration may 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 may be isotonic with the blood of the recipient. Isotonic agents that can be included in liquid preparations include sugars, buffers, and sodium chloride. A solution of the anti-RSV antibody or antigen-binding fragment thereof may be prepared in water. In certain embodiments, the solution of the anti-RSV antibody or antigen-binding fragment thereof prepared in water may be mixed with a non-toxic surfactant.

[0155] Dispersions of RSV RNA vaccines can be prepared, for example, in water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, glycerol esters, and mixtures thereof. The final dosage form can, in some embodiments, be sterile, fluid, and stable under the conditions of manufacture and storage. The required fluidity can be achieved, for example, by using liposomes, by using an appropriate particle size in the case of dispersions, or by using surfactants. Sterilization of liquid preparations can be achieved by any convenient method that preserves the biological activity of the anti-RSV antibody or antigen-binding fragment thereof, for example, filter sterilization. Methods for preparing powders include vacuum drying and lyophilization of sterile injectable solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, such as antibacterial, antiviral, and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of the anti-RSV antibody or antigen-binding fragment thereof can be achieved by including agents that delay absorption, for example, aluminum monostearate and gelatin.

[0156] 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 as a 0.5 mL dose containing 10 micrograms, 30 micrograms, 75 micrograms, or 110 micrograms of mRNA. In a specific embodiment, the RSV RNA vaccine is diluted with 2.2x PBS (2°C to 8°C).

[0157] Adjuvants 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 function to enhance the immune response (humoral and / or cellular) elicited in a subject administered 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.

[0158] In some embodiments, the RSV RNA vaccine composition comprises an oil-in-water emulsion and at least one surfactant.

[0159] In some embodiments, the RSV RNA vaccine composition comprises one or more tocopherols.

[0160] In some embodiments, the RSV RNA vaccine composition comprises tocopherol and squalene, hi some embodiments, the oil content ranges from 2-20% (by volume).

[0161] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a mineral-containing composition comprising a calcium salt and an aluminum salt (or a mixture thereof). Calcium salts include calcium phosphate. Aluminum salts include hydroxides, phosphates, sulfates, etc., and the salts may take any suitable form (e.g., gel, crystalline, amorphous, etc.). The mineral-containing composition may also be formulated as particles of metal salts.

[0162] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising one or more saponins, which are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots, and flowers of various plant species. Saponins from the bark of the Quillaja saponaria Molina tree have been widely studied as adjuvants. Saponins can also be commercially obtained from Smilax ornata (sarsaparilla), Gypsophilla paniculate (brides veil), and Saponaria officinalis.

[0163] Saponin adjuvant formulations include purified preparations such as QS21 and lipid formulations such as ISCOMs. QS21 is commercially available as STIMULON®. Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs). In some embodiments, ISCOMs contain phospholipids such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. In an exemplary embodiment, the ISCOMs include one or more of QuilA, QHA, and QHC.

[0164] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a fatty adjuvant.

[0165] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant containing a bacterial ADP-ribosylating toxin (e.g., E. coli heat-labile enterotoxin "LT", cholera toxin "CT", or pertussis toxin "PT") and its detoxified derivatives, such as the mutant toxins known as LT-K63 and LT-R72.

[0166] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant, including bioadhesives and mucoadhesives, such as esterified hyaluronic acid microspheres or chitosan and its derivatives.

[0167] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a cytokine-inducing agent.

[0168] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a liposome.

[0169] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a polyoxyethylene ether and / or a polyoxyethylene ester. Such formulations further include a polyoxyethylene sorbitan ester surfactant in combination with octoxynol, and a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant, such as octoxynol. Exemplary polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steolyl ether, polyoxyethylene-8-steolyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

[0170] In some embodiments, the RSV RNA vaccine composition comprises an adjuvant comprising a muramyl peptide, such as N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP or THERAMIDE™), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0171] The RSV RNA vaccine composition can include one or more adjuvants, such as two, three, four, or more adjuvants. For example, the RSV RNA vaccine composition can include both an oil-in-water emulsion and a cytokine-inducing agent.

[0172] IV. Vaccination Methods The RSV vaccines disclosed herein can be administered to a subject to induce an immune response against the RSV F protein, and the subject's anti-antigen antibody titer is increased after vaccination compared to the anti-antigen antibody titer of a subject not vaccinated with the RSV vaccine disclosed herein or compared to an alternative vaccine against RSV. An "anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0173] In one aspect, the present disclosure provides a method for inducing an immune response against RSV or protecting a subject against RSV infection, the method comprising administering a RSV vaccine described herein to a subject.The present disclosure also provides a RSV vaccine described herein for use in inducing an immune response against RSV or protecting a subject against RSV infection.The present disclosure also provides a RSV mRNA described herein for use in producing a vaccine for inducing an immune response against RSV or protecting a subject against RSV infection.

[0174] In a specific embodiment, the subject has a higher serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO: 1.

[0175] In certain embodiments, the subject has a comparable serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a RSV protein vaccine co-administered with an adjuvant.

[0176] In certain embodiments, the RSV vaccine increases the serum concentration of antibodies with binding specificity to site Φ of the RSV F protein.

[0177] In a specific embodiment, the subject has a lower serum concentration of antibodies having binding specificity for site I or site II of the RSV F protein after administration of the RSV vaccine compared to a subject receiving a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO: 2.

[0178] In certain embodiments, the RSV vaccine increases serum concentrations of neutralizing antibodies in subjects with pre-existing RSV immunity.

[0179] V. RSV F protein In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen.

[0180] In some embodiments, the ORF is codon-optimized, which refers to the introduction of specific codons (in place of the respective wild-type codons encoding the same amino acids), which may be more favorable with respect to RNA stability and / or with respect to codon usage in a subject.

[0181] In some embodiments, an epitope of the RSV F protein shared between Pre-F and Post-F is blocked. Blocking the epitope reduces or eliminates the production of antibodies against the epitope when RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject. 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 targeting site Φ). Since F has a pre-fusion conformation in viruses that have not yet entered cells, increasing the proportion of antibodies targeting Pre-F can provide a greater degree of 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, an N-glycosylation site not present in wild-type F can be added, for example, by mutating the appropriate residue 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 topologically overlap with the blocked epitope are also blocked. The blocked epitope may be an epitope of antigenic site I of RSV F.

[0182] In some embodiments, the RSV F polypeptide contains an asparagine substitution at one or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 1 (i.e., E328N, S348N, or R507N). In some embodiments, the RSV F polypeptide contains an asparagine substitution at two or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 1 (i.e., E328N, S348N, or R507N). In some embodiments, the RSV F polypeptide contains an asparagine substitution at positions 328, 348, and 507 of SEQ ID NO: 1 (i.e., E328N, S348N, and R507N).

[0183] Asparagine may function as a glycosylation site (see WO 2019 / 195291, incorporated herein by reference). Furthermore, without wishing to be bound by any particular theory, glycans at these sites may inhibit the development of antibodies against nearby epitopes, including epitopes common to pre-fusion and post-fusion RSV F proteins, when RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject. In some embodiments, glycosylation of asparagine 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 development of antibodies against epitopes common to pre-fusion and post-fusion RSV F proteins may be beneficial because it can direct the development of antibodies against epitopes specific to the pre-fusion RSV F protein, such as the site Φ epitope, which may have more effective neutralizing activity than antibodies against other RSV F epitopes. The site Φ epitope includes amino acid residues 62-69 and 196-209 of SEQ ID NO: 1. Thus, in some embodiments, the RSV F polypeptide includes amino acid residues 62-69 and 196-209 of SEQ ID NO: 1.

[0184] The RSV F polypeptides described herein may have deletions or substitutions of different lengths compared 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 deletion 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 deletion 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.

[0185] In general, 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 description of structural alignment provided herein as an alternative approach to identifying corresponding positions.

[0186] In some embodiments, the RSV F polypeptide comprises a mutation that adds a glycan to block an epitope on a line fusion antigen that is structurally similar to an epitope on the surface of post-fusion RSV F. In some embodiments, the glycan is added to specifically block an epitope that may be present in the post-fusion conformation of RSV F. In some embodiments, a glycan is added that blocks an epitope that may be present in the post-fusion conformation of RSV F but does not affect one or more epitopes present in the line fusion conformation of RSV F, such as the site Φ epitope.

[0187] 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 set forth in SEQ ID NO:1.

[0188] 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 set forth in SEQ ID NO:2.

[0189] 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 set forth in SEQ ID NO:3.

[0190] In some embodiments, the RSV F polypeptide comprises a DS-CAV1 amino acid substitution (e.g., as described in McLellan et al., Science, 342(6158):592-598, 2013) with further modifications including at least one, two, or three of the above asparagines. The CAV1 mutations are S190F and V207L ​​compared to SEQ ID NO: 1. The DS mutations are S155C and S290C relative to SEQ ID NO: 1.

[0191] In some embodiments, the amino acid substitution or pair of amino acid substitutions is an inter-protomer stabilizing substitution. Exemplary substitutions that may be inter-protomer stabilizing are V207L; N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L, using the position numbering of SEQ ID NO: 1.

[0192] In some embodiments, the amino acid substitution or pair of amino acid substitutions is intraprotomer stabilizing. Exemplary substitutions that may be intraprotomer stabilizing are V220I; and A74L and Q81L (using the position numbering of SEQ ID NO: 1).

[0193] In some embodiments, the amino acid substitutions are predicted to be helix-stabilizing, i.e., stabilize the helical domain of RSV F. Stabilization of the helical domain may generally contribute to the stability of the site Φ epitope and pre-fusion conformation of RSV F. Exemplary substitutions that may be helix-stabilizing are N216P or I217P, using the position numbering of SEQ ID NO: 1. Position 217 of SEQ ID NO: 1 corresponds to position 177 of SEQ ID NO: 3.

