WO2022221336A1

WO2022221336A1 - Respiratory syncytial virus mrna vaccines

Info

Publication number: WO2022221336A1
Application number: PCT/US2022/024498
Authority: WO
Inventors: Christine SHAW; Allison AUGUST
Original assignee: Modernatx, Inc.
Priority date: 2021-04-13
Filing date: 2022-04-12
Publication date: 2022-10-20

Abstract

The disclosure provides human respiratory syncytial virus (RSV) mRNA vaccines, as well as methods of treating a human subject using the vaccines and compositions comprising the vaccines. The vaccines comprise an mRNA comprising an open reading frame encoding a stabilized trimeric prefusion form of the RSV F glycoprotein, with an additional deletion of its cytoplasmic tail domain. The mRNA is formulated in a lipid nanoparticle comprising 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 ml% PEG- modifled lipid, and 45-55 mol% ionizable amino lipid of Compound I (mRNA-1345).

Description

RESPIRATORY SYNCYTIAL VIRUS MRNA VACCINES RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/174,320, filed April 13, 2021, U.S. provisional application number 63/241,848, filed September 8, 2021, and U.S. provisional application number 63/247,431, filed September 23, 2021, each of which are incorporated by reference herein in their entirety.

BACKGROUND

Respiratory syncytial virus (RSV) is a common negative- sense, single-stranded ribonucleic acid (RNA) virus of the family Pneumovirinae. RSV infection usually causes mild, cold-like symptoms, with most people recovering in a week or two, but RSV can be serious, especially for infants and older adults. RSV is the leading cause of unaddressed severe lower respiratory tract disease and hospitalization in infants and young children worldwide. This virus is the most common cause of respiratory tract illness, including bronchiolitis (inflammation of the small airways in the lung) and pneumonia (infection of the lungs), with most children infected at least once by two years of age. The virus is transmitted primarily via contamination of environmental surfaces with infectious secretions, and symptoms typically begin within several days of exposure. The illness may manifest as wheezing, bronchiolitis, pneumonia, hospitalization or even death. In the United States, it is estimated that over two million children younger than five years of age receive medical attention and more than 86,000 are hospitalized due to RSV infection annually. Globally, it is estimated that RSV is responsible for over approximately 33 million episodes of acute lower-respiratory tract infection, 3.2 million hospitalizations and as many as 118,000 deaths per year in children younger than five years of age. Infections with RSV follow a seasonal pattern, occurring primarily in the Northern Hemisphere between the months of November and April, and primarily in the Southern Hemisphere between the months of March and October.

The continuing health problems associated with RSV are of concern internationally, reinforcing the importance of developing an effective and safe vaccine against this virus.

SUMMARY

The present disclosure provides, in some aspects, a messenger RNA (mRNA) vaccine that includes an mRNA encoding a membrane- anchored version of the stabilized prefusion F glycoprotein (“F protein”), the main target of potently neutralizing and protective antibodies. The mRNA vaccine described herein, formulated in a lipid nanoparticle, was engineered for increased expression and immunogenicity relative to other mRNA vaccines in development. The interim Phase 1 data described below shows that this vaccine is both safe and effective, eliciting high titers of neutralizing antibodies following just a single dose of the vaccine.

Some aspects of the present disclosure provide a method comprising: administering to a human subject a dose of a vaccine comprising a lipid nanoparticle that comprises a messenger ribonucleic acid (mRNA), wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human respiratory syncytial virus (RSV) F glycoprotein that comprises a deletion of a cytoplasmic tail domain, the dose of the vaccine comprises 12.5 pg to 200 pg or 50 pg to 100 pg or 50 pg to 200 pg of the mRNA, and the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 3-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

In some embodiments, the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 4-fold higher than the neutralizing antibody titers against RSV- A and/or RSV-B in the subject at baseline.

In some embodiments, the geometric mean titer (GMT) of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 29 post administration of the dose is at least 15,000.

In some embodiments, the geometric mean (GM) of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 15,000 IU/mL. In some embodiments, the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 11,000 IU/mL.

In some embodiments, the geometric mean fold rise (GMFR) of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 12.

In some embodiments, the GMFR of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 20.

In some embodiments, the GMFR of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 10.

In some embodiments, the dose comprises 12.5 pg of the mRNA. In some embodiments, the dose comprises 25 pg of the mRNA. In some embodiments, the dose comprises 50 pg of the mRNA. In other embodiments, the dose comprises 100 pg of the mRNA. In some embodiments, the dose comprises 200 pg of the mRNA. In some embodiments, the method further comprises administering to the subject a second dose of the vaccine. In some embodiments, the second dose of the vaccine is administered at least 2 months after the first dose is administered.

In some embodiments, the GMT of neutralizing antibody titers induced against RSV-A in the subject at Day 141 post administration of the first dose is at least 5,000. In some embodiments, the GMT of neutralizing antibody titers induced against RSV-B in the subject at Day 141 post administration of the first dose is at least 11,000.

In some embodiments, the GM of neutralizing antibody titers induced against RSV-A in the subject at Day 141 post administration of the first dose is at least 5,000 IU/mL. In some embodiments, the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 141 post administration of the first dose is at least 8,000 IU/mL.

In some embodiments, the GMFR of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 141 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 7. In some embodiments, the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 141 post administration of the first dose are at least 4-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

In some embodiments, the method further comprises administering to the subject a third dose of the vaccine. In some embodiments, the third dose of the vaccine is administered at least 2 months after the second dose is administered.

In some embodiments, the second dose comprises 50 pg of the mRNA. In other embodiments, the second dose comprises 100 pg of the mRNA.

In some embodiments, each of the second dose and the third dose comprises 50 pg of the mRNA. In other embodiments, each of the second dose and the third dose comprises 100 pg of the mRNA.

In some embodiments, the subject is an 18-49 year old subject. In other embodiments, the subject is a pediatric subject under the age of 5 years (e.g., 2 to 59, 6 to 59 or 12 to 59 months).

In still embodiments, the subject is an elderly subject at least 50, at least 55, at least 60, or at least 65 years of age.

In some embodiments, the subject is an older adult subject (e.g., 65-79 years old). In some embodiments, the GMT of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 12,000. In some embodiments, the GMT of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 11,000. In some embodiments, the GM of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 12,500 IU/mL. In some embodiments, the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post admini tration of the dose is at least 7,500 IU/mL.

In some embodiments, the GMFR of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 9.5. In some embodiments, the GMFR of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 5.

In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F protein (preF) in the subject at Day 29 post administration of the dose is at least 55,500. In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F protein (postF) in the subject at Day 29 post administration of the dose is at least 48,000.

In some embodiments, the GMFR of binding antibody titers induced against RSV preF in the subject at Day 29 post administration of the dose is at least 7.0. In some embodiments, the GMFR of binding antibody titers induced against RSV postF in the subject at Day 29 post administration of the dose is at least 4.5.

In some embodiments, administration of any one of the vaccines described herein produces an immune response with an RSV preF bias (e.g., more antibodies against the RSV F protein in its prefusion conformation are produced that antibodies against the RSV F protein in its postfusion conformation).

In some embodiments, the subject is a woman aged 18-40. In some embodiments, the dose is 12.5 pg. In some embodiments, the dose is 25 pg. In some embodiments, the dose is 50 pg. In some embodiments, the GMT of binding antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 8,200. In some embodiments, the GMT of binding antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 10,000. In some embodiments, the GMFR of binding antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 9.5. In some embodiments, the GMFR of binding antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 6.4. In some embodiments, the GMT of binding antibody titers induced against RSV preF in the subject at Day 29 post administration of the dose is at least 50,000. In some embodiments, the GMT of binding antibody titers induced against RSV postF in the subject at Day 29 post administration of the dose is at least 58,000. In some embodiments, the GMFR of binding antibody titers induced against RSV preF in the subject at Day 29 post administration of the dose is at least 8.5. In some embodiments, the GMFR of binding antibody titers induced against RSV postF in the subject at Day 29 post admini tration of the dose is at least 7.2.

In some embodiments, the F glycoprotein encoded by the ORF of the mRNA is membrane anchored. In some embodiments, the F glycoprotein encoded by the ORF of the mRNA comprises interprotomer disulfide stabilizing mutations.

In some embodiments, the F glycoprotein encoded by the ORF of the mRNA has at least 95% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the F glycoprotein encoded by the open reading frame of the mRNA comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the F glycoprotein encoded by the open reading frame of the mRNA consists of the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the mRNA comprises a 5’ 7mG(5’)ppp(5’)NlmpNp cap and a 3’ poly A tail.

In some embodiments, the mRNA comprises a 1 -methylpseudourine chemical modification.

In some embodiments, the lipid nanoparticle comprises 45-55 mol% ionizable amino lipid, 15-20 mol% neutral lipid, 35-45 mol% cholesterol, and 0.5-5 mol% PEG-modified lipid.

In some embodiments, the ionizable amino lipid is Compound I:

(Compound I).

In some embodiments, the lipid nanoparticle comprises 50 mol% ionizable amino lipid.

In other embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid. In still other embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid.

In some embodiments, the vaccine is administered intramuscularly.

Other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 18-49 years old, and the dose comprises 50 pg of the mRNA. Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 18-49 years old, and the dose comprises 100 pg of the mRNA.

Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 18-49 years old, and each of the three doses comprises 100 pg of the mRNA.

Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 18-49 years old, and the dose comprises 200 pg of the mRNA.

Other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 65-79 years old, and the dose comprises 50 pg of the mRNA.

Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 65-79 years old, and the dose comprises 100 pg of the mRNA.

Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 65-79 years old, and the dose comprises 200 pg of the mRNA.

Other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 65-79 years old, and the dose comprises 12.5 pg of the mRNA.

Yet other aspects of the present disclosure provide a method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I, the human subject is 65-79 years old, and the dose comprises 25 pg of the mRNA.

In some embodiments, the use of any one of the vaccines described herein is provided. In some embodiments, the use of any one of the vaccines described herein in the manufacture of a medicament for inducing an immune response in a subject, is provided. In some embodiments, a composition (e.g., any one of the vaccines described herein) for use in any one of the methods described herein is provided. The entire contents of International Application No. PCT/US2016/058327 (Publication No. W02017/07062) and International Application No. PCT/US2017/065408 (Publication No. W02018/107088) are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a schematic of the human RSV F glycoprotein encoded by the mRNA vaccine of the present disclosure.

FIG. 2 includes graphs showing the Geometric Mean Titer (GMT) against RSV-A (top) and RSV-B (bottom) at baseline (BL) and Month 1 (Ml) post vaccination in the younger adult cohorts.

FIG. 3 includes graphs showing the Geometric Mean Fold rise (GMFR) for RSV-A (top) and RSV-B (bottom) at Ml post vaccination in the younger adult cohorts.

FIG. 4 includes graphs showing the GMT against RSV-A (top) and RSV-B (bottom) at BL and Ml post vaccination, including the GMFR relative to baseline, in the younger adult cohorts.

FIG. 5 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), GMFR (bottom) against RSV-A at BL and Months 1, 2, 3, 4, and 5 post-vaccination, in the younger adult cohorts.

FIG. 6 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), and GMFR (bottom) against RSV-B at BL and Months 1, 2, 3, 4, and 5 post-vaccination, in the younger adult cohorts.

FIG. 7 includes graphs showing the GMT against RSV-A (top) and RSV-B (bottom) at BL and Month 1 post-vaccination. The treatment group includes pooled data from older adult subjects who received 50 pg, 100 pg, or 200 pg of the human RSV F glycoprotein encoded by the mRNA vaccine described herein (mRNA-1345).

FIG. 8 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), and GMFR (bottom) neutralization titers against RSV-A at BL and Ml post-vaccination in the older adult cohorts.

FIG. 9 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), and GMFR (bottom) neutralization titers against RSV-B at BL and Ml post-vaccination in the older adult cohorts.

FIG. 10 includes graphs showing the GMT (top) and GMFR (bottom) of binding antibodies against RSV prefusion protein (preF, left) and RSV postfusion protein (postF, right) in the older adult cohorts. FIG. 11 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), and GMFR (bottom) neutralization titers against RSV-A at BL and Ml post-vaccination in the women of childbearing age (18-40) cohorts.

FIG. 12 includes graphs showing the GMT (top), Geometric Mean (IU/mL) (middle), and GMFR (bottom) neutralization titers against RSV-B at BL and Ml post-vaccination in the women of childbearing age (18-40) cohorts.

FIG. 13 includes graphs showing the GMT (top) and GMFR (bottom) of binding antibodies against RSV prefusion protein at BL and Ml post-vaccination in the women of childbearing age (18-40) cohorts.

FIG. 14 includes graphs showing the GMT (top) and GMFR (bottom) of binding antibodies against RSV postfusion protein at BL and Ml post-vaccination in the women of childbearing age (18-40) cohorts.

DETAILED DESCRIPTION

The present disclosure provides an mRNA vaccine against respiratory syncytial virus (RSV) encoding a prefusion F protein that elicits a superior neutralizing antibody response compared to the post-fusion state and compared to other mRNA RSV vaccines in development. The interim data provided herein is from a Phase I, randomized, observer-blind, placebo- controlled, dose-ranging trial, the purpose of which is to assess in three patient populations the safety and immunogenicity (e.g., humoral immune response) following vaccination.

Surprisingly, interim results from the trial show that a single dose of the mRNA RSV vaccine in the younger adult population (18-49 years of age) boosted neutralizing antibody titers against RSV (both A and B lineages) at two evaluated dose levels (50 pg and 100 pg) with no apparent dose response. Even more surprising, there was still no apparent dose response with the third dose level tested (200 pg). At Month 1 for the 50 pg 1-dose, 100 pg 1-dose, 200 pg 1-dose, and 100 pg 3-dose groups, the geometric mean titers (GMTs) were 19040, 15776, 22189, and 18068 for RSV-A and 16562, 18370, 18665, and 19459 for RSV-B; the geometric means (GMs) (IU/mL) were 20141, 16688, 23471, and 19112 for RSV-A and 11488, 12742, 12946, and 13497 for RSV-B; and the geometric mean fold rises (GMFRs, defined as the ratio of post baseline/baseline titers) were 20.5, 22.3, 20.0, and 23.5 for RSV-A and 14.4, 11.7, 14.1, and 16.0 for RSV-B, respectively. Even more surprisingly, the percentages of participants with a greater than 4-fold increase in titer over baseline values were 100.0%, 100.0%, 100%, and 100.0% for RSV-A and 94.4%, 89.5%, 93.8%, and 89.5% for RSV-B. At Month 5 for the 50 pg, 100 pg 1- dose, and 100 pg 3-dose groups, the GMTs were 9039, 5278, and 11584 for RSV-A and 12224, 11563, and 22041 for RSV-B; the GMs (IU/mL) were 9562, 5583, and 12254 for RSV-A and 8479, 8020, and 15288 for RSV-B; and GMFRs were 9.7, 7.4, and 17.7 for RSV-A and 10.7,

7.4, and 23.0 for RSV-B. Moreover, the percentages of participants with a greater than 4-fold increase in titer over baseline values were 77.8%, 75.7%, and 100% for RSV-A and 83.3%, 68.4%, and 100% for RSV-B.

Further interim results from the trial show that a single dose of the mRNA RSV vaccine in the older adult population (65-79 years of age) boosted neutralizing antibody titers against RSV (both A and B lineages) at three evaluated dose levels (50 pg, 100 pg, and 200 pg). At Month 1, pooled results for the 50 pg, 100 pg, and 200 pg groups showed a geometric mean fold rise (GMFRs, defined as the ratio of post-baseline/baseline titers) of 14.2 for RSV-A and 10.1 for RSV-B. Unexpectedly, additional interim results from the trial show that a single dose of the mRNA RSV vaccine in the older adult population boosted neutralizing antibody titers against RSV (both A and B lineages) at five evaluated dose levels (12.5 pg, 25 pg, 50 pg, 100 pg, and 200 pg) with minimal dose response. The GMTs were lowest with the 12.5 pg dose (12,033 for RSV-A and 11,239 for RSV-B), similar for the 25 pg, 50 pg, and 100 pg doses (13,175 to 17,546 for RSV-A and 12,788 to 14,685 for RSV-B, respectively), and highest for the 200 pg dose (29,470 for RSV-A and 26,685 for RSV-B). The neutralizing antibody geometric mean (IU/mL) and GMFR followed a similar trend as GMT. The neutralizing antibody GMFR was at least 9.75 (range 9.75 to 16.86) for RSV-A and at least 5.30 (range 5.30 to 12.29) for RSV-B. RSV prefusion F protein (preF) and postfusion F protein (postF) binding antibody responses followed a similar trend as the RSV neutralizing antibody, and the response to mRNA-1345 was found to be preF-biased.

Additional interim results from the trial show that a single dose of the mRNA RSV vaccine in the population of women, aged 18-40, boosted neutralizing antibody titers against RSV (both A and B lineages) at three evaluated dose levels (12.5 pg, 25 pg, and 50 pg). The GMT at Month 1 was lowest with the 12.5 pg dose, and similar with 25 pg and 50 pg doses of vaccine. Vaccination also boosted PreF- and PostF-binding antibodies at all 3 dose levels evaluated. The preF GMT at Month 1 was the lowest with the 12.5 pg dose, and similar with 25 pg and 50 pg doses. The postF GMT at Month 1 was similar between the 12.5 pg and 25 pg groups, and highest in the 50 pg group. The PreF GMFR was greater than the PostF GMFR for each dose level, suggesting that the vaccine preferentially boosts antibodies to the PreF conformation (i.e., the response is preF-biased).

Additional patient populations enrolled in this trial children (12-59 months of age). All patients enrolled exhibit serologic evidence of prior exposure to RSV.

Respiratory syncytial virus is a common negative- sense, single-stranded RNA virus of the family Pneumovirinae present in at least two antigenic subgroups, known as Group A and Group B, primarily resulting from differences in the surface G glycoproteins. The taxonomy of RSV was recently reclassified and renamed to Family Pneumoviridae , Genus Orthopneumovirus and species Human Orthopneumovirus by the International Committee on Taxonomy of Viruses (ICTV). See J. Gen. Virol. 98:2912-2913 (2017). However, the RSV name will be used for Human Orthropneumovirus throughout to avoid confusion.

The envelope of human RSV contains three surface glycoproteins: F, G, and SH. The G and F proteins are protective antigens and targets of neutralizing antibodies. The two proteins mediate attachment with and to cells of the respiratory epithelium. F surface glycoproteins mediate coalescence of neighboring cells, resulting in the formation of syncytial cells. The F protein is a type I fusion glycoprotein that is well conserved among clinical isolates, including between the RSV-A and RSV-B antigenic subgroups. The F protein transitions between prefusion and more stable postfusion states, thereby facilitating entry into target cells. It is initially synthesized as an F0 precursor protein, which then folds into a trimer that is activated by furin cleavage into the mature prefusion protein comprising FI and F2 subunits (Bolt, et al., Virus Res., 68:25, 2000). Although targets for neutralizing monoclonal antibodies exist on the postfusion conformation of F protein, the neutralizing antibody response primarily targets the F protein prefusion conformation in people naturally infected with RSV (Magro M et al., Proc Natl Acad Sci USA 2012; 109(8): 3089-94; Ngwuta IO et al., Sci Transl Med 2015; 7(309): 309ral62). Consistent with this, RSV F protein stabilized in the prefusion conformation produces a greater neutralizing immune response in animal models than that observed with RSV F protein stabilized in the postfusion conformation (McLellan et al., Science, 342: 592-598, 2013). Thus, stabilized prefusion RSV F proteins are good candidates for inclusion in an RSV vaccine.

Stabilized prefusion RSV F proteins exist in a labile, high-energy state, as a result of mutations (e.g., stabilizing mutations) that prevent the transition of the protein into its postfusion conformation. There are several examples of stabilized prefusion forms of the RSV protein. For example,

For example, the RSV F protein designated as DS-Cavl is composed of the RSV F protein ectodomain assembled as a trimer stabilized in its prefusion native conformation with a foldon trimerization domain at the C-terminus and four (4) internal mutations, relative to wild- type RSV F protein: S155C and S290C, resulting in the formation of a disulfide bond; and two cavity-filling mutations, S190F and V207L. Other examples of stabilized prefusion RSV F proteins include SC-TM, which includes N67I, S215P, and E487Q substitutions, and a processed variant with an optimized apex, PR-DM, which includes the N67I and S215P substitutions.