[0194] In some embodiments, the amino acid substitution is a helix capping. In some embodiments, the amino acid substitution is a helix PRO capping. Helix capping is based on the biophysical observation that mutation of a proline residue located in an alpha helix can disrupt helix formation, but that a proline at the N-terminus of a helical region can help induce helix formation by stabilizing the PHI / PSI bond angle. Exemplary substitutions that may be helix capping are N216P or I217P, using the position numbering of SEQ ID NO: 1.

[0195] In some embodiments, the amino acid substitution replaces the disulfide mutation of DS-CAV1. In some embodiments, the engineered disulfide of DS-CAV1 is restored to the wild type (C69S and / or C212S mutation of DS-CAV1 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 disulfide bonds. In some embodiments, the RSV F polypeptide contains both C69S and C212S using the position numbering of SEQ ID NO: 1. In some embodiments, the substitution of such cysteines, thereby eliminating disulfide bonds, blocks the 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 substitutions at C69 and / or C212.

[0196] In some embodiments, the amino acid substitutions prevent proteolysis by trypsin or trypsin-like proteases. In some embodiments, the amino acid substitutions that prevent such proteolysis are located within the RSV F heptad repeat region B (HRB) region.

[0197] The appearance of fragments consistent with proteolysis of the RSV F polypeptide containing the wild-type HRB region suggested that lysine or arginine in this region is the target of proteolysis. Amino acid substitutions to remove K or R residues can be referred to as knockouts (KOs). In some embodiments, K or R is replaced with L or Q. In some embodiments, K is replaced 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.

[0198] In some embodiments, the amino acid substitutions add glycans. In some embodiments, the amino acid substitutions increase glycosylation by adding glycans to the RSV F polypeptide. Substitutions that add glycans can also be referred to as engineered glycosylation, compared to native glycosylation (no additional glycans).

[0199] In some embodiments, the amino acid substitution to add a glycan is with N. In some embodiments, the amino acid substitution with N allows for N-linked glycosylation. In some embodiments, the substitution with N is accompanied by a substitution with T or S at the second amino acid position C-terminal to N, forming an NxT / S glycosylation motif. In some embodiments, N is surface-exposed.

[0200] Each of the above substitutions and mutations in the RSV F polypeptide is described in more detail in International Publication No. WO 2019 / 195291, which is incorporated herein by reference.

[0201] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the RSV F protein antigen comprises one or more of the following substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1: 1) amino acid positions 98 to 146 of SEQ ID NO: 1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO: 16); 2) amino acid substitutions S190F and V207L; 3) amino acid substitution I217P; 4) amino acid substitutions E328N, S348N, and R507N; 5) amino acid substitution L373R; 6) the amino acid substitution K498L; and 7) Amino acid substitution K508Q.

[0202] In another aspect, the present disclosure provides a RSV vaccine comprising an mRNA including an ORF encoding a RSV F protein antigen, wherein the RSV F protein antigen includes each of the following substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1: 1) amino acid positions 98 to 146 of SEQ ID NO: 1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO: 16); 2) amino acid substitutions S190F and V207L; 3) amino acid substitution I217P; 4) amino acid substitutions E328N, S348N, and R507N; 5) amino acid substitution L373R; 6) the amino acid substitution K498L; and 7) Amino acid substitution K508Q.

[0203] In a specific embodiment, the RSV F protein antigen comprises the transmembrane domain and cytoplasmic tail amino acid sequence of IMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 17).

[0204] 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 set forth 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 set forth in any one of SEQ ID NOs: 12-14.

[0205] VI. RSV RNA The RSV vaccines of the present disclosure may include at least one ribonucleic acid (RNA) comprising an ORF encoding a RSV F protein antigen.

[0206] In certain embodiments, the RNA is an mRNA comprising an ORF encoding a 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.

[0207] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:12.

[0208] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:13.

[0209] In some embodiments, the RSV mRNA has at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:14.

[0210] II.A.5' Cap The 5' cap on an mRNA may confer resistance to nucleases found in most eukaryotic cells and may promote translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m 7 The nucleotide sequence of the nucleotide sequence of the transcribed nucleotide (also referred to as "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide through a 5'-5'-triphosphate bond.

[0211] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, generating a 5'5'5 triphosphate linkage; and 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. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0212] 5'-Capping of polynucleotides can be simultaneously completed during in vitro transcription reactions using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 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 RNAs can be completed post-transcriptionally using vaccinia virus capping enzyme to generate the Cap0 structure: m7G(5')ppp(5')G. The Cap1 structure can be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. The Cap2 structure can be generated from the Cap1 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using 2'-O-methyltransferase. The Cap3 structure can be generated from the Cap2 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using 2'-O-methyltransferase.

[0213] In certain embodiments, an mRNA of the 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.

[0214] In certain embodiments, the mRNA of the present disclosure comprises the following 5' cap: [ka]

[0215] II.B. Untranslated Regions (UTRs) In some embodiments, mRNAs of the present disclosure include 5' and / or 3' untranslated regions (UTRs). In an mRNA, the 5' UTR begins at the transcription initiation site and continues up to, but not including, the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0216] In some embodiments, the mRNAs disclosed herein may comprise a 5' UTR that contains one or more elements that affect mRNA stability or translation. In some embodiments, the 5' UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, the 5' UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR may be at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, or about The length is 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.

[0217] In some embodiments, the mRNAs disclosed herein may include a 3' UTR that includes one or more of 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 an miRNA. In some embodiments, the 3' UTR may be 50 to 5,000 or more nucleotides in length. In some embodiments, the 3' UTR may be 50 to 1,000 or more nucleotides in length. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0218] In some embodiments, the mRNAs disclosed herein may include a 5' or 3' UTR that is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0219] In certain embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to enhance mRNA stability. For example, the 5' UTR sequence may include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve mRNA half-life. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof in the 3' end or untranslated region of the mRNA. Generally, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) compared to the unmodified counterpart, including modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0220] Exemplary 5'UTRs include sequences from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 18) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference).

[0221] 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. Furthermore, 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. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0222] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra).

[0223] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0224] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0225] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0226] 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. 5' UTRs and 3' UTRs are described in more detail in WO 2012 / 075040, which is incorporated herein by reference.

[0227] II.C. Polyadenylation Tail As used herein, the terms "poly(A) sequence," "poly(A) tail," and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. A poly(A) tail may confer stability to an mRNA and protect it from exonuclease degradation. A poly(A) tail may enhance translation. In some embodiments, a poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of essentially 100 nucleotides. In certain embodiments, a poly(A) tail may be interrupted by at least one nucleotide that is different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides that is different from an adenosine nucleotide). In certain embodiments, a poly(A) tail may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides that is different from an adenosine nucleotide). [ka] Includes:

[0228] As used herein, "poly(A) tail" typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0229] 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.

[0230] 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 common chemical synthesis methods without being transcribed 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, for example, using the methods and procedures described in WO 2016 / 174271.

[0231] The nucleic acid may include a poly(A) tail obtained by enzymatic polyadenylation, with the majority of the nucleic acid molecule comprising from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0232] In some embodiments, a nucleic acid can include a poly(A) tail derived from a template DNA, e.g., as described in WO 2016 / 091391, and can additionally include at least one additional poly(A) tail generated by enzymatic polyadenylation. In certain embodiments, a nucleic acid includes at least one polyadenylation signal. In various embodiments, a nucleic acid can include at least one poly(C) sequence. As used herein, the term "poly(C) sequence" refers to a sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence includes about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence includes about 30 cytosine nucleotides.

[0233] II.D. Chemical modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically improve RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may 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 mRNAs may contain modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-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, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxymethylaminomethyl) ... hydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0234] In some embodiments, the disclosed mRNAs can comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-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-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0235] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0236] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0237] 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 uracil nucleotides in the mRNA are chemically modified.

[0238] 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 uracil nucleotides in the ORF are chemically modified.

[0239] The preparation of such analogs is described, for example, in U.S. Pat. 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.

[0240] II.E. mRNA Synthesis The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, 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 performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions may vary depending on the specific application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. In some embodiments, mRNA provided from an in vitro transcription reaction may be desired, although other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0241] In certain embodiments, the mRNA comprises the following structural elements: (i) a structure: [ka] a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of 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 of SEQ ID NO: 11, and (v) poly(A) tail Includes:

[0242] In certain embodiments, the poly(A) tail has a length of about 10 to about 500 adenosine nucleotides.

[0243] VII. Lipid Nanoparticles (LNPs) LNPs of the present disclosure may include four categories of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids (e.g., cholesterol), and (iv) helper lipids.

[0244] A. Cationic lipids Ionizable lipids can promote the encapsulation of mRNA and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH, promoting the efficient encapsulation of negatively charged mRNA drug substances. Exemplary cationic lipids are shown in Table 1 below.

[0245] [Table 1]

[0246] [Table 2]

[0247] [Table 3]

[0248] [Table 4]

[0249] The cationic lipids were [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (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); di((Z)-non-2-en-1-yl)9-((4 -(Dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(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-[di(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-Cho l); tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionate (306Oi10); decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)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); Hexa(octa) N-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (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(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); IM-001; and combinations thereof.

[0250] In certain embodiments, the cationic lipid is biodegradable.

[0251] In various embodiments, the cationic lipid is not biodegradable.

[0252] In some embodiments, the cationic lipid is cleavable.

[0253] In certain embodiments, the cationic lipid is not cleavable.

[0254] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Pat. Nos. 9,512,073; and 10,201,618, each of which is incorporated herein by reference.

[0255] B. PEGylated lipids PEGylated lipid components can provide control over nanoparticle size and stability. The addition of such components can prevent complex aggregation, extend circulation life, and provide a means to increase delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al. FEBS Letters 268(1):235-7. 1990). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0256] Contemplated PEGylated lipids include C6-C 20 (e.g., C8, C 10 , C 12 , C 14 , C 16 or C 18Examples of suitable ceramides include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)), covalently attached to lipids having alkyl chains of up to 5 kDa in length. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-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-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyloxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0257] 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 lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0258] C. Cholesterol-based lipids 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-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., Biochem Biophys Res Comm. (1991) 179:280), and 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280). al., BioTechniques (1997) 23:139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,2 5-dihydrolanosterol); zymosterol (5α-cholesta-8,24-dien-3β-ol); lasosterol (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-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0259] D. Helper lipids Helper lipids can improve the structural stability of LNPs and assist LNPs in endosomal escape. Helper lipids can improve uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids with fusogenic properties to improve uptake and release of drug payloads. 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-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0260] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebroside, ganglioside, 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.

[0261] In various embodiments, the LNP 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.

[0262] E. Molar ratio of lipid components The molar ratios of the above components may play a role in the effectiveness of LNPs in delivering mRNA. The molar ratio of 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 cationic lipid to total lipid in the LNP (i.e., A) is 35-55%, e.g., 35-50% (e.g., 38-42%, e.g., 40% or 45-50%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50% (e.g., 27-30%, e.g., 28.5% or 38-43%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35% (e.g., 28-32%, e.g., 30% or 8-12%, e.g., 10%). In some embodiments, the PEGylated lipid + cholesterol component has the same molar amount as the helper lipid. In some embodiments, the LNP contains a molar ratio of cationic lipid to helper lipid that is greater than 1.

[0263] In certain embodiments, the LNPs of the disclosure are a molar ratio of 35% to 55% or 40% to 50% cationic lipid (e.g., 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% cationic lipid); polyethylene glycol (PEG)-conjugated (PEGylated) lipids at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); Cholesterol-based lipids at a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids at 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 A helper lipid at a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., a helper lipid at 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%) All molar ratios are relative to the total lipid content of the LNP.

[0264] In certain embodiments, the LNP comprises a 40% molar ratio of cationic lipid; a 1.5% molar ratio of PEGylated lipid; a 28.5% molar ratio of cholesterol-based lipid; and a 30% molar ratio of helper lipid.

[0265] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0266] In various embodiments, the cholesterol-based lipid is cholesterol.

[0267] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0268] In certain embodiments, the LNP comprises OF-02 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%.

[0269] In certain embodiments, the LNP comprises cKK-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%.

[0270] In certain embodiments, the LNPs comprise 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%.

[0271] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-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%.

[0272] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-3-E14 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%.

[0273] In certain embodiments, the LNPs comprise 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%.

[0274] In certain embodiments, the LNPs comprise 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%.

[0275] In certain embodiments, the LNPs comprise a 40% molar ratio of OF-02, a 1.5% molar ratio of DMG-PEG2000, a 28.5% molar ratio of cholesterol, and a 30% molar ratio of DOPE. This LNP formulation is referred to herein as "Lipid A."

[0276] In certain embodiments, the LNPs comprise 40% molar cKK-E10, 1.5% molar DMG-PEG2000, 28.5% molar cholesterol, and 30% molar DOPE. This LNP formulation is referred to herein as "Lipid B."

[0277] In certain embodiments, the LNPs comprise 40% GL-HEPES-E3-E10-DS-3-E18-1, 1.5% DMG-PEG2000, 28.5% cholesterol, and 30% DOPE. This LNP formulation is referred to herein as "Lipid C."

[0278] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 (40% molar ratio; DMG-PEG2000 at 1.5% molar ratio; cholesterol at 28.5% molar ratio; and DOPE at 30% molar ratio. This LNP formulation is referred to herein as "Lipid D."

[0279] In certain embodiments, the LNPs comprise 40% GL-HEPES-E3-E12-DS-3-E14, 1.5% DMG-PEG2000, 28.5% cholesterol, and 30% DOPE. This LNP formulation is referred to herein as "Lipid E."

[0280] In certain embodiments, the LNPs comprise a 50% molar ratio of 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); a 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 38.5% molar ratio of cholesterol; and a 1.5% molar ratio of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0281] In certain embodiments, the LNPs comprise a 46.3% molar ratio of (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); a 9.4% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 42.7% molar ratio of cholesterol; and a 1.6% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0282] In certain embodiments, the LNPs comprise a 47.4% molar ratio of (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); a 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 40.9% molar ratio of cholesterol; and a 1.7% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0283] In certain embodiments, the LNPs comprise 35% to 55% molar ratio of IM-001; 0.25% to 2.75% molar ratio of polyethylene glycol (PEG)-conjugated (PEGylated) lipids; 20% to 45% molar ratio of cholesterol-based lipids; and 5% to 35% molar ratio of helper lipids, all molar ratios being relative to the total lipid content of the LNPs.

[0284] In certain embodiments, the LNP comprises a 40% molar ratio of IM-001; a 1.5% molar ratio of a PEGylated lipid; a 28.5% molar ratio of a cholesterol-based lipid; and a 30% molar ratio of a helper lipid, all molar ratios being relative to the total lipid content of the LNP.

[0285] In certain embodiments, the LNP comprises 40% molar IM-001; 1.5% molar DMG-PEG2000; 28.5% molar cholesterol-based lipid; and 30% molar DOPE, all molar ratios relative to the total lipid content of the LNP.

[0286] To calculate the actual amount of each lipid to be included 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, based on the molar amount of the cationic lipid and the selected molar ratio, the molar amounts of each of the other lipids can be calculated. These molar amounts can then be converted to weight using the molecular weight of each lipid.

[0287] F. Buffers and Other Components To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine product), facilitate administration of the LNP pharmaceutical composition, and / or enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients include, but are not limited to, parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0288] The LNP compositions of the present disclosure can be provided in a frozen liquid form or a lyophilized form. Various cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains, for example, 5-30% (e.g., 10%) (w / v) trehalose. When formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at temperatures between -20°C and -80°C.

[0289] The LNP compositions can be provided to the patient in an aqueous buffer solution (thawed if previously frozen, or reconstituted in an aqueous buffer solution at the bedside if previously lyophilized). The buffer solution can be isotonic, e.g., suitable for intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0290] VIII. Vector In one aspect, vectors containing the mRNA compositions disclosed herein are provided herein. RNA sequences encoding proteins of interest (e.g., mRNA encoding RSV F protein) can be cloned into several 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 generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0291] In certain embodiments, this vector can be used to express mRNA in a host cell. In various embodiments, this vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0292] In some embodiments, the vectors disclosed herein may include, from 5' to 3', at least the following: an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may include a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0293] Various RNA polymerase promoters are known. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7 promoter, T3 promoter, and SP6 promoter are known.

[0294] Also disclosed herein are host cells (eg, mammalian cells, eg, human cells) comprising the vectors or RNA compositions disclosed herein.

[0295] Polynucleotides can be introduced into target cells using any of a number of different methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), cationic liposome-mediated transfection using (ECM 830(BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg, Germany), lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems, such as "gene guns" (see, e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70), or the TransIT-RNA transfection Kit (Mirus, Madison, WI).

[0296] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0297] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of the mRNA sequence in the host cells.

[0298] IX. Self-replicating and trans-replicating RNA Self-replicating RNA: In one embodiment, a self-replicating RNA encoding a RSV F protein is disclosed herein.

[0299] Self-replicating RNA can be produced, for example, by using replication elements derived from alphaviruses to replace structural viral proteins with nucleotide sequences encoding proteins of interest (e.g., RSV F protein). Self-replicating RNAs are typically positive-strand molecules that can be directly translated after delivery to cells; this translation then provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs and collinear subgenomic transcripts can be translated to provide in situ expression of the encoded antigen (i.e., RSV F protein antigen), or can be transcribed to provide additional transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is a large amplification of the number of introduced replicon RNAs, so that the encoded antigen becomes the major polypeptide product of the cell.

[0300] One suitable system for achieving such self-replication is the use of alphavirus-based replicons. These replicons are positive-strand (positive-sense) RNAs that, after delivery to cells, result in the translation of a replicase (or replicase transcriptase). The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that generates genomic copies of the positive-strand delivered RNA. These negative-strand transcripts can themselves be transcribed to provide additional copies of the positive-strand parent RNA and further to provide subgenomic transcripts encoding antigens. Translation of the subgenomic transcripts thus results in in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See the following reference: WO 2005 / 113782, incorporated herein by reference.

[0301] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an RSV F protein antigen. The polymerase may be, for example, an alphavirus replicase comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. While native alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, while a self-replicating RNA may produce its own genomic RNA copies in cells, it does not produce RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist by itself in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are absent in the self-replicating RNA of the present disclosure; their place is taken by a gene encoding the immunogen of interest, resulting in the subgenomic transcript encoding the immunogen rather than the structural alphavirus virion proteins. Self-replicating RNA is described in further detail in WO2011005799, which is incorporated herein by reference.

[0302] Trans-replicating RNA: In one embodiment, a trans-replicating RNA encoding a RSV F protein is disclosed herein.

[0303] Trans-replicating RNA has similar elements to the self-replicating RNA described above. However, in trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the RNA replicase (e.g., an alphavirus replicase), and the second RNA molecule encodes a protein of interest (e.g., an RSV F protein antigen). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby significantly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNA is described in more detail in International Publication No. WO 2017162265, which is incorporated herein by reference.

[0304] X. Process for Making LNP Vaccines The LNPs of the present invention can be prepared by various techniques. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, for example, by dissolving the lipids in a suitable solvent, depositing the selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. MLVs can then be formed by adding an aqueous phase to the vessel with a vortex motion. Unilamellar vesicles (ULVs) can then be formed by homogenizing, sonicating, or extruding the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0305] Various methods are described in U.S. Patent Application Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and 2021 / 0046192 and can be used to make LNP vaccines. One exemplary process involves encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing preformed LNPs with the mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0306] In some embodiments, a process for preparing mRNA-loaded LNPs includes heating one or more solutions to a temperature above ambient temperature, where one or more solutions is a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the process includes heating one or both of the mRNA solution and the preformed LNP solution before the mixing step. In some embodiments, the process includes heating one or more of the solution containing preformed LNPs, the solution containing mRNA, and the solution containing LNP-encapsulated mRNA during the mixing step. In some embodiments, the process includes heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is greater than or equal to about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0307] Various methods can be used to prepare mRNA solutions suitable for the present disclosure. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA in water or a buffer at a concentration of 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 or greater.