FIG. 1 provides a schematic of the RSV F glycoprotein variant encoded by the mRNA vaccine of the present disclosure. This variant includes the four DS-Cavl mutations, S155C and S290C (forming a disulfide bond), and S190F and V207L (cavity-filling mutations), as well as the interprotomer mutations, A149C and Y458C. Additionally, the variant provided herein includes a deletion of amino acid residues 104-144, replaced by a GS linker, and a deletion of the cytoplasmic tail.

The RSV mRNA vaccines described herein are superior to current vaccines in several ways. For example, the lipid nanoparticle (LNP) delivery system used herein increases the efficacy of RNA vaccines in comparison to other formulations, including a protamine-based approach described in the literature. The use of this LNP delivery system enables the effective delivery of chemically-modified RNA vaccines or unmodified RNA vaccines, without requiring additional adjuvant to produce a therapeutic result (e.g., production neutralizing antibody titer).

In some embodiments, the RSV mRNA vaccines disclosed herein are superior to conventional vaccines by a factor of at least 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1,000- fold when administered intramuscularly (IM) or intradermally (ID). These results can be achieved even when significantly lower doses of the mRNA are administered in comparison with RNA doses used in other classes of lipid-based formulations.

Further, unlike self-replicating RNA vaccines, which rely on viral replication pathways to deliver enough RNA to a cell to produce an immunogenic response, the vaccines of the present disclosure do not require viral replication to produce enough protein to result in a strong immune response. Thus, the vaccines of the present disclosure do not include self-replicating RNA and do not include components necessary for viral replication.

It should be understood that the mRNA of the vaccines of the present disclosure are not naturally occurring. That is, the mRNA encoding the RSV F protein, as provided herein, does not occur in nature. It should also be understood that the vaccines described herein exclude viruses (i.e., the vaccines are not, nor do they contain, viruses).

RSV Antigens

Antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). Herein, use of the term “antigen” encompasses immunogenic/antigenic proteins and immunogenic/antigenic fragments (e.g., an immunogenic/antigenic fragment that induces (or is capable of inducing) an immune response to human RSV). It should also be understood that the term “protein” encompasses full length proteins, truncated proteins, modified proteins, and peptides.

Exemplary nucleic acid and amino acids sequences of the RSV F protein of the vaccine provided herein are provided in Table 1. In some embodiments, an RSV F protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8. For example, an RSV F protein may comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, an RSV F protein comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, an RSV F protein consists essentially of the amino acid sequence of SEQ ID NO: 8. In some embodiments, an RSV F protein consists of the amino acid sequence of SEQ ID NO: 8.

It should be understood that the mRNA F protein described herein may or may not comprise a signal sequence.

RSV Nucleic Acids

The vaccines of the present disclosure comprise a (at least one) mRNA having an open reading frame (ORF) encoding an RSV antigen. In some embodiments, the mRNA further comprises a 5' UTR, 3' UTR, a poly(A) tail and/or a 5' cap analog.

It should also be understood that the hRSV and/or hMPV mRNA vaccine of the present disclosure may include any 5' untranslated region (UTR) and/or any 3' UTR. Exemplary UTR sequences are provided in the Sequence Listing ( e.g ., SEQ ID NOs: 2-5); however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein. UTRs may also be omitted from the mRNA polynucleotides provided herein.

Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a b-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2 '-ami no- a-LNA having a 2'- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and/or chimeras and/or combinations thereof.

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

An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5' and 3' UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA polynucleotide of the present disclosure.

Variants

In some embodiments, the vaccines of the present disclosure include mRNA that encodes an RSV antigen variant. Antigen variants or other polypeptide variants refers to molecules that differ in their amino acid sequence from a wild-type, native, or reference sequence. The antigen/polypeptide variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a wild-type, native, or reference sequence.

Variant antigens/polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, enhance their expression, and/or improve their stability or PK/PD properties in a subject. Variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art and exemplary such assays are set forth in the Examples section. Similarly, PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response. The stability of protein(s) encoded by a variant nucleic acid may be measured by assaying thermal stability or stability upon urea denaturation or may be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art.

In some embodiments, a vaccine comprises an mRNA or an mRNA open reading frame that comprises a nucleotide sequence of any one of the sequences provided herein (see, e.g. , Sequence Listing and Table 1), or comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of any one of the sequences provided herein. The term “identity” refers to a relationship between the sequences of two or more polypeptides ( e.g ., antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related antigens or nucleic acids can be readily calculated by known methods. “Percent (%) identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” I. Mol. Biol. 48:443-453). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an mRNA vaccine.

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

In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that lacks a cytoplasmic tail. In some embodiments, the cytoplasmic tail comprises the C-terminal 20-30, 20-25, 15-30, 15-25, 15-20, 10-30, 10-25, 10- 20, 10-15, 5-30, 5-25, 5-20, or 5-15 amino acids of the of the hRSV F glycoprotein variant. In some embodiments, the cytoplasmic tail comprises the C-terminal 25 amino acids (e.g., CKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 25)) of the hRSV F glycoprotein. In some embodiments, the cytoplasmic tail comprises the C-terminal 20 amino acids (e.g.,

TP VTLS KDQLS GINNIAF S N (SEQ ID NO: 26)) of the hRSV F glycoprotein. In some embodiments, the cytoplasmic tail comprises the C-terminal 15 amino acids (e.g., SKDQLSGINNIAFSN (SEQ ID NO: 27)) of the hRSV F glycoprotein. In some embodiments, the cytoplasmic tail comprises the C-terminal 10 amino acids ( e.g ., SGINNIAFSN (SEQ ID NO: 28)) of the hRSV F glycoprotein.

In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that lacks a cytoplasmic tail, wherein the RSV F glycoprotein variant has at least 80%, at least 85%, at least 90%, at least 95% identity to a wild- type hRSV F glycoprotein (e.g., a wild-type hRSV F glycoprotein comprising the sequence of SEQ ID NO: 1) or a wild-type hRSV F glycoprotein that lacks a cytoplasmic tail. In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that lacks a cytoplasmic tail, wherein the RSV F glycoprotein variant has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 8. In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that comprises the sequence of SEQ ID NO: 8.

In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that that lacks a cytoplasmic tail, wherein the mRNA comprises an ORF sequence that has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 7. In some embodiments, a vaccine comprises an mRNA encoding a stabilized prefusion form of a hRSV F glycoprotein variant that lacks a cytoplasmic tail, wherein the mRNA comprises a sequence that has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 15.

In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a modification, relative to the wild-type hRSV F glycoprotein (e.g., SEQ ID NO: 1), selected from the group consisting of: a P102X substitution, a substitution of amino acids 104-144 with a linker molecule, an A149X substitution, an S155X substitution, an S190X substitution, a V207X substitution, an S290X substitution, a L373X substitution, an I379X substitution, an M447X substitution, and a Y458X substitution, wherein X is any amino acid (e.g., A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a modification, relative to the wild-type hRSV F glycoprotein, selected from the group consisting of: a P102A substitution, a substitution of amino acids 104-144 with a linker molecule, an A149C substitution, an S155C substitution, an S190F substitution, a V207L substitution, an S290C substitution, a L373R substitution, an I379V substitution, an M447V substitution, and a Y458C substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a P102A substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a substitution of amino acids 104-144 with a linker molecule. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an A149C substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an S155C substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an S190F substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a V207L substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an S290C substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an L373R substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an I379V substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises an M447V substitution. In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises a Y458C substitution.

In some embodiments, an hRSV F glycoprotein variant that lacks a cytoplasmic tail further comprises the following modifications, relative to the wild-type hRSV F glycoprotein: a P102A substitution, a substitution of amino acids 104-144 with a linker molecule, an A149C substitution, an S155C substitution, an S190F substitution, a V207L substitution, an S290C substitution, a L373R substitution, an I379V substitution, an M447V substitution, and a Y458C substitution.

In some embodiments, a vaccine further comprises an mRNA encoding an hMPV F glycoprotein variant that has at least 80%, at least 85%, at least 90%, at least 95% identity to a wild-type hMPV F glycoprotein. In some embodiments, a vaccine further comprises an RNA encoding an hMPV F glycoprotein variant has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 11. In some embodiments, a vaccine further comprises an mRNA encoding an hMPV F glycoprotein variant that comprises the sequence of SEQ ID NO: 11.

In some embodiments, a vaccine further comprises an RNA encoding an hMPV F glycoprotein variant, wherein the mRNA comprises an open reading frame sequence that has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 10. In some embodiments, a vaccine further comprises an mRNA encoding an hMPV F glycoprotein variant, wherein the mRNA comprises a sequence that has at least 80%, at least 85%, at least 90%, at least 95% identity to the sequence of SEQ ID NO: 16. Stabilizing Elements

Naturally occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5'-end (5' UTR) and/or at their 3'-end (3' UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5' UTR and the 3' UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5 '-cap and the 3 '-poly (A) tail are usually added to the transcribed (premature) mRNA during mRNA processing.

In some embodiments, a vaccine includes an mRNA having an open reading frame encoding at least one antigenic polypeptide having at least one modification, at least one 5' terminal cap, and is formulated within a lipid nanoparticle. 5 '-capping of polynucleotides may be completed concomitantly during the in vzYro-transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-0-Me-m7G(5')ppp(5') G [the ARC A cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of mRNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5’)ppp(5')G-2'-0-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl- transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0- methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived from a recombinant source.

The 3 '-poly (A) tail is typically a stretch of adenine nucleotides added to the 3 '-end of the transcribed mRNA. It can, in some instances, comprise up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.

In some embodiments, a vaccine includes a stabilizing element. Stabilizing elements may include for instance a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3’-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The mRNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem- loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5' and two nucleotides 3' relative to the stem- loop.

In some embodiments, an mRNA includes a coding region, at least one histone stem- loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein ( e.g . Luciferase, GFP, EGFP, b-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha- Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

In some embodiments, an mRNA does not include a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not include an intron.

An mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base vaccine of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

In some embodiments, an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3’UTR.

The AURES may be removed from the mRNA vaccines. Alternatively, the AURES may remain in the mRNA vaccine. Signal Peptides

In some embodiments, a vaccine comprises an mRNA having an open reading frame that encodes a signal peptide fused to the RSV antigen. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by a ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane.

A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

Signal peptides from heterologous genes (which regulate expression of genes other than RSV antigens in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure. In some embodiments, the signal peptide may comprise one of the following sequences: MDS KGS S QKGS RLLLLLV V S NLLLPQG V V G (SEQ ID NO: 18), MD WTWILFL V A A ATRVHS (SEQ ID NO: 19); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 20); MLGSNS GQR V VFTILLLL V AP A Y S (SEQ ID NO: 21); MKCLLYLAFLFIGVNCA (SEQ ID NO: 22); MWLVSLAIVTACAGA (SEQ ID NO: 23).

Fusion Proteins

In some embodiments, a vaccine of the present disclosure includes an mRNA encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together. In some embodiments, the mRNA encodes a hMPV F glycoprotein fused to a hRSV glycoprotein. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the RSV antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.

Scaffold Moieties

The mRNA vaccines as provided herein, in some embodiments, encode fusion proteins that comprise RSV antigens linked to scaffold moieties. In some embodiments, such scaffold moieties impart desired properties to an antigen encoded by a nucleic acid of the disclosure. For example, scaffold proteins may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner.

In some embodiments, the scaffold moiety is protein that can self-assemble into protein nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10- 150 nm, a highly suitable size range for optimal interactions with various cells of the immune system. In some embodiments, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold moiety is a hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of ~22 nm and which lacked nucleic acid and hence are non-inf ectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58-68). In some embodiments, the scaffold moiety is a hepatitis B core antigen (HBcAg) self-assembles into particles of 24-31 nm diameter, which resembled the viral cores obtained from HBV-infected human liver. HBcAg produced in self-assembles into two classes of differently sized nanoparticles of 300 A and 360 A diameter, corresponding to 180 or 240 protomers. In some embodiments, the RSV antigen is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the RSV antigen.

In some embodiments, bacterial protein platforms may be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.

Ferritin is a protein whose main function is intracellular iron storage. Ferritin is made of 24 subunits, each composed of a four- alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009;390:83 98). Several high- resolution stmctures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003;8:105-111; Lawson D.M. et al. Nature. 1991;349:541-544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Thus, the ferritin nanoparticle is well-suited to carry and expose antigens. Lumazine synthase (LS) is also well- suited as a nanoparticle platform for antigen display. LS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The LS monomer is 150 amino acids long and consists of beta-sheets along with tandem alpha-helices flanking its sides. A number of different quaternary structures have been reported for LS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 A diameter. Even LS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol. 2006;362:753-770).

Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, may also be used as a platform to present antigens on the surface of self-assembling nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated as a cellular compartment that encapsulates proteins such as DyP (Dye decolorizing peroxidase) and Flp (Ferritin like protein), which are involved in oxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104).

Linkers and Cleavable Peptides

In some embodiments, the mRNAs of the disclosure encode more than one polypeptide, referred to herein as fusion proteins. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker can be, for example, a cleavable linker or protease- sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6:el8556). In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain.

Cleavable linkers known in the art may be used in connection with the disclosure. Exemplary such linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750). The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the mRNAs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will likewise appreciate that other polycistronic mRNA encoding more than one antigen/polypeptide separately within the same molecule) may be suitable for use as provided herein.

Sequence Optimization

In some embodiments, an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary stmctures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally occurring or wild-type sequence ORF (e.g., a naturally occurring or wild- type mRNA sequence encoding an RSV antigen). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen).

In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally occurring or wild-type sequence ( e.g ., a naturally occurring or wild-type mRNA sequence encoding an RSV antigen).

In some embodiments, a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than an RSV antigen encoded by a non-codon-optimized sequence.

When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.

In some embodiments, a codon optimized mRNA may be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the mRNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than mRNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater mRNA stability without changing the resulting amino acid. The approach is limited to coding regions of the mRNA.

Chemically Unmodified Nucleotides

In some embodiments, an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed mRNA (e.g., A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).

Chemical Modifications

The vaccines of the present disclosure comprise, in some embodiments, an mRNA having an open reading frame encoding an RSV antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art. In some embodiments, a naturally occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177;

PCT/US 2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/036773; PCT/US2015/036759; PCT/US2015/036771; or PCT/IB2017/051367 all of which are incorporated by reference herein.

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, a modified mRNA, introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified mRNA, introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified. The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid ( e.g ., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1 -methyl-pseudouridine (ihΐy), 1 -ethyl-pseudouridine (eΐy), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5 -methoxy methyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

In some embodiments, a mRNA of the disclosure comprises 1 -methyl-pseudouridine (ml\|/) substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises 1 -methyl-pseudouridine (hiΐy) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid..

In some embodiments, a mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1 -methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1 -methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly (A) tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i. e. , any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

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

Untranslated Regions (UTRs)

The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where mRNAs are designed to encode at least one antigen of interest, the nucleic may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5' UTR and 3' UTR sequences are known and available in the art.

A 5' UTR is region of an mRNA that is directly upstream (5¹) from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5¹ UTR does not encode a protein (is non-coding). Natural 5' UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO: 29), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ’G’. 5' UTRs also have been known to form secondary structures which are involved in elongation factor binding. In some embodiments of the disclosure, a 5' UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF. In another embodiment, a 5' UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5' UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, 9012219). CMV immediate-early 1 (IE1) gene (US20140206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 30) (WO2014144196) may also be used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO/2015101414, W02015101415, WO/2015/062738, WO2015024667, WO2015024667; 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, W02015101415, WO/2015/062738), 5' UTR element derived from the 5'UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

In some embodiments, a 5' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 3 and SEQ ID NO: 4.

A 3' UTR is region of an mRNA that is directly downstream (3¹) from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3' UTR does not encode a protein (is non-coding). Natural or wild type 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) (SEQ ID NO: 31) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.

Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of nucleic acids ( e.g ., mRNA) of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, and/or 7 days post-transfection.

3' UTRs may be heterologous or synthetic. With respect to 3' UTRs, globin UTRs, including Xenopus b-globin UTRs and human b-globin UTRs are known in the art (8278063, 9012219, US20110086907). A nucleic acid (e.g., mRNA) encoding a modified b-globin with enhanced stability in some cell types by cloning two sequential human b-globin 3' UTRs head to tail has been developed and is well known in the art (US2012/0195936, WO2014/071963). In addition, a2-globin, a 1 -globin, UTRs and mutants thereof are also known in the art (W02015101415, WO2015024667). Other 3' UTRs described in the mRNA in the non-patent literature include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other exemplary 3' UTRs include that of bovine or human growth hormone (wild type or modified) (WO2013/185069, US20140206753, WO2014152774), rabbit b globin and hepatitis B vims (HBY), a-globin 3' UTR and Viral VEEV 3' UTR sequences are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014144196) is used. In some embodiments, 3' UTRs of human and mouse ribosomal protein are used. Other examples include rps93' UTR (W02015101414), FIG4 (W02015101415), and human albumin 7 (W02015101415).

In some embodiments, a 3¹ UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5.

Those of ordinary skill in the art will understand that 5' UTRs that are heterologous or synthetic may be used with any desired 3' UTR sequence. For example, a heterologous 5' UTR may be used with a synthetic 3' UTR with a heterologous 3' UTR.

Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels. Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5 UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5 UTRs described in US Patent Application Publication No. 20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety.

It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' UTR or 5' UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5' UTR or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.

It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

The untranslated region may also include translation enhancer elements (TEE). As a non limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art.

In vitro Transcription of mRNA cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of mRNA is known in the art and is described in International Publication WO/2014/152027, which is incorporated by reference herein in its entirety.

In some embodiments, the mRNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the mRNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a mRNA, for example, but not limited to RSV mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5' to and operably linked to the gene of interest.

In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a poly(A) tail. The particular nucleic acid sequence and length of an in vitro transcription template will depend on the mRNA encoded by the template.

A “5' untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. When mRNA transcripts are being generated, the 5 ' UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.

A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide. An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,

220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo ) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.

In some embodiments, a nucleic acid includes 200 to 3,000 nucleotides. For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).

An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.

The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.

Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase.

In some embodiments, the mRNA transcript is capped via enzymatic capping. In some embodiments, the mRNA comprises 5' terminal cap, for example, 7mG(5')ppp(5')NlmpNp.

Chemical Synthesis

Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.

Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.

Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone.

Ligation of Nucleic Acid Regions or Subregions

Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5' and 3' ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5' phosphoryl group and another with a free 3' hydroxyl group, serve as substrates for a DNA ligase.

Purification

Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

Quantification

In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheo alveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

Assays may be performed using antigen- specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications.

In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred. Degradation of the nucleic acid may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

Lipid Nanoparticles (LNPs)

In some embodiments, the mRNA of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable amino lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551 ; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.

Vaccines of the present disclosure are typically formulated in lipid nanoparticles. The vaccines can be made, for example, using mixing processes such as microfluidics and T- junction mixing of two fluid streams, one of which contains the mRNA and the other has the lipid components. In some embodiments, the vaccines are prepared by combining an ionizable amino lipid, a phospholipid (such as DOPE or DSPC), a PEG lipid (such as 1,2-dimyristoyl-OT- glycerol methoxypoly ethylene glycol, also known as PEG-DMG), and a structural lipid (such as cholesterol) in an alcohol (e.g., ethanol). The lipids may be combined to yield desired molar ratios and diluted with water and alcohol (e.g., ethanol) to a final lipid concentration of between about 5.5 mM and about 25 mM, for example.

Vaccines including mRNA and a lipid component may be prepared, for example, by combining a lipid solution with an mRNA solution at lipid component to mRNA wt:wt ratios of between about 5:1 and about 50:1. The lipid solution may be rapidly injected using a microfluidic based system (e.g., NanoAssemblr) at flow rates between about 10 ml/min and about 18 ml/min, for example, into the mRNA solution to produce a suspension (e.g., with a water to alcohol ratio between about 1:1 and about 4:1).

Vaccines can be processed by dialysis to remove the alcohol (e.g., ethanol) and achieve buffer exchange. Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, for example, at volumes greater than that of the primary product (e.g., using Slide- A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL)) with a molecular weight cutoff of 10 kD, for example. The forgoing exemplary method induces nanoprecipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nanoprecipitation.