[0308] In some embodiments, the mRNA stock solution is mixed with the buffer solution 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 faster rate than the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than 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 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 or greater.

[0309] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of 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 or greater.

[0310] The process of incorporating desired mRNA into lipid nanoparticles is called "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The nucleic acid incorporated into LNPs can be completely or partially within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation", and the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0311] Suitable LNPs can be made in various sizes.In some embodiments, the size reduction of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of appropriate LNP size can take into account the target cell or tissue site and the application for which lipid nanoparticles are made.

[0312] Various methods are available for sizing lipid nanoparticle populations. In various embodiments, the methods herein utilize a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed in the Zetasizer instrument. The z-average diameter (nm) or cumulant average is considered to be the average size of LNPs in the sample. The Zetasizer instrument 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) evaluation to guide efficient lipid nanoparticle synthesis.

[0313] 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 to 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 to 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).

[0314] In certain embodiments, the LNPs have an average diameter of between 30 and 200 nm.

[0315] In various embodiments, the LNPs have an average diameter of 80 to 150 nm.

[0316] In some embodiments, the LNPs in the composition have an average size of 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.

[0317] In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99% of the LNPs in the composition have a size in the range of 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), making them suitable for pulmonary delivery via nebulization.

[0318] In some embodiments, the dispersity or molecular size heterogeneity measure (PDI) of the LNPs in the pharmaceutical compositions provided by the present disclosure is less than about 0.5. In some embodiments, the LNPs have a PDI of 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 instrument, as described above.

[0319] 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 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 mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99% or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0320] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio of greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In certain embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0321] In some embodiments, a 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, a pharmaceutical composition contains between about 0.1 μg and 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.

[0322] In some embodiments, mRNA can be produced by chemical synthesis or by in vitro transcription (IVT) of a DNA template. In this process, i.e., the IVT process, a cDNA template is used to generate mRNA transcripts, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding a cap and tail, and the modified RNA is again purified by depth filtration and TFF.

[0323] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of 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 to 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 at pH 4.5.

[0324] An exemplary, non-limiting process for producing mRNA-LNP compositions involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, homogeneous manner, with the lipid:mRNA ratio maintained throughout the mixing process. In this illustrative example, the mRNA is present in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a 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 ethanolic lipid solution are mixed in a 4:1 volumetric ratio in a "T" mixer equipped with a nearly "pulseless" pump system. 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 buffer exchange the nascent LNPs obtained immediately after formation via the T mixing process. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

[0325] XI. Packaging and Use of mRNA-LNP RSV Vaccines The mRNA-LNP vaccine can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or 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 an extemporaneous formulation, in which the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine composition can also be shipped and provided in the form of an aqueous or frozen aqueous solution, and can be administered directly to a subject without reconstitution (after thawing, if previously frozen).

[0326] Thus, the present disclosure provides products such as kits that provide an mRNA-LNP vaccine in a single container, or that 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 containers may contain single-use doses or multi-use doses. The containers may be pre-processed glass vials or ampoules. The products may also include instructions for use.

[0327] In certain embodiments, mRNA-LNP vaccines are provided for use by intramuscular (IM) injection. The vaccine can be injected into a subject, for example, in the deltoid muscle of the 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 by inhalation and is provided in a pre-filled pump, aerosol generator, or inhaler.

[0328] The mRNA-LNP vaccine can be administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient period of time (e.g., 1 year, 2 years, 5 years, 10 years, or a lifetime). Sufficient immune protection can be, for example, prevention or alleviation of symptoms associated with infection by the pathogen. 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 primary dose and a booster dose) can be separated by intervals of at least, for example, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year (i.e., 12 months), 2 years, 5 years, or 10 years.

[0329] In order that this invention may be better understood, 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. [Example]

[0330] The foregoing description of specific embodiments sufficiently reveals the general nature of the present disclosure so that others, by applying knowledge within the skill of those skilled in the art, can readily modify and / or adapt such specific embodiments to various applications without departing from the general concepts of the present disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be 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 phrases or terminology herein are intended to be descriptive, rather than limiting, as the terms or phrases herein would be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

[0331] Example 1: A Phase I / II Randomized, Double-Blind, Placebo-Controlled, Multi-Arm Dose-Finding Study to Evaluate the Safety and Immunogenicity of RSV mRNA Vaccine Candidates Using Either LNP cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10 in Adult Participants Aged 18-50 Years Old (Sentinel Cohort) in Study A and 60 Years Old and Older (Primary and Booster Cohorts) in Study B and Booster Studies Introduction background Currently, there is no vaccine available for the prevention of RSV in the elderly, and no effective antiviral treatments. Thus, there is an unmet medical need to address the prevention of respiratory disease in the elderly while improving the quality of life of patients.

[0332] This example outlines the parameters used to evaluate the efficacy, safety, and immunogenicity of the RSV mRNA LNP vaccines described herein in adults (ages 18-50 and 60+), which may prevent LRTD caused by RSV in older adults.

[0333] Rationale for the study The clinical trial described herein will test the safety and immunogenicity of an RSV mRNA LNP vaccine containing mRNA encoding the RSV pre-fusion (pre-F) antigen in one of two encapsulated LNP formulations (i.e., LNPs containing either cKK-E10 (non-biodegradable) or GL-HEPES-E3-E12-DS-4-E10 (biodegradable)) administered at three different doses (i.e., low dose (10 μg), medium dose (30 μg), or high dose (75 μg)) in healthy adults aged 18-50 years (Study A, i.e., Sentinel Cohort) and 60 years and older (Study B and Booster Studies, i.e., Primary and Booster Cohorts).

[0334] Research overview and research design Number of participants, details of intervention group, and frequency of visits The total expected number of participants across the two studies (Study A and Study B) is approximately 790 randomized participants.

[0335] The first clinical trial, Study A (titled "Sentinel Cohort"), was a smaller randomized, double-blind, dose-escalation safety study (i.e., approximately 90 participants total; see Table 2 below), followed by a larger Study B (titled "Primary Cohort"; approximately 700 participants total; see Table 3 below) to evaluate the safety and immunogenicity of the LNP-encapsulated RSV mRNA vaccine in healthy adult participants (18-50 years old in Study A; 60 years old or older in Study B). Timeline graphs for Studies A and B are shown in Figures 1 and 2, respectively.

[0336] Studies A and B will include the same six experimental subcohorts and a placebo control group. The six experimental subcohorts will receive three different doses (i.e., low dose (10 μg), medium dose (30 μg), and high dose (75 μg)) of the RSV messenger mRNA vaccine candidate (listed as SEQ ID NO: 14) encapsulated in one of two different lipid nanoparticle (LNP)-based formulations (i.e., LNP containing cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10). Both Studies A and B will also have a placebo control group that will receive 0.9% saline. The vaccine will be administered intramuscularly (upper deltoid muscle) at a dose level of 0.5 ml per dose. The vaccine will be stored at -80°C ± 10°C and diluted in 2.2x PBS at the research facility.

[0337] At the time of vaccination, participants in Studies A and B will be followed for 12 months post-vaccination. Study A will have seven planned site visits, in addition to the initial screening participant visit, occurring on Day -14 (D) (-D14), D01, D04, D08, D29, Month 3, Month 6, and Month 12. Study B will have six planned site visits, in addition to the initial screening participant visit, occurring on Month -D14, D01, D08, D29, Month 3, Month 6, and Month 12.

[0338] It is also planned that 140 participants enrolled in Study B (i.e., 20 participants per treatment group) will be selected for inclusion in the cell-mediated immunity (CMI) subset. Participants in the CMI subset will be enrolled from a limited number of selected centers. Study B is also planned to enroll participants with a minimum of 6% Japanese ancestry (i.e., a minimum of approximately 42 participants; 6 participants per treatment group).

[0339] [Table 5]

[0340] [Table 6]

[0341] In addition, these clinical trials will evaluate the safety and immunogenicity of a booster vaccination administered 12 months after the primary vaccination in a subset of the study population (titled the "Booster Cohort"; approximately 200 participants from Study B; see Table 4 below). The booster vaccination will be administered 12 months after the primary vaccination in a subset of the study population from Study B. For the booster vaccination, a single vaccine formulation will be used based on evaluation of the candidate formulation after the first injection. The participation period for each participant will be 24 months across the subset of participants enrolled in the Booster Cohort. As in Study B, participants will have six scheduled visits plus a screening visit. Participants will receive a booster vaccination 12 months after the primary vaccination at Visit 8, which may occur on the same day as the 12-month follow-up visit for Study B (i.e., Visit 7). After the booster vaccination, participants will return to the site at D08, D29, 3 months, 6 months, and 12 months.

[0342] [Table 7]

[0343] Definition of research completion Participants were considered to have completed the study if they completed the last planned contact in the scheduled activity. The scheduled activities for Studies A, B, and the booster cohort are shown in Figures 4-6. The end of the study was defined as the last contact date for the last participant in the study. However, for routine safety reporting, the study was considered completed when the clinical study report was completed.

[0344] Administration 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 LNPs containing cKK-E10 or LNPs containing GL-HEPES-E3-E12-DS-4-E10. The vaccine is formulated as a single dose of mRNA-LNP complex diluted to the required mRNA dose at the research site using buffer diluent (diluent = 2.2x PBS at 2°C to 8°C). The sentinel and primary cohorts will receive one intramuscular injection. The booster cohort will receive two intramuscular injections, with the second injection administered 12 months after the first. Each dose (vial) of vaccine is provided in an individual box. Each dose (vial) of vaccine is stored at -80°C + / - 10°C.