In some embodiments, the lipid nanoparticle comprises at least one ionizable amino (cationic) lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable amino lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% non- cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25- 35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG- modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5- 5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

In some embodiments, an ionizable amino lipid of the disclosure comprises a compound of Formula (I):

or a salt or isomer thereof, wherein: Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)_nQ, -(CH2)_nCHQR, -CHQR, -CQ(R)2, and unsubstituted Ci-₆ alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -0(CH₂)_nN(R)₂, -C(0)0R, -0C(0)R, -CX3, -CX₂H, -CXH₂, -CN, -N(R)₂, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)RS, -0(CH₂)nOR, -N(R)C(=NRg)N(R)2, -N(R)C(=CHR₉)N(R)₂, -0C(0)N(R)¾ -N(R)C(0)0R, -N(0R)C(0)R, -N(0R)S(0)₂R, -N(0R)C(0)0R, -N(0R)C(0)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(0)N(R)0R, and -C(R)N(R)₂C(0)0R, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

-N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0)₂-, -S-S-, an aryl group, and a heteroaryl group;

R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

Rg is selected from the group consisting of C3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO2, Ci-₆ alkyl, -OR, -S(0)₂R, -S(0)₂N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, a subset of compounds of Formula (I) includes those in which when R₄ is -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, or -CQ(R)₂, then (i) Q is not -N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includes those in which

Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C_2-i4 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

R4 is selected from the group consisting of a C3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted Ci-₆ alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)_nN(R)₂, -C(0)OR, -OC(0)R, -CX3, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)₂, -N(R)C(S)N(R)₂, -CRN(R)₂C(0)OR, -N(R)R_S, -0(CH₂)„OR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(0)N(R)₂, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(OR)C(0)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(0)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, mono- or di-alkylamino, and Ci-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C₂-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C1-3 alkyl, C₂-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R7 is selected from the group consisting of C1-3 alkyl, C₂-3 alkenyl, and H;

Rs is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, N0₂, Ci-₆ alkyl, -OR, -S(0)₂R, -S(0)₂N(R)₂, C_2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Cws alkyl, C_2-is alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

R4 is selected from the group consisting of a C3-6 carbocycle, -(CFh)_nQ, -(CFhl_nCHQR, -CHQR, -CQ(R)2, and unsubstituted Ci-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)nN(R)₂, -C(0)OR, -OC(0)R, -CX3, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)₂C(0)OR, -N(R)RS, -0(CH₂)„OR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -0C(0)N(R)2, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(0R)C(0)N(R)2, -N(0R)C(S)N(R)₂, -N(0R)C(=NR₉)N(R)₂, -N(0R)C(=CHR₉)N(R)2, -C(=NR₉)R, -C(0)N(R)OR, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is -(CFh)_nQ in which n is 1 or 2, or (ii) R4 is -(CFh)_nCFIQR in which n is 1, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

-N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0)₂-, -S-S-, an aryl group, and a heteroaryl group; R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

R9 is selected from the group consisting of H, CN, NO2, Ci-₆ alkyl, -OR, -S(0)2R, -S(0)₂N(R)₂, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Cws alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C_2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

Ri is selected from the group consisting of C_5-30 alkyl, C_5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R4 is selected from the group consisting of a C3-6 carbocycle, -(CFh)nQ, -(CFhlnCHQR, -CHQR, -CQ(R)2, and unsubstituted Ci-₆ alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)nN(R)₂, -C(0)OR, -OC(0)R, -CX₃, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)₂, -CRN(R)₂C(0)OR, -N(R)RS, -0(CH₂)nOR, -N(R)C(=NR₉)N(R)2, -N(R)C(=CHR₉)N(R)2, -0C(0)N(R)2, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(0R)C(0)N(R)2, -N(0R)C(S)N(R)₂, -N(0R)C(=NR₉)N(R)2, -N(0R)C(=CHR₉)N(R)2, -C(=NR₉)R, -C(0)N(R)OR, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; each Rs is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R7 is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, Ci-₆ alkyl, -OR, -S(0)₂R, -S(0)₂N(R)₂, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-₁₂ alkyl and C_2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R₂ and R₃ are independently selected from the group consisting of H, C_2-14 alkyl, C_2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R₄ is -(CH₂)_IIQ or -(CH2)nCHQR, where Q is -N(R)₂, and n is selected from 3, 4, and 5; each Rs is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Cws alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-₁₂ alkyl and Ci-₁₂ alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R2 and R3 are independently selected from the group consisting of Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R₄ is selected from the group consisting of -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, and -CQ(R)₂, where Q is -N(R)₂, and n is selected from 1, 2, 3, 4, and 5; each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and

H; each R’ is independently selected from the group consisting of C_HS alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula

(IA):

(IA), or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’ ; R4 is unsubstituted C1-3 alkyl, or -(CFh)_nQ, in which Q is OH, -NHC(S)N(R)₂, -NHC(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)Rg, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -0C(0)N(R)₂, -N(R)C(0)0R, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-, -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C_2-i4 alkenyl.

(P):

(II) or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R4 is unsubstituted C1-3 alkyl, or -(CH2)_nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)_¾ -NHC(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)Rs, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -0C(0)N(R)₂, -N(R)C(0)0R, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-, -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (Ila), (lib), (lie), or (He):

or a salt or isomer thereof, wherein R4 is as described herein.

(lid):

(lid), or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R’ , R”, and R2 through R₆ are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having stmcture:

In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure:

In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG. In some embodiments, a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In some embodiments, a LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mole percent ionizable amino lipid.

In some embodiments, the lipid nanoparticle comprises 5 - 15 mole percent DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mole percent DSPC.

In some embodiments, the lipid nanoparticle comprises 35 - 40 mole percent cholesterol. For example, the lipid nanoparticle may comprise 35, 36, 37, 38, 39, or 40 mole percent cholesterol.

In some embodiments, the lipid nanoparticle comprises 1 - 2 mole percent DMG-PEG. For example, the lipid nanoparticle may comprise 1, 1.5, or 2 mole percent DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 50 mole percent ionizable amino lipid, 10 mole percent DSPC, 38.5 mole percent cholesterol, and 1.5 mole percent DMG-PEG.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of from about 2: 1 to about 30:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 6:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 3:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the mRNA of from about 10:1 to about 100:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the mRNA of about 20: 1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the mRNA of about 10:1.

In some embodiments, a LNP of the disclosure has a mean diameter from about 50 nm to about 150 nm.

In some embodiments, a LNP of the disclosure has a mean diameter from about 70 nm to about 120 nm.

Combination Vaccines

The vaccines, as provided herein, may include an mRNA or multiple mRNAs encoding two or more antigens of the same or different viral strains. Also provided herein are combination vaccines that include mRNA encoding one or more hRSV antigen(s) and one or more antigen(s) of a different organism, such as hMPV (see Table 1 for exemplary sequences). Thus, the vaccines of the present disclosure may be combination vaccines that target one or more antigens of the same strain/species, or one or more antigens of different strains/species, e.g., antigens which induce immunity to organisms which are found in the same geographic areas where the risk of RSV infection is high or organisms to which an individual is likely to be exposed to when exposed to a respiratory virus.

Vaccine Formulations and Dosing Schedules

Provided herein, in some aspects, are vaccines, methods, kits and reagents for prevention or treatment of RSV infection in humans. The vaccines provided herein can be used as prophylactic or therapeutic agents to prevent or treat RSV infection or disease progression. A vaccine may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of mRNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

A vaccine may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. mRNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines.

In some embodiments, the RSV vaccine containing mRNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the mRNA is translated and expressed in vivo to produce an RSV F protein, which then stimulates an immune response in the subject. In some embodiments, an mRNA vaccine is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific (e.g., RSV-specific) immune response.

Formulation/Dose

A vaccine comprising mRNA and LNP may or may not further comprise one or more other components. For example, a vaccine may include other components including, but not limited to, adjuvants and/or excipients. In some embodiments, a vaccine does not include an adjuvant (they are adjuvant free).

Vaccines may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccines, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

Formulations of the vaccines described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient ( e.g ., mRNA) into association with an excipient and/or one or more other accessory ingredients (e.g., lipid nanoparticle components described herein), and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient (i.e., mRNA), the pharmaceutically acceptable excipient (e.g., LNP components), and/or any additional ingredients in a vaccine in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the vaccine is to be administered. By way of example, the vaccine may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) mRNA.

In some embodiments, an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo: and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the mRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

An effective amount (e.g., effective dose) prevents infection by the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. An effective amount (e.g., effective dose) of a vaccine (e.g., comprising mRNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the mRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a vaccine provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount of the vaccine containing mRNA having at least one chemical modification is more efficient than a vaccine containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the mRNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

An effective amount of the vaccine may depend on the age of the subject being vaccinated. In some embodiments, the subject is pediatric subject having an age of 2- 60 months old. For example, a pediatric subject may be 6-60, or 12-60 (e.g., 12-59) months old. In other embodiments, the subject is a young adult having an age of 18-50 (e.g., 18-49) years old. In some embodiments, the subject is a woman having an age of 18-40. In yet other embodiments, the subject is a 60-80 year old subject. For example, an older adult subject may be 60-70, 60-75, or 65-80 (e.g., 65-79) years old.

The effective amount of the mRNA, as provided herein, ranges from 12.5 pg to 200 pg, in depending at least in part on the age of the subject being vaccinated and the vaccine dosing schedule. In some embodiments, the vaccine is administered as a single dose. For example, the vaccine may be administered as a single dose of 12.5 pg, 25 pg, 30 pg, 50 pg, 100 pg, or 200 pg. In other some embodiments, one or more booster dose(s) of the vaccine is administered. For example, a booster dose of the vaccine may be a dose of 12.5 pg, 25 pg, 30 pg, 50 pg, 100 pg, or 200 pg. Thus, in some embodiments, a second dose is administered. In some embodiments, a third dose is also administered. In some embodiments, the third dose of the vaccine may be a dose of 12.5 pg, 25 pg, 30 pg, 50 pg, 100 pg, or 200 pg.

It should be understood that a “total dose” refers to the total amount of mRNA administered, either as a single vaccination or cumulative of an initial dose and any booster dose(s). Thus, if a vaccine is administered as a single dose of 50 pg of mRNA, then the total dose is 50 pg of mRNA. If a vaccine is administered as an initial dose of 50 pg of mRNA then later as a booster dose of 50 pg of mRNA, then the total dose is 100 pg of mRNA. In some embodiments, a total dose of 12.5 pg, 30 pg, 50 pg, 100 pg, or 200 pg of mRNA is administered to a subject. In other embodiments, a total dose of 25 pg, 50 pg, 60 pg, 100 pg, 200 pg, or 400 pg of mRNA is administered to a subject. In other embodiments, a total dose of 37.5 pg, 90 pg, 150 pg, or 300 pg of mRNA is administered to a subject.