[0345] the purpose Primary Objective: The primary objective is to evaluate the safety and immunogenicity profiles of three different dose levels (i.e., low dose (10 μg), medium dose (30 μg), and high dose (75 μg)) of the RSV mRNA vaccine described herein encapsulated in either LNPs containing cKK-E10 or LNPs containing GL-HEPES-E3-E12-DS-4-E10.

[0346] Secondary Objectives. The secondary objectives are to evaluate: (1) the safety profile of a booster vaccination given 12 months after primary vaccination in a subset of participants; (2) the durability of the immune response at 3, 6, and 12 months after primary vaccination prior to vaccination (D01); and (3) the durability of the immune response after a booster vaccination 12 months after primary vaccination in a subset of participants.

[0347] Table 5 below summarizes the primary objectives and corresponding endpoints. Table 6 below summarizes the secondary objectives and corresponding endpoints. Table 7 below summarizes the exploratory objectives and corresponding endpoints.

[0348] [Table 8]

[0349] [Table 9]

[0350] [Table 10]

[0351] Study population Inclusion and Exclusion Criteria Inclusion Criteria. For all studies (Studies A, B, and the Booster Cohort), participants were eligible for inclusion only if they met all of the following criteria at screening and the first visit (Day 01; Visit 01): (I1) Participants must be (1) between 18 and 50 years of age on the day of enrollment in Study A (i.e., "18 years of age" means after their 18th birthday) or (2) 60 years of age or older on the day of enrollment in Study B and the Booster Cohort (i.e., "60 years of age or older" means after their 60th birthday). (I2) Female participants were eligible if they were not pregnant or breastfeeding and of no childbearing potential (to be considered of no childbearing potential, women must be at least 1 year postmenopausal or surgically sterilized). In the Sentinel Cohort, a urine or serum pregnancy test was performed in women of childbearing potential before vaccination, and (I3) participants must be able to attend all scheduled visits and comply with all study procedures. A fourth additional criterion at screening is that participants also sign and date an informed consent form.

[0352] Exclusion Criteria. For all studies (Studies A, B, and Booster Cohort), participants were ineligible if any of the following criteria applied: (E1) known or suspected congenital or acquired immunodeficiency; or receiving immunosuppressive therapy such as anticancer chemotherapy or radiotherapy within the past 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 of the study intervention components (e.g., polyethylene glycol, polysorbate); life-threatening reaction to the study intervention used in the study or to products containing any of the same substances, mRNA (E3) History of any allergic reaction (e.g., anaphylaxis) after administration of a COVID-19 vaccine; (E4) History of clinically, serologically, or microbiologically diagnosed RSV-associated disease within the past 12 months; (E5) Past medical history of myocarditis, pericarditis, and / or myopericarditis; (E6) Bleeding disorder or administration of anticoagulants within 3 weeks prior to enrollment that contraindicates intramuscular injection in the investigator's opinion; (E7) History of any bleeding disorder or administration of anticoagulants within 3 weeks prior to enrollment that contraindicates intramuscular injection in the investigator's opinion. (E8) any chronic disease at a stage that may interfere with the conduct or completion of the study (e.g., cardiac, renal, autoimmune, diabetes, psychiatric, or chronic infectious disease); (E9) any vaccine within 4 weeks before or scheduled to be administered within 4 weeks after administration of any study intervention (if a participant is enrolled and wishes to be vaccinated with an approved influenza or non-mRNA COVID-19 vaccine outside the study, the participant is encouraged to proactively discuss this intention with the investigator and will be permitted to receive the approved vaccine at least 28 days after administration of the study vaccine and any time thereafter); (E10) any mRNA vaccine within 60 days before or scheduled to be administered within 60 days after administration of any study intervention; (E11) previous vaccination against RSV with an investigational vaccine; (E12) administration of immune globulin, blood, or blood-derived products in the past 3 months;(E13) Administration of oral or injectable antibiotic therapy within 72 hours prior to the first blood draw; (E14) Participation at the time of study enrollment (or within 4 weeks prior to administration of the first study intervention) or planned participation during the study in another clinical study investigating a vaccine, drug, medical device, or medical procedure; (E15) Deprivation of liberty or involuntary hospitalization by administrative or court order or under emergency circumstances; (E16) Self-reported or documented positivity for human immunodeficiency virus (HIV), hepatitis B virus surface antigen (HbsAg), hepatitis B core antibody (HbcAb), or hepatitis C virus antibody (HCV Abs) detected by any FDA-approved / validated test, or SARS-CoV-2 RT-PCR or antigen test; or (E17) Identified as an investigator or employee of the investigator or research center directly involved in the proposed study, or identified as an immediate family member (i.e., parent, spouse, natural child, or adopted child) of an investigator or employee directly involved in the proposed study. ;

[0353] Exclusion criteria E1-E17 will be checked at the participant's initial screening visit. In addition to E1-E17, three additional exclusion criteria E18-E20 will be checked at the first visit (Visit 1; Day 1). E18 is a screening electrocardiogram showing clinically relevant abnormalities consistent with possible myocarditis, pericarditis, and / or myopericarditis, or that, in the investigator's opinion, may affect the participant's safety or study outcomes. E19 is moderate or severe acute illness / infection (based on the investigator's judgment) or febrile illness (temperature 38.0°C) on the day of study intervention administration. Potential participants should not be enrolled in the study until the condition has resolved or the febrile event has subsided. E20 is any screening laboratory parameter with a laboratory abnormality greater than Grade 1 or deemed clinically significant in the investigator's opinion.

[0354] If a participant has a primary physician who is not the investigator, the site should contact this physician, with the participant's consent, to inform him or her of the participant's participation in the study. In addition, the site should ask this physician to verify exclusion criteria related to previous treatments, such as administration of blood products or previous vaccines.

[0355] Study interventions and concomitant therapies How participants were randomly assigned to intervention groups A randomized participant is defined as a participant assigned to a randomized intervention (i.e., a participant enrolled by the IRT), regardless of whether treatment was administered. Participants cannot be randomized more than once during the study.

[0356] Participants who sign informed consent 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 arms at Visit 1 according to the following: Study A Cohort 1 (Sentinel Cohort 1): In a 1:1:1 ratio, low-dose RSV mRNA vaccine containing LNP cKK-E10, low-dose RSV mRNA vaccine containing LNP GL-HEPES-E3-E12-DS-4-E10, or placebo. Study A Cohort 2 (Sentinel Cohort 2): In a 1:1:1 ratio, receive a medium-dose RSV mRNA vaccine containing LNP cKK-E10, a medium-dose RSV mRNA vaccine containing LNP GL-HEPES-E3-E12-DS-4-E10, or placebo. Study A Cohort 3 (Sentinel Cohort 3): In a 1:1:1 ratio, 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. Study B (primary cohort): In a 1:1:1:1:1:1:1:1 ratio, 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.

[0357] At Visit 1, randomization will be stratified by cohort (sentinel cohort 1, 2, 3 or primary cohort), CMI subset (yes or no), and Japanese origin status (yes or no).

[0358] Participants from the placebo and selected formulation groups (i.e., both dose levels and LNP formulations) of Study B (primary cohort) will be eligible to receive a booster dose 12 months after their first vaccination.

[0359] At Visit 8, participants who meet the eligibility criteria, including the booster screening visit (Screening Visit 8), will be randomly assigned in a 1:1 ratio to receive the RSV mRNA vaccine formulation of choice or placebo. At Visit 8, randomization will be stratified by the study intervention group randomized at Visit 1.

[0360] Site staff connect to the interactive response technology (IRT) and respond to IRT prompts to enter identification and security information and verify a minimal amount of data. The IRT then provides group assignment for confirmation by site staff. The IRT also informs site staff if the participant has been assigned to a CMI subset (i.e., 20 participants in each study intervention group in the primary cohort recruited from a limited number of selected sites). If the participant is not eligible for study participation, the information is recorded only in the participant recruitment log. Participant numbers should not be reassigned for any reason. The randomization code is securely stored within the IRT.

[0361] Concomitant Therapy - Reportable Agents Any medications that participants received prior to the day of vaccination, are receiving at the time of enrollment, or will receive during the study must be reported by the investigator if the medication may affect the interpretation of safety data (e.g., antipyretics or analgesics that may reduce the intensity or frequency of adverse events) or may interfere with the development or measurement of immune responses (e.g., use of immunosuppressants, immunomodulators, or some antibiotics that may affect the effect of certain bioassays). Steroid medications may affect both safety assessments and immune responses to the vaccine.

[0362] Below is a list of reportable drug categories: Medications that affect or may affect the safety assessment (e.g., antipyretics, analgesics, and nonsteroidal anti-inflammatory drugs (NSAIDs), systemic steroids / corticosteroids). NOTE: Topical analgesics should not be applied at the injection site of the study intervention, but if these are applied inadvertently it should be recorded. Drugs that affect or may affect the immune response (e.g. other vaccines, blood products, classes of antibiotics, systemic steroids / corticosteroids, immunosuppressants, immunomodulators with immunosuppressive properties, antiproliferative agents such as DNA synthesis inhibitors that may interfere with the bioassays used by Sanofi Pasteur laboratories or other testing laboratories). Medications that affect or may affect both safety and immune response (e.g., systemic steroids / corticosteroids).

[0363] Reportable medications will be collected on the Case Report Form (CRF) until the end of the unsolicited follow-up period (i.e., 28 days after vaccination). Medications that may affect the immune response or may affect both safety and immune response will be collected throughout the study. For mRNA vaccines, reports will be collected throughout the study, including 28 days after vaccination.

[0364] Dosage and route of administration, homeopathic medications, topical and inhaled steroids and topical, ophthalmic and otic treatments will not be recorded (except for topical analgesics applied at the injection site of the study intervention).

[0365] Medications administered in response to an adverse event are recorded only in the "Actions Taken" section of the case report form. Unless the medication administered belongs to one of the pre-listed categories, details are not recorded on the concomitant medication form of the CRF. Medications are coded. Information about previous medications, including previous influenza vaccinations and previous mRNA vaccinations / products the participant has received, is recorded on the participant's eCRF.

[0366] Rescue medication In the event of anaphylaxis, vasovagal reaction, or other immediate allergic reaction, appropriate medical equipment and emergency medications, including epinephrine (1:1000), must be available at the research facility.

[0367] Discontinuation of study intervention Participants will be permanently discontinued from the study intervention, i.e., will lose eligibility to receive a booster dose, if they experience at least one of the 13 critical contraindications listed below: Additional unscheduled visits may be conducted for safety reasons, and information will be reported in the source documents.

[0368] Three temporary contraindications If a participant experiences one of the three conditions listed herein, the investigator will postpone further vaccination (i.e., administration of a booster dose for eligible participants enrolled in a booster cohort) until the condition resolves. The postponement must be within the vaccination window indicated in the scheduled activity. (1) Febrile illness (temperature ≥ 38°C [≥ 100.4°F]) or moderate or severe acute illness / infection on the day of vaccination, as determined by the investigator. (2) Scheduled administration of any vaccine (other than a study mRNA vaccine) within 4 weeks prior to administration of any study vaccination intervention or scheduled administration of any vaccine other than a mRNA vaccine within 4 weeks after administration of any study intervention. (3) Scheduled administration of any mRNA vaccine within 60 days prior to administration of any study vaccination intervention or scheduled administration of any mRNA vaccine within 60 days after administration of any study intervention.

[0369] 13 critical contraindications for withdrawal from the study The investigator will definitively discontinue vaccination if a participant experiences at least one of the conditions listed herein: (1) anaphylaxis or allergic reaction to the previous dose of vaccine; (2) abnormal laboratory parameters that are Grade 2 or 3 and are assessed by the investigator as related to the previous dose of vaccine; (3) an SAE assessed as related to the study vaccine after the previous dose of vaccine, based on the investigator's judgment; (4) myocarditis, pericarditis, and / or myopericarditis; (5) clinically, serologically, or microbiologically diagnosed RSV-associated disease; (6) thrombocytopenia or bleeding disorder; (7) a chronic disease at a stage that, in the investigator's opinion, may interfere with the conduct or completion of the study (e.g., cardiac disorder, renal disorder, autoimmune disorder, diabetes, psychiatric disorder, or chronic infection); (8) known or suspected (9) administration of anticoagulants in the 3 weeks prior to the booster injection; (10) administration of immune globulin, blood, or blood-derived products in the past 3 months; (11) administration of oral or injectable antibiotic therapy within 72 hours prior to the pre-booster vaccination blood draw at Visit 7 (BL0005); (12) participation or planned participation in another clinical study investigating a vaccine, drug, medical device, or medical procedure during the booster phase of the study; (13) self-reported or documented seropositivity for human immunodeficiency virus (HIV) antigens and / or antibodies, hepatitis B virus surface antigen (hBsAg), hepatitis B core antibody (hBcAb), or hepatitis C virus antibody (HCV Ab).

[0370] If a local or national immunization program with pandemic influenza vaccine or any other vaccine is implemented as appropriate, participants who receive that vaccine at any time during the study will not be withdrawn from the study.

[0371] Participants may withdraw from the study at any time upon their own request or at the investigator's discretion for safety, behavioral, or compliance reasons. If a participant withdraws consent, they will be permanently discontinued from both the study intervention and the study at that time. Withdrawn participants will not be replaced.

[0372] Study Assessment and Procedures Study assessment data collected at each visit, including routine clinical management (e.g., blood counts, electrocardiogram, physical examination), will be obtained as shown in the table for scheduled activities for Study A (sentinel cohort) shown in Figure 4, the table for Study B (primary cohort) shown in Figure 5, and the table for the booster cohort shown in Figure 6.

[0373] blood sample Blood samples will be collected at visits according to the schedule of scheduled activities for each cohort shown in Figures 4-6 and will be used for safety and immunogenicity assessments, as well as serology testing for HIV, hepatitis B, and hepatitis C. The maximum amount of blood collected from each participant during the study will not exceed 255 mL, including any additional assessments that may be required. The amount of blood collected at each visit will range from 15 mL to 50 mL, as shown in Tables 8, 9, and 10 below. Repeat or unscheduled samples may be taken for safety reasons or technical issues with the samples.

[0374] [Table 11]

[0375] [Table 12]

[0376] [Table 13]

[0377] Immunogenicity assessment RSV anti-F IgG ELISA Antibodies to the RSV-F antigen are measured using an RSV anti-F IgG ELISA. Briefly, RSV-F antigen is coated onto a microtiter plate, and serial two-fold dilutions of human serum samples are added and incubated to allow binding to the RSV-F antigen. A horseradish peroxidase (HRP)-conjugated anti-human IgG detection antibody is then added, followed by a colorimetric substrate. The concentration of IgG antibodies to the RSV-F antigen is calculated using six serial dilutions against a qualified internal standard calibrated to the WHO International Standard (the First International Standard for RSV Antisera) with an assigned value (international units / mL).

[0378] Evaluation of RSV neutralizing antibodies RSV neutralizing antibodies are measured using the microneutralization (MN) assay, Nexelis PRNT A2 assay, or A Long assay. Serial two-fold dilutions of serum samples are heat-inactivated and then mixed with a fixed concentration of RSV A2 strain (ATCC VR-1540). The mixture is seeded into wells of a 96-well microplate containing permissive hEp-2 cells (ATCC CCL-23) and incubated for two days. Reductions in viral infectivity (viral antigen production) due to neutralization by antibodies present in the serum samples are detected by ELISA. After washing and fixation, RSV antigen production in the cells is detected by sequential incubation with a mouse anti-RSV-specific monoclonal antibody, an HRP anti-mouse IgG conjugate, and a chromogenic substrate. The resulting optical density is measured using a microplate reader. Reductions in RSV infectivity compared to those in virus control wells indicate a positive neutralization reaction, indicating the presence of neutralizing antibodies in the serum sample.

[0379] cell-mediated immunity T helper cell responses are assessed using fresh whole blood (TruCulture). Analysis of T cells in whole blood collected using the RBM TruCulture Whole Blood Collection and Culture System allows for consistent and reliable assessment of T helper cell polarization. Triculture tubes containing the selected stimulant or antigen allow for near-instant stimulation of cells in the presence of all blood components after blood is drawn into the tube, thus minimizing variability that can result from blood handling and manipulation, including peripheral blood mononuclear cell (PBMC) processing. Preliminary results and previous reports in the literature demonstrate the robustness of the method. (Duffy et al. (2017), Clin Immunol, 183:325-335). This assay relies on the fact that T helper cytokines are secreted into a single tube after stimulation, and the supernatant is collected at 24 or 48 hours. A panel of cytokines using the RBM TruCulture's Luminex xMAP technology is measured to determine the state of T helper cell polarization.

[0380] Safety evaluation The planned time points for all safety assessments are provided in the tables of scheduled activities for each cohort shown in Figures 4-6. Prior to enrollment, participants will be evaluated for pre-existing conditions and diseases, both past and ongoing, and such conditions will be documented. Significant (clinically relevant) medical history (reported as diagnoses), including conditions / diseases for which the participant is currently or has been followed by a physician, or conditions / diseases that may recur during the study or lead to SAEs or repeat outpatient visits, will be reported in the case report form. Additionally, a history of administration of the mRNA-based vaccine will be recorded.

[0381] Physical examination and vital signs At the screening visit, the investigator or designee will conduct a complete physical examination. A complete physical examination will also be performed at Visit 1. Targeted / abbreviated physical examinations will also be performed at all subsequent visits at the times specified in the tables of scheduled activities for each cohort shown in Figures 4-6. Oral or axillary pre-vaccination temperatures will be systematically collected by the investigator. Tympanic membrane, skin, and temporal artery thermometers should not be used.

[0382] electro-cardiogram An ECG will be performed at the screening visit to serve as a baseline and to exclude participants with probable or possible myocarditis, pericarditis, and / or myopericarditis, as well as to identify participants with clinically relevant abnormalities that may affect participant safety or study outcomes. If a participant develops symptoms of myocarditis, pericarditis, and / or myopericarditis during the conduct of the study, an additional ECG will be performed as soon as possible at an unscheduled visit, if necessary. ECGs will be recorded, and any evaluation will be based on standard medical care.

[0383] Clinical Safety Test Evaluation Table 11 below lists the clinical laboratory tests. Laboratory tests will be performed during the time points specified in the tables of scheduled activities for each cohort shown in Figures 4-6. The investigator will review the laboratory reports and record any clinically significant changes that occur after either the primary vaccination or the booster vaccination as an adverse event.

[0384] All laboratory tests with values ​​considered to be clinically significantly abnormal after either primary or booster vaccination 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 time period deemed reasonable by the investigator, the cause should be identified and the sponsor notified.

[0385] [Table 14]

[0386] Nasal swab collection Nasal swab specimens for the detection of RSV and respiratory pathogens (including COVID-19) will be collected from participants with any respiratory illness episode. If participants visit any other off-study physician / hospital at any time during the study, nasal swab specimens will be obtained at the study site immediately upon subject discharge, if deemed appropriate by the investigator. All nasal swab specimens will be collected in recommended viral transport media tubes and stored at -60°C to -80°C until ready for shipment. If deemed appropriate by the investigator, the requirement for a sick visit or illness clinic visit will be initially assessed via video call to allow for remote assessment of severity and remote management of mild (Grade 1) illness.