In some embodiments, the effective amount is a total dose of 90 pg (e.g., administered as three 30 pg doses in pediatric patients, 12-59 months of age). In some embodiments, the effective amount is a total dose of 12.5 pg (e.g., administered as a single 12.5 pg dose in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 25 pg (e.g., administered as a single 25 pg dose or two 12.5 pg doses in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 50 mg (e.g., administered as a single 50 pg dose or two 25 pg doses in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 100 pg (e.g., administered as a single 100 pg dose or two 50 pg doses in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 200 pg (e.g., administered as a single 200 pg dose or two 100 pg doses in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 400 pg (e.g., administered as two 200 pg doses in adult patients, 18-79 years of age). In some embodiments, the effective amount is a total dose of 300 pg (e.g., administered as three 100 pg doses in adult patients, 18-79 years of age).

In some embodiments, a vaccine may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art. In preferred embodiments, the vaccine is administered intramuscularly.

The RNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

Dosing Schedule

Prophylactic protection from RSV infection can be achieved following administration of an mRNA vaccine of the present disclosure. Vaccines can be admini tered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster). It is also possible to administer a vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

A booster dose refers to an extra administration of the vaccine. A booster (or booster dose or booster vaccine) may be given after an earlier administration of the vaccine. Any two doses of an mRNA vaccine (e.g., an initial dose and a booster dose, or a booster dose and a second booster dose) may be administered, for example, at least one month (-28, 29, 30, or 31 days) apart, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart. In some embodiments, a booster dose is administered yearly.

In some embodiments, a booster dose (e.g., a first, second or third booster dose) is administered at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months after the initial dose or after another booster dose. In some embodiments, a booster dose is administered one month, two months, three months, four months, five months, or six months after the initial dose or after another booster dose. In some embodiments, a second booster dose is admini tered at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months after a first booster dose. In some embodiments, a second booster dose is administered one month, two months, three months, four months, five months, or six months after a first booster dose. In some embodiments, a booster dose is administered one year or at least one year after the initial dose. In some embodiments, a second booster dose is administered one year or at least one year after a first booster dose.

A method of eliciting an immune response in a subject against an RSV antigen (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, the method involves administering to the subject a vaccine comprising a mRNA having an open reading frame encoding an RSV F protein, thereby inducing in the subject an immune response specific to the RSV F protein, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen (e.g., RSV F protein).

An anti-antigen antibody is a serum antibody the binds specifically to the antigen (e.g., RSV F protein). In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the RSV or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the RSV or an unvaccinated subject.

A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the RSV at 2 times to 100 times the dosage level relative to the vaccine. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to a vaccine of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to a vaccine of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to a vaccine of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to a vaccine of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to a vaccine of the present disclosure.

In some embodiments, the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactic ally effective dose of a traditional vaccine against the respiratory virus. In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

In some embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake or reduce RSV transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of the mRNA vaccines, wherein the mRNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an RSV antigen). “An effective amount” is a dose of the mRNA effective to produce an antigen- specific immune response. Also provided herein are methods of inducing an antigen- specific immune response in a subject.

As used herein, an immune response to a vaccine or LNP of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more)

RSV protein(s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T- lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T- cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells.

In some embodiments, the antigen- specific immune response is characterized by measuring an anti-RSV antigen antibody titer produced in a subject administered a vaccine as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen ( e.g ., an anti-RSV F glycoprotein, including an anti-RSV prefusion F glycoprotein or an anti-RSV postfusion F glycoprotein) or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by an mRNA vaccine.

In some embodiments, an anti-RSV antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-RSV antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-RSV antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti- RSV antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-RSV antigen antibody titer produced in a subject may be increased by 1- 1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-RSV antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-RSV antigen n antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control.

In some embodiments, the anti-RSV antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-RSV antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the anti-RSV antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7,

5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

In some embodiments, an antigen- specific immune response is measured as a ratio of geometric mean titer (GMT), referred to as a geometric mean ratio (GMR), of serum neutralizing antibody titers to hRSV. A geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data.

In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following the first dose of the vaccine, is 15,000-20,000 or 15,000-25,000, 10,000-30,000, or 5,000-12,000 e.g., 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,5000, 26,000,

26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, and 30,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 8,000-12,000 (e.g., approximately 8,000 or 12,000). In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 11,000-17,500 (e.g., approximately 11,500 or 17,500). In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 50 pg first dose of the vaccine, is approximately 11,000-20,000 (e.g., approximately 11,500, 13,000, or 19,000). In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 15,000-18,000, e.g., 15,000, 16,000, or 18,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 200 pg first dose of the vaccine, is approximately 20,000-30,000 (e.g., approximately 22,000 or 29,500).

In some embodiments, the geometric mean (GM) (IU/mL) of neutralizing antibody induced against RSV-A in a subject at Day 29, following the first dose of the vaccine, is 15,000-

25,000, or 10,000-35,000, e.g., 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,5000, 26,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, 30,000, 30,500,

31,000, 31,500, 32,000, 33,000, 34,000, and 35,000. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 12.5 mg first dose of the vaccine, is approximately 12,500. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 18,500. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 50 pg first dose of the vaccine, is approximately 13,000-21,000 (e.g., approximately 14,000 or 20,000). In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 16,000-20,000, e.g., approximately 16,000, 16,500, or 19,000. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 200 pg first dose of the vaccine, is approximately 22,000-32,000 (e.g., approximately 23,500 or 31,000).

In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following the first dose of the vaccine, is 15,000-20,000 or 10,000-20,000, e.g., 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000,

15.500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 10,000-11,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 14,500-17,000 (e.g., approximately 14,500 or 16,500. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 50 pg first dose of the vaccine, is approximately 13,000-17,000 (e.g., approximately 13,500 or 16,500). In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 12,000-20,000 or 18,000-19,500, e.g., approximately

12.500, 18,500, or 19,500. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 200 pg first dose of the vaccine, is approximately 18,000-27,000 (e.g., approximately 18,500 or 26,500).

In some embodiments, the GM (IU/mL) of neutralizing antibody induced against RSV-B in a subject at Day 29, following the first dose of the vaccine, is 7,000-20,000 or 11,000-15,000, e.g., 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,00015,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 7,500. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 10,000. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 50 m first dose of the vaccine, is approximately 9,000-12,000 (e.g., approximately 9,500 or 11,500). In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 8,000-14,000 or 12,000-14,000, e.g., approximately 8,500, or 12,500, or 13,500.

In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 200 pg first dose of the vaccine, is approximately 12,000-19,000 (e.g., approximately 13,000 or 18,500).

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against RSV-A in a subject at Day 29, following the first dose of the vaccine, is 9-35 or 15-30 or 20-24, e.g., 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. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 9.5 or 10. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 11 or 12.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 50 pg first dose of the vaccine, is approximately 11-21 (e.g., approximately 11, 12, or 20). In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 12-24 or 20-24, e.g., approximately 13.5 or 21 or 23.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 29, following a 200 pg first dose of the vaccine, is approximately 15-21 (e.g., approximately 17 or 20).

In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following the first dose of the vaccine, is 3-20 or 10-20, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 5.5-8 (e.g., approximately 5.5 or 7.5). In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 25 pg first dose of the vaccine, is approximately 6.5 or 7. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 50 pg first dose of the vaccine, is approximately 8-15 (e.g., approximately 9 or 14.5). In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 100 pg first dose of the vaccine, is approximately 8-16 or 11-16, e.g., approximately 9 or 11.5 or 16. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 29, following a 200 mg first dose of the vaccine, is approximately 11-15 (e.g., approximately 12 or 14.0).

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer against RSV-A by Month 1 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer against RSV-B by Month 1 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer against RSV-A by Month 1 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer against RSV-B by Month 1 following vaccination, relative to baseline.

In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following the first dose of the vaccine, is 55,000-120,000, e.g., 55,000, 56,000, 57,000, 58,000, 59,000, 60,000, 61,000, 62,000, 63,000, 64,000, 65,000, 66,000, 67,000, 68,000, 69,000, 70,000, 71,000, 72,000, 73,000, 74,000, 75,000, 76,000, 77,000, 78,000, 79,000, 80,000, 81,000, 82,000, 83,000, 84,000, 85,000, 86,000, 87,000, 88,000, 89,000, 90,000, 91,000, 92,000, 93,000, 94,000, 95,000, 96,000, 97,000, 98,000, 99,000, 100,000, 101,000, 102,000, 103,000, 104,000, 105,000, 106,000, 107,000, 108,000, 109,000, 110,000, 111,000, 112,000, 113,000, 114,000, 115,000, 116,000, 117,000, 118,000, 119,000, or 120,000.

In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 50,000.

In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 25 pg first dose of the vaccine, is approximately 58,000- 67,000. In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 50 pg first dose of the vaccine, is approximately 60,000-69,000. In some embodiments, the GMT binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 100 pg first dose of the vaccine, is approximately 77,000. In some embodiments, the GMT of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 200 pg first dose of the vaccine, is approximately 112,000.

In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following the first dose of the vaccine, is 40,000-100,000, e.g., 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000, 56,000, 57,000, 58,000, 59,000, 60,000, 61,000, 62,000,

63,000, 64,000, 65,000, 66,000, 67,000, 68,000, 69,000, 70,000, 71,000, 72,000, 73,000, 74,000,

75,000, 76,000, 77,000, 78,000, 79,000, 80,000, 81,000, 82,000, 83,000, 84,000, 85,000, 86,000,

87,000, 88,000, 89,000, 90,000, 91,000, 92,000, 93,000, 94,000, 95,000, 96,000, 97,000, 98,000,

99,000, or 100,000. In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 48,000-58,000. In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 25 pg first dose of the vaccine, is approximately 58,000-84,000. In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 50 pg first dose of the vaccine, is approximately 68,000-72,500. In some embodiments, the GMT binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 100 pg first dose of the vaccine, is approximately 80,000. In some embodiments, the GMT of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 200 pg first dose of the vaccine, is approximately 96,000.

In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following the first dose of the vaccine, is 5-15, e.g., 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 7.0-8.5. In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 25 pg first dose of the vaccine, is approximately 7.2-9.5. In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 50 pg first dose of the vaccine, is approximately 7.6-9.8. In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 100 pg first dose of the vaccine, is approximately 11.0. In some embodiments, the GMFR of binding antibody titers induced against RSV prefusion F glycoprotein at Day 29, following a 200 pg first dose of the vaccine, is approximately 12.0.