[0387] Lower Respiratory Tract and Acute Respiratory Disease Assessment If RSV is confirmed by RT-PCR, the following RSV disease categories are used: (1) RSV acute respiratory disease (ARD) is any respiratory symptom including nasal congestion, sore throat, hoarseness, new or worsening cough, sputum production, and dyspnea with or without fever; (2) severe RSV ARD is acute respiratory illness confirmed by RT-PCR with fever or a history of fever measured at 38°C or higher, cough onset within the past 10 days, and requiring hospitalization; (3) non-medical care RSV lower respiratory tract disease (LRTD) is ARD with 10 days of ARD symptom onset and RSV confirmed by RT-PCR, and one or more symptoms of lower respiratory tract disease (including lower respiratory tract: tracheal, bronchial, and pulmonary involvement, which may be complicated, i.e., bronchopneumonia, tracheobronchitis); (4) RSV requiring medical attention LRTD is a medically treated ARD with 10 days of ARD symptom onset and confirmed by RT-PCR, with one or more symptoms of lower respiratory tract disease (this includes lower respiratory tract: tracheal, bronchial, and pulmonary involvement, which may lead to complications, i.e., bronchopneumonia, tracheobronchitis). Medical treatments are categorized as emergency room, hospitalization, and outpatient clinic visits.

[0388] Example 2: Interim Analysis 2 - Potency and Safety Data The planned sample size (IA2) was 790, including 90 in the sentinel cohort and 700 in the primary cohort. The IA2 sample size (partially the primary cohort) was 667 participants for analyzing immunogenicity (DO and D29) and 698 participants for analyzing safety data (up to day 29 (D29)). The demographic characteristics of the primary cohort are shown in Figure 7.

[0389] Titer Summary results (PPAS-1) of RSV A neutralizing antibody (NAb) geometric mean titers (GMTs) and neutralizing antibody geometric mean titer ratios (GMTRs) for the full primary cohort (ages 60 years and older) on D29 / D01 after primary vaccination are shown in Figure 8. The cKK-E10 + RSV mRNA 75mcg group had a slightly higher GMT at D29 with a GMTR of 5.66 compared to the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 75mcg group (GMTR of 5.44), followed by the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 30mcg group with a GMTR of 4.74.

[0390] Fold increase data in RSV-A neutralizing antibodies after primary vaccination for the full primary cohort are shown in Figure 9. The percentage of participants with at least a 4-fold increase ranged from 38.4% to 69.6%, depending on dose and LNP.

[0391] Summary results of the partial primary cohort (ages 60 years and older) geometric mean IgG antibody titers and IgG antibody GMTR after primary vaccination are shown in Figure 10. The GL-HEPES-E3-E12-DS-4-E10+75mcg group showed higher IgG GM at D29 with the highest fold increase (10.4), followed by the cKK-E10+75mcg group (10.3) and the GL-HEPES-E3-E12-DS-4-E10+30mcg group (8.29). These data correlated with the group showing the best RSV-A GMT response.

[0392] The GMT results (PPAS-1) of RSV A neutralizing antibody titers after primary vaccination of the sentinel cohort (aged 18-50 years) and the neutralizing antibody geometric mean titer ratio (GMTR) on D29 / D01 are summarized in Figure 11. In the sentinel cohort, the GL-HEPES-E3-E12-DS-4-E10+75mcg group showed a higher GMT on D29 with a GMTR of 11.5, followed by the cKK-E10+75mcg group with a GMTR of 8.64 and the cKK-E10 and GL-HEPES-E3-E12-DS-4-E10+30mcg groups with GMTRs of 6.43 and 6.06, respectively.

[0393] Fold increase data in RSV-A neutralizing antibodies after primary vaccination for the sentinel cohort are shown in Figure 12. The percentage of participants with at least a 4-fold increase ranged from 55.6% to 90.0%, depending on dose and LNP.

[0394] A summary of the sentinel cohort for RSV-A neutralizing antibody titers after primary vaccination is shown in Figures 13A-13B.

[0395] Immunogenicity conclusions The GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 75 mcg group showed higher GMTs at D29 with a GMTR of 5.22, followed by the cKK-E10 + RSV mRNA 75 mcg group with a GMTR of 4.56 and the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 30 mcg group with a GMTR of 4.4.

[0396] IgG results correlated with RSV-A neutralizing antibody responses (higher titers and fold increase in the same groups).

[0397] A higher dose of mRNA (75 mcg) demonstrated greater immunogenicity.

[0398] 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).

[0399] safety Safety data in the elderly showed that all RSV mRNA products were generally well tolerated.

[0400] The GL-HEPES-E3-E12-DS-4-E10 group showed a trend toward better reactogenicity compared with cKK-E10, particularly injection site pain and muscle pain (the most frequently reported induced reactions). Induced reactions were generally mild to moderate, with few grade 3 reactions. A dose-response was observed across mRNA groups.

[0401] Spontaneous adverse effects (AEs) were generally balanced between the mRNA groups (with a tendency to be slightly more commonly reported in the mRNA group compared to placebo). No dose response was observed.

[0402] An increase from baseline in cardiac biomarkers (troponin I levels) on day 8 was observed in five participants (all in the mRNA group, with normal baselines) (this observation was not considered significant at this stage). One elevated troponin level was reported as an associated serious adverse effect (SAE) (silent myocardial injury; adjudicated by the Cardiac Adjudication Committee). Strenuous exercise was determined as the potential cause of the elevated troponin.

[0403] A summary of induced reactions (%) within 7 days after primary vaccination is shown in Figure 14. Most induced reactions were mild to moderate in severity and short in duration. A summary of injection site reactions (%) within 7 days after primary vaccination is shown in Figure 15. Most injection site reactions were mild to moderate in severity and short in duration. A summary of induced systemic reactions (%) within 7 days after primary vaccination is shown in Figure 16. Most induced systemic reactions were mild to moderate in severity and short in duration.

[0404] A summary of safety after primary vaccination is shown in Figure 17.

[0405] Overview of reactogenicity Overall, injection site pain was the most frequently reported induced injection site reaction across the six mRNA treatment groups (39.4%–71.0% in the cKK-E10 group; 28.7%–53.1% in the GL-HEPES-E3-E12-DS-4-E10 group).

[0406] Myalgia, fatigue, and headache were the most frequently reported induced systemic reactions across the six mRNA treatment groups. Myalgia (range 17.2%-41.0% in the cKK-E10 group), fatigue (13.1-29.0% in the cKK-E10 group), and headache (16.2-23.0% in the cKK-E10 group, 23% at the mid-dose) were the most frequently reported induced systemic reactions in the cKK-E10 group, closely followed by arthralgia and chills at the highest dose (21.0% and 15.0%, respectively). The same trends were observed in the GL-HEPES-E3-E12-DS-4-E10 group (myalgia ranging from 19.8 to 27.6%; fatigue from 15.8 to 26.0% (26.0% at the mid-dose); and headache from 14.9 to 25.5%), with the exception of joint pain and chills, which were similarly likely to be reported as in the placebo group (8.2% and 9.2%, respectively, even at the highest dose).

[0407] Most induced reactions began between D01 and D04 and were short-lived (lasting 1-3 days). Induced reactions were generally mild to moderate, with few grade 3 reactions (less than 4.1% in the GL-HEPES-E3-E12-DS-4-E10 group and less than 7.0% in the cKK-E10 group), and all grade 3 reactions lasted 1-3 days at maximum intensity (except for one outlier with injection site erythema lasting 5 days at maximum intensity).

[0408] A dose response was observed across mRNA groups, with a trend toward better reactogenicity in the GL-HEPES-E3-E12-DS-4-E10 group compared with the cKK-E10 group (GL-HEPES-E3-E12-DS-4-E10 high dose ≈ cKK-E10 medium dose).

[0409] No delayed injection site reactions were observed.

[0410] A summary of the induced responses within 7 days after primary vaccination is shown in FIG.

[0411] Spontaneous adverse effects (AEs) Two subjects, both in the high-dose mRNA group, reported immediate spontaneous AEs / ARs: hypertension (SOC: Vascular disorders) assessed as related to IMP (both Grade 1).

[0412] Spontaneous AEs tended to be reported slightly more frequently in the mRNA group compared with placebo, with an overall balance observed between the mRNA groups: 25.7% of subjects reported at least one AE in the pooled cKK-E10 group, compared with 22.7% in the pooled GL-HEPES-E3-E12-DS-4-E10 group and 17.2% in the placebo group. No dose response was observed. The slight imbalance observed was not significant and was thought to be driven by musculoskeletal and connective tissue disorders (e.g., arthralgia, myalgia, muscle cramps) and gastrointestinal disorders (e.g., abdominal pain, diarrhea, nausea). Spontaneous ARs (AEs assessed as related to IMPs) were rare: 5% of subjects in the pooled cKK-E10 group, 4% in the pooled GL-HEPES-E3-E12-DS-4-E10 group, and 2% in the placebo group.

[0413] Twenty-three subjects had a medically required adverse event (MAAE). MAAEs were well balanced between groups.

[0414] Five subjects experienced serious adverse events (SAEs) within 28 days, all in the mRNA group and evenly distributed between treatment groups (two in the LNP cKK-E10 group and three in the LNP GL-HEPES-E3-E12-DS-4-E10 group). Only one was assessed as related to the investigational medical product IMP (mRNA LNP cKK-E10 low dose) (silent myocardial injury).

[0415] No adverse events of particular interest (AESIs) were observed (i.e., anaphylactic reactions (including bronchospasm and pharyngeal spasm), myocarditis, pericarditis, and myopericarditis). AESIs in the biostatistical table were downgraded to non-AESIs by the investigator after the cutoff date (the diagnosis was changed from myocarditis to silent myocardial injury). No AEs led to study discontinuation, and no deaths were reported.

[0416] A summary of spontaneous AEs is shown in Figure 19. A summary of spontaneous AEs due to gastrointestinal, musculoskeletal, and connective tissue disorders is shown in Figure 20.

[0417] Overall conclusion The GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 75 mcg group showed higher GMTs at D29 with a GMTR of 5.22, followed by the cKK-E10 + RSV mRNA 75 mcg group with a GMTR of 4.56 and the GL-HEPES-E3-E12-DS-4-E10 + RSV mRNA 30 mcg group with a GMTR of 4.4.

[0418] A higher dose of mRNA (75 mcg) demonstrated greater immunogenicity.

[0419] 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 better reactogenicity compared to cKK-E10 (GL-HEPES-E3-E12-DS-4-E10 high dose ≈ cKK-E10 medium dose).

[0420] GL-HEPES-E3-E12-DS-4-E10+mRNA 30mcg has the best overall safety profile for grade 3 and grade 2 induced and special events.

[0421] Example 3: Phase IIb / III - Stage II XII. Rationale for the Study RSV is a major viral agent causing severe respiratory tract disease in older adults worldwide. There is a medical need to improve patient quality of life by preventing lower respiratory tract disease (LRTD). The clinical trials described in Example 1, Study A (i.e., the sentinel cohort) and Study B (i.e., the primary cohort), include a subsequent Stage 2, Phase IIb / III study enrolling approximately 13,482 adults aged 60 years or older to evaluate the efficacy, immunogenicity, and safety of LNPs selected in Stage 1 (i.e., Phase I / IIa described in Examples 1 and 2) using a 110 μg dose of RSV mRNA vaccine for the prevention of LRTD caused by RSV.

[0422] During Stage 1, a 75 μg dose of RSV mRNA vaccine combined with selected LNPs demonstrated the highest immune response with a favorable safety profile. Therefore, Example 3 tests a 110 μg dose of RSV mRNA vaccine with selected LNPs. Phase IIb / III of this study evaluates the safety of a 110 μg dose of RSV mRNA vaccine containing selected LNPs and is primarily designed to demonstrate the clinical efficacy of selected RSV mRNA vaccine candidates for preventing RSV-LRTD.

[0423] A. Research Overview and Research Design Number of participants, details of intervention group and period Number of Participants. As shown in Table 12 below, a total of 13,482 participants are planned to be randomized, including 50 participants in the Phase IIb Sentinel Cohort (25 participants per treatment group) and 5190 participants in the Phase IIb Pivotal Cohort (2595 participants per treatment group).

[0424] [Table 15]

[0425] An additional 8,242 participants (4,121 participants per treatment group) are planned to be randomized in the Phase III cohort. Approximately 2,000 participants (1,000 per treatment group) from the Phase IIb cohort and 4,000 participants (2,000 per treatment group) from the Phase III cohort will be included in the reactogenicity subset to collect elicited injection site and systemic reactions occurring up to 7 days after vaccination. Approximately 500 participants (250 per treatment group) in each of the Phase IIb and Phase III cohorts will also be included in the immunogenicity subset to evaluate the durability of neutralizing antibody responses. Approximately 100 participants (50 per treatment group) in the Phase III cohort will also be selected for inclusion in the CMI subset. Participants in the CMI subset will be enrolled from a limited number of select centers, chosen based on their past experience in collecting and processing blood samples for CMI assays.

[0426] Intervention Group. The Stage 2 intervention group is eligible participants enrolled in a 1:1 ratio to receive a single intramuscular (IM) dose of the RSV mRNA vaccine candidate or placebo.

[0427] Expected Duration: The expected total duration of the study is approximately 6 months for participants in the Phase IIb Sentinel Cohort and approximately 12 months for participants in the Phase IIb Pivotal Cohort / Phase III Cohort.

[0428] Composition: Stage 2 participants will receive either one dose (0.5 mL) in a vial containing 110 μg of RSV pre-F mRNA with the selected LNP in a 2.2x diluent of PBS or one dose (0.5 mL) in a vial containing 0.9% normal saline via IM injection.

[0429] B. Purpose Primary Objectives: The primary objectives are to evaluate the safety of a 110 μg dose of RSV mRNA vaccine containing selected LNPs and to demonstrate the same clinical efficacy of mRNA RSV vaccine candidates for preventing RSV-LRTD during the first pandemic period occurring 14 days or later after vaccination.

[0430] Secondary Objective. The secondary objective is to demonstrate the clinical efficacy of the mRNA RSV vaccine candidate in preventing RSV-ARD (RSV-Acute Respiratory Illness) and RSV-MAARD (RSV-Medicare-Required Acute Respiratory Illness) during the first pandemic occurring 14 days or more after vaccination.

[0431] Table 13 below summarizes the primary objectives and corresponding endpoints. Table 14 below summarizes the secondary objectives and corresponding endpoints. Table 15 below summarizes the immunogenicity objectives and corresponding endpoints. Table 16 below summarizes the safety objectives and corresponding endpoints. Table 17 below summarizes the exploratory objectives and corresponding endpoints.

[0432] [Table 16]

[0433] [Table 17]

[0434] [Table 18]

[0435] [Table 19]

[0436] [Table 20]

[0437] [Table 21]

[0438] [Table 22]

[0439] [Table 23]

[0440] [Table 24]

[0441] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

[0442] All patents and publications cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for inducing an immune response against respiratory syncytial virus (RSV) in a subject, comprising administering to the subject a prophylactically effective amount of an RSV vaccine comprising 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 or consisting of the amino acid sequence of SEQ ID NO:

3.

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

3. A method for inducing an immune response against respiratory syncytial virus (RSV) in a subject, 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 or consisting of the nucleic acid sequence of SEQ ID NO:

14.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

24. 19. The method of any one of claims 1-18, wherein the subject receives an initial dose of the RSV vaccine and a booster dose of the RSV vaccine.

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

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

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

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

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

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

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

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

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

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

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

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

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

38. 1. A method for preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, comprising administering to the subject a prophylactically effective amount of an RSV vaccine comprising 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 or consisting of the amino acid sequence of SEQ ID NO:

3.

39. 39. The method of 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, 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 or consisting of the nucleic acid sequence of SEQ ID NO:

14.

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

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

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

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

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

46. 46. ​​The method of any one of claims 38 to 45, wherein the mRNA is formulated in a lipid nanoparticle (LNP).

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

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

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

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

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

52. 48. The method of 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. 53. The method of claim 52, wherein said at least one cationic lipid is cKK-E10.

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

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

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

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

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

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

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

61. 56. The method of any one of claims 38-55, wherein the subject receives an initial dose of the RSV vaccine and a booster dose of the RSV vaccine.

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

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

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

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

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

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

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

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

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

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

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

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

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

75. 75. The method of any one of claims 38 to 74, wherein the RSV vaccine is administered with a device suitable for cutaneous injection.

76. 75. The method of 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-induced acute respiratory disease (MAARD), severe ARD, lower respiratory tract disease (LRTD) not requiring medical attention, medically-induced LRTD, congestion, runny nose, cough, fever, sore throat, headache, pneumonia, bronchiolitis, bronchopneumonia, and tracheobronchitis.

77. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; administering to the subject a prophylactically effective amount of an RSV vaccine comprising 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 or consisting of the amino acid sequence of SEQ ID NO:3; A method comprising:

78. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; and A method comprising:

79. 1. A method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, comprising: selecting a subject who is at least 60 years old; administering to the subject a prophylactically effective amount of an RSV vaccine comprising 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 or consisting of the amino acid sequence of SEQ ID NO:3; A method comprising:

80. 1. A method of preventing respiratory syncytial virus (RSV) infection or reducing one or more symptoms of RSV infection in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; and A method comprising:

81. 1. A respiratory syncytial virus (RSV) vaccine for use in eliciting an immune response to RSV in a subject, the vaccine comprising 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 or consists of the amino acid sequence of SEQ ID NO:3, and the RSV F protein antigen is a pre-fusion protein.

82. 1. A respiratory syncytial virus (RSV) vaccine for use in eliciting an immune response to RSV in a subject, the vaccine comprising 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 or consists of the amino acid sequence of SEQ ID NO:

3.

83. A respiratory syncytial virus (RSV) vaccine for use in inducing an immune response to RSV in a subject, the vaccine comprising messenger RNA (mRNA), the mRNA comprising 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.

84. 1. A respiratory syncytial virus (RSV) vaccine for use in preventing or reducing one or more symptoms of RSV infection in a subject, the vaccine comprising 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 or consisting of the amino acid sequence of SEQ ID NO:

3.

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

14.

86. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in lipid nanoparticles (LNPs) comprising GL-HEPES-E3-E12-DS-4-E10; The method wherein the RSV vaccine is administered at a dose of about 110 micrograms.

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

88. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in lipid nanoparticles (LNPs) comprising GL-HEPES-E3-E12-DS-4-E10; The method wherein the RSV vaccine is administered in a dose of about 30 micrograms.

89. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in lipid nanoparticles (LNPs) containing cKK-E10; The method wherein the RSV vaccine is administered in a dose of about 30 micrograms.

90. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in lipid nanoparticles (LNPs) containing cKK-E10; The method wherein the RSV vaccine is administered in a dose of about 75 micrograms.

91. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in lipid nanoparticles (LNPs) containing cKK-E10; The method wherein the RSV vaccine is administered at a dose of about 110 micrograms.

92. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in a lipid nanoparticle (LNP) containing IM-001; The method wherein the RSV vaccine is administered in a dose of about 30 micrograms.

93. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in a lipid nanoparticle (LNP) containing IM-001; The method wherein the RSV vaccine is administered in a dose of about 75 micrograms.

94. 1. A method of eliciting an immune response to respiratory syncytial virus (RSV) in a subject, comprising: selecting a subject who is at least 60 years old; 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, or consists of, the nucleic acid sequence of SEQ ID NO:14; Including, the mRNA is formulated in a lipid nanoparticle (LNP) containing IM-001; The method wherein the RSV vaccine is administered at a dose of about 110 micrograms.

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