In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following the first dose of the vaccine, is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 12.5 pg first dose of the vaccine, is approximately 4.5-7.8. In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 25 pg first dose of the vaccine, is approximately 6.0-7.2. In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 50 pg first dose of the vaccine, is approximately 6.0-7.6. In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 100 pg first dose of the vaccine, is approximately 7.5. In some embodiments, the GMFR of binding antibody titers induced against RSV postfusion F glycoprotein at Day 29, following a 200 pg first dose of the vaccine, is approximately 9.0.

In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 141, following the first dose (and/or second dose) of the vaccine, is 5,000-15,000, e.g., 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, and 15,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 50 pg first dose of the vaccine, is approximately 9,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 5,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-A in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 11,500.

In some embodiments, the GM (IU/mL) of neutralizing antibody induced against RSV-A in a subject at Day 141, following the first dose (and/or second dose) of the vaccine, is 5,000- 15,000, e.g., 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, and 15,000. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 50 pg first dose of the vaccine, is approximately 9,000. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 5,500. In some embodiments, the GM of neutralizing antibody induced against RSV-A in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 12,000.

In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 141, following the first dose (and/or second dose) of the vaccine, is 10,000- 25,000, e.g., 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, or 25,000. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 50 mg first dose of the vaccine, is approximately 12,500. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 11,500. In some embodiments, the GMT of neutralizing antibody induced against RSV-B in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 22,000.

In some embodiments, the GM (IU/mL) of neutralizing antibody induced against RSV-B in a subject at Day 141, following the first dose (and/or second dose) of the vaccine, is 7,000- 16,000, e.g., 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, and 16,000. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 50 pg first dose of the vaccine, is approximately 8,500. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 8,000. In some embodiments, the GM of neutralizing antibody induced against RSV-B in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 15,000.

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against RSV-A in a subject at Day 141, following the first dose (and/or second) of the vaccine, is 5-20 or 7-18, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, or 20. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 50 pg first dose of the vaccine, is approximately 9.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 7.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-A in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 17.5.

In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 141, following the first dose (and/or second) of the vaccine, is 8-25, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 50 pg first dose of the vaccine, is approximately 10.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 141, following a 100 pg first dose of the vaccine, is approximately 7.5. In some embodiments, the GMFR of neutralizing antibody induced against RSV-B in a subject at Day 141, following two 100 pg doses of the vaccine, is approximately 23.0. In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer against RSV-A by Month 5 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer against RSV-B by Month 5 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer against RSV-A by Month 5 following vaccination, relative to baseline.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer against RSV-B by Month 5 following vaccination, relative to baseline.

A control, in some embodiments, is an anti-RSV antigen antibody titer produced in a subject who has not been administered an mRNA vaccine. In some embodiments, a control is an anti-RSV antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine. Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism.

In some embodiments, the ability of an mRNA vaccine to be effective is measured in a murine model. For example, a vaccine may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies may also be used to assess the efficacy of a vaccine of the present disclosure. For example, a vaccine may be administered to a murine model, the murine model challenged with vims, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).

In some embodiments, an effective amount of an mRNA vaccine is a dose that is reduced compared to the standard of care dose of a recombinant protein vaccine. A “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent RSV infection or a related condition, while following the standard of care guideline for treating or preventing RSV infection or a related condition.

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

Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1;201(11): 1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:

Efficacy - (ARU - ARV)/ARU x 100; and

Efficacy = (1-RR) x 100.

Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 lun 1;201(11): 1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine -related factors that influence the ‘real- world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination:

Effectiveness = (1 - OR) x 100.

In some embodiments, efficacy of the vaccine is at least 60% relative to unvaccinated control subjects. For example, efficacy of the vaccine may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects. Sterilizing Immunity. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of a vaccine of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, the effective amount of a vaccine of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, the effective amount of a vaccine of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control. For example, the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.

Detectable Antigen. In some embodiments, the effective amount of a vaccine of the present disclosure is sufficient to produce detectable levels of RSV antigen as measured in serum of the subject at 1-72 hours post administration.

Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-RSV antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

In some embodiments, the effective amount of a vaccine of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the RSV antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 1,000- 5,000 neutralizing antibody titer produced by neutralizing antibody against the RSV antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the RSV antigen as measured in serum of the subject at 1-72 hours post administration.

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

In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL. In some embodiments, an anti-RSV antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, an anti-RSV antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control.

In some embodiments, an anti-RSV antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, an anti-RSV antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control.

In some embodiments, a geometric mean, which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.

A control may be, for example, an unvaccinated subject, or a subject administered a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit vaccine.

Additional Embodiments

Additional embodiments of the present disclosure are encompassed by the following numbered paragraphs.

1. A method comprising: administering to a human subject a dose of a vaccine comprising a lipid nanoparticle that comprises a messenger ribonucleic acid (mRNA), wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human respiratory syncytial vims (RSV) F glycoprotein that comprises a deletion of a cytoplasmic tail domain, the dose of the vaccine comprises 12.5 pg to 200 pg of the mRNA, and the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 3-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

2. The method of paragraph 1, wherein the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 4-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

3. The method of any one of the preceding paragraphs, wherein the geometric mean titer (GMT) of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 29 post administration of the dose is at least 15,000.

4. The method of paragraph 3, wherein the geometric mean titer (GMT) of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 29 post administration of the dose is 15,000-20,000. 5. The method of any one of the preceding paragraphs, wherein the geometric mean fold rise (GMFR) of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is 15-25 and/or the GMFR of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is 10-20.

6. The method of any one of paragraphs 1-5, wherein the dose comprises 12.5 pg of the mRNA.

7. The method of any one of paragraphs 1-5, wherein the dose comprises 30 pg of the mRNA.

8. The method of any one of paragraphs 1-5, wherein the dose comprises 50 pg of the mRNA.

9. The method of any one of paragraphs 1-5, wherein the dose comprises 100 pg of the mRNA.

10. The method of any one of paragraphs 1-5, wherein the dose comprises 200 pg of the mRNA.

11. The method of any one of the preceding paragraphs, further comprising administering to the subject a second dose of the vaccine.

12. The method of paragraph 11, wherein the second dose of the vaccine is administered at least 2 months after the first dose is administered.

13. The method of paragraph 11 or 12, further comprising administering to the subject a third dose of the vaccine.

14. The method of paragraph 13, wherein the third dose of the vaccine is administered at least 2 months after the second dose is administered.

15. The method of paragraph 11 or 12, wherein the second dose comprises 12.5 pg of the mRNA.

16. The method of paragraph 11 or 12, wherein the second dose comprises 30 pg of the mRNA.

17. The method of paragraph 11 or 12, wherein the second dose comprises 50 pg of the mRNA.

18. The method of paragraph 11 or 12, wherein the second dose comprises 100 pg of the mRNA.

19. The method of paragraph 11 or 12, wherein the second dose comprises 200 pg of the mRNA. 20. The method of paragraph 13 or 14, wherein each of the second dose and the third dose comprises 12.5 pg of the mRNA.

21. The method of paragraph 13 or 14, wherein each of the second dose and the third dose comprises 30 pg of the mRNA.

22. The method of paragraph 13 or 14, wherein each of the second dose and the third dose comprises 50 pg of the mRNA.

23. The method of paragraph 10 or 11, wherein each of the second dose and the third dose comprises 100 pg of the mRNA.

24. The method of paragraph 13 or 14, wherein each of the second dose and the third dose comprises 100 pg of the mRNA.

25. The method of any one of the preceding paragraphs, wherein the subject is a pediatric subject under the age of 5 years (e.g., 2 to 59, 6 to 59 or 12 to 59 months).

26. The method of any one of the preceding paragraphs, wherein the subject is an 18- 49 year old subject.

27. The method of any one of the preceding paragraphs, wherein the subject is an elderly subject at least 50, at least 55, at least 60 (e.g., 60-79), or at least 65 years of age.

28. The method of any one of the preceding paragraphs, wherein the F glycoprotein encoded by the ORF of the mRNA is membrane anchored.

29. The method of any one of the preceding paragraphs, wherein the F glycoprotein encoded by the ORF of the mRNA comprises interprotomer disulfide stabilizing mutations.

30. The method of any one of the preceding paragraphs, wherein the F glycoprotein encoded by the ORF of the mRNA has at least 95% identity to the amino acid sequence of SEQ ID NO: 8.

31. The method of paragraph 30, wherein the F glycoprotein encoded by the open reading frame of the mRNA comprises the amino acid sequence of SEQ ID NO: 8.

32. The method of paragraph 31, wherein the F glycoprotein encoded by the open reading frame of the mRNA consists of the amino acid sequence of SEQ ID NO: 8.

33. The method of any one of the preceding paragraphs, wherein the mRNA comprises a 5’ 7mG(5’)ppp(5’)NlmpNp cap and a 3’ polyA tail.

34. The method of any one of the preceding paragraphs, wherein the mRNA comprises a 1-methylpseudourine chemical modification.

35. The method of any one of the preceding paragraphs, wherein the lipid nanoparticle comprises 45-55 mol% ionizable amino lipid, 15-20 mol% neutral lipid, 35-45 mol% cholesterol, and 0.5-5 mol% PEG-modified lipid. 36. The method of any one of the preceding paragraphs, wherein the ionizable amino lipid is Compound I:

(Compound I).

37. The method of any one of paragraphs 1-36, wherein the lipid nanoparticle comprises 50 mol% ionizable amino lipid.

38. The method of any one of paragraphs 1-36, wherein the lipid nanoparticle comprises 49 mol% ionizable amino lipid.

39. The method of any one of paragraphs 1-36, wherein the lipid nanoparticle comprises 48 mol% ionizable amino lipid. 40. The method of any one of the preceding paragraphs, wherein the vaccine is administered intramuscularly.

41. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and the dose comprises 50 pg of the mRNA.

42. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I: (Compound I), the human subject is 18-49 years old, and the dose comprises 100 pg of the mRNA.

43. A method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and each of the three doses comprises 100 pg of the mRNA.

44. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and the dose comprises 200 pg of the mRNA. 45. A method comprising: administering to a human subject intramuscularly two doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

the human subject is 65-79 years old, and each of the two doses comprises 50 pg of the mRNA.

46. A method comprising: administering to a human subject intramuscularly two doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 65-79 years old, and each of the two doses comprises 100 pg of the mRNA.

47. A method comprising: administering to a human subject intramuscularly two doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 65-79 years old, and each of the two doses comprises 200 pg of the mRNA.

48. A method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 12-59 months old, and each of the three doses comprises 30 pg of the mRNA.

49. A method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 12-59 months old, and each of the three doses comprises 100 pg of the mRNA.

EXAMPLES

Example 1 - Phase I Study

This is a Phase 1, randomized, observer-blind, placebo-controlled, dose-ranging trial to assess the safety of the mRNA vaccine (RSV vaccine encoding SEQ ID NO: 8 and formulated in a lipid nanoparticle) in younger adults (18-49 years of age), older adults (65 to 79 years of age), and children (12-59 months of age) with serologic evidence of prior exposure to RSV. The secondary purpose of the study is to assess the humoral immune responses in each of these three populations.

Healthy younger adult participants ( 18 to 49 years of age) were enrolled in 4 younger adult cohorts and randomized via Interactive Response Technology (IRT). In the younger adult cohorts (Cohorts 1, 2, and 4), 3 escalating dose levels administered in a single dose of the mRNA vaccine (50 pg, 100 pg, or 200 pg) or placebo were evaluated. In addition, 1 younger adult cohort (Cohort 3) administered in a 3 dose series of mRNA 1345 (100 pg) or placebo was evaluated.

The primary objectives of the study were: to evaluate the tolerability and reactogenicity of a single injection of 1 of 3 dose levels of mRNA 1345 in younger and older adults; to evaluate the tolerability and reactogenicity of 3 injections of the middle dose level of mRNA 1345 given 56 days apart in younger adults; to evaluate the tolerability and reactogenicity of a booster injection of the mRNA vaccine given approximately 12 months after the primary injection in older adults; and to evaluate the tolerability and reactogenicity of 3 injections of 1 of 2 dose levels of the mRNA vaccine given 56 days apart in RSV- seropositive children. The primary endpoints were: solicited local and systemic adverse reactions (ARs) through 7 days after each vaccination; unsolicited adverse events (AEs) through 28 days after last vaccination; and serious adverse events (SAEs) and medically attended AEs throughout the entire study period.

The secondary objectives of the study were: to evaluate the antibody response to each vaccine dose level in healthy younger and older adults; to evaluate the antibody response to both 1 and 3 vaccine injections in healthy younger adults at the middle dose level of mRNA- 1345; to evaluate the antibody response to a vaccine booster injection given approximately 12 months after the primary injection in older adults; and to evaluate the antibody response to each vaccine dose level in RSV seropositive children. Secondary endpoints included: geometric mean titer of serum RSV neutralizing and binding antibodies across prespecified study time points; geometric mean fold-rise of postbaseline/baseline antibody titers; and proportion of participants with > 2- fold and > 4 fold increases in antibody titers from baseline. Immunogenicity was assessed by measuring serum neutralizing antibody titers against RSV (both A and B lineages) in microneutralization assays. All immunogenicity analyses were performed on the Per Protocol (PP) Set and results are presented as geometric mean titer (GMT) and geometric mean fold rise (GMFR, defined as the ratio of post-baseline/baseline titers) (FIGs. 2-4). These analyses were conducted with samples collected at Baseline (Day 1, prior to vaccination), Month 1 (Day 29), Month 2 (57), Month 3 (Day 85), Month 4 (Day 113), and Month 5 (Day 141). Neutralizing antibodies against RSV were confirmed to be present at Baseline (prior to vaccination) in all subjects, as was expected. The Baseline GMTs were generally comparable across treatment groups; the RSV-A GMTs were within 2.1-fold across groups and the RSV-B GMTs were within 1.4-fold across groups (when comparing groups with n > 4 individuals). A single vaccination of 50 pg or 100 pg or 200 pg in younger adults boosted neutralizing antibody titers against RSV with no apparent dose response (FIGs. 2 and 4). At Month 1 , the geometric mean fold rise (GMFR) in neutralizing antibody relative to baseline was at least 20.5 for RSV-A and at least 11.7 for RSV-B, regardless of the dose administered (FIG. 3). The data is shown in FIGs. 2-4 and in Table 2 below.

Table 2. Geometric Mean Titer and Geometric Mean Fold Rise at Month 1

After a single vaccination of 50 pg or 100 pg RSV vaccine, the RSV-A neutralizing antibody declined from Month 1 through Month 5 (FIG. 5). The RSV-B neutralizing antibody response was stable from Month 1 to Month 2 and declined less rapidly through Month 5 (FIG. 6). The GMFR at Month 5 was at least 7.4 for both RSV-A and RSV-B, a reduction from the peak GMFR of 2-3-fold for RSV-A and less than 2-fold for RSV-B. Also, a second and third 100 pg injection did not further boost neutralizing antibody titers, but did help maintain peak titers through Month 5, relative a single 100 pg injection. This data is shown in Table 3 below. Table 3. Geometric Mean Titer and Geometric Mean Fold Rise at Month 5

Note that there was a minimal change in RSV-A or RSV-B neutralizing antibody titer in the placebo 1-dose from Baseline through Month 5 and in the placebo 3-dose from Baseline through Month 5 (GMFR of approximately 1 for both).

Healthy older adult participants (65 to 79 years of age) were enrolled in 6 older adult cohorts and randomized via IRT. In the older adult cohorts (Cohorts 7, 8, 9, 10, and 11), 5 dose levels administered in a single dose of the mRNA vaccine (12.5 pg, 25 pg, 50 pg, 100 pg, or 200 pg) or placebo were evaluated. Older adult participants dosed with the mRNA vaccine on Day 1 were randomized to receive the same dose level of mRNA- 1345 or placebo as a booster injection at Month 12, and those participants dosed with placebo on Day 1 received a placebo injection at Month 12. Older adult participants were randomized based on the randomization ratio 2:2: 1 to the following treatment sequences (Day 1/Month 12): vaccine/vaccine: vaccine/placebo: placebo/placebo. A single dose of 12.5 ug, 25 ug, 50 ug, 100 ug, or 200 ug of mRNA-1345 was generally well- tolerated in older adults.

Immunogenicity was assessed by measuring serum neutralizing antibody titers against RSV (both A and B lineages) in microneutralization assays. All immunogenicity analyses were performed on the Per Protocol (PP) Set and results are presented as geometric mean titer (GMT) and geometric mean fold rise (GMFR, defined as the ratio of post-baseline/baseline titers). These analyses were conducted with samples collected at Baseline (Day 1, prior to vaccination), Month 1 (Day 29), Month 2 (57), Month 3 (Day 85), Month 4 (Day 113), and Month 5 (Day 141). Neutralizing antibodies against RSV were confirmed to be present at Baseline (prior to vaccination) in all subjects, as was expected. A single vaccination of 50 pg, 100 pg, or 200 pg in older adults boosted neutralizing antibody titers against RSV-A approximately 14-fold and against RSV-B approximately 10-fold (FIG. 7) and was well-tolerated. Note that the data were pooled across dose levels because there was not a significant difference between doses. In all, there were 34 subjects in the placebo group and 135 subjects in the treatment groups (approximately 45 subjects for each of the three dosage levels). Additional neutralization data was collected. Neutralizing antibodies against RSV were confirmed to be present at Baseline (prior to vaccination) in all subjects, as was expected. The Baseline GMTs were generally comparable across treatment groups. A single vaccination of 12.5 pg or 25 pg or 50 pg or 100 pg or 200 pg in older adults boosted neutralizing antibody titers against RSV with minimal dose response (FIGs. 8 and 9). Across all dose groups, the RSV neutralizing antibody GMTs differed less than 2.5-fold. In general, the titers were lowest with 12.5 pg, similar for 25, 50 and 100 pg, and highest for 200 pgAt Month 1, the RSV-A GMFR was at least 9.75 (range 9.75 to 16.86) and the RSV-B GMFR was at least 5.30 (range 5.30 to 12.29). (FIGs. 8 and 9). There was minimal change in RSV-A or RSV-B neutralizing antibody titer in the placebo group from Baseline through Month 1, represented by a GMFR of approximately 1. The data is shown Tables 4 and 5 below.

Table 4. RSV-A Neutralization (older adult cohorts)

Table 5. RSV-B Neutralization (older adult cohorts)

Binding antibodies against prefusion F protein (preF) and postfusion F protein (postF) were confirmed to be present at Baseline (prior to vaccination) in all subjects, as was expected. The Baseline GMTs were similar across treatment groups. A single vaccination was found to boost preF and postF binding antibodies at all dose levels evaluated (12.5 pg, 25 pg, 50 pg, 100 pg, and 200 pg). There was minimal dose response at Month 1, as the preF and postF GMT and GMFRs differed less than 2.0-fold across the dose groups. At Month 1, the preF GMFR was at least 7.11 (range, 7.11 to 12.06) and the postF GMFR was at least 4.50 (range, 4.50 to 9.14). At every dose level, the preF GMFR was greater than the postF GMFR, suggesting that the mRNA vaccine preferentially boosts antibodies to the preF conformation. There was minimal change in preF or postF binding antibody titer in the placebo group from Baseline through Month 1, represented by a GMFR of approximately 1. The data is shown in FIG. 10 and Tables 6 and 7 below. Table 6. PreF Binding Antibody

Table 7. PostF Binding Antibody

Interim Results: Women (18-40 years of age)

Described herein are top line results of the planned interim analysis (IA) of immunogenicity from baseline through Month 1 (Day 29) for the women of child-bearing potential. The women of child-bearing potential cohorts each include 60 participants aged 18 to 40 years, randomized 5:1 to receive a single dose of 12.5 pg mRNA-1345 or placebo (Cohort 12), a single dose of 25 pg mRNA-1345 or placebo (Cohort 13), or a single dose of 50 pg mRNA-1345 or placebo (Cohort 14).

Immunogenicity was assessed by measuring serum neutralizing antibody titers against RSV-A, RSV-B, and binding antibody titers against the RSV F protein (both PreF and PostF conformations). Neutralizing antibodies were measured in qualified microneutralization assays and results are presented as GMT, GMT in international units per milliliter (GMT IU/mL), and GMFR (defined as the ratio of the post baseline/baseline titers). The IU/mL was calculated using a reference standard serum in each assay that had been calibrated against the established international standard antiserum to RSV-A and RSV-B from the World Health Organization (NIBSC code 16/284; to convert titer to IU/mL; RSV-A titers are multiplied by 1.0578 and RSV- B titers by 0.6936). PreF and PostF binding (immunoglobulin G) antibodies were measured in a qualified multiplex Luminex assay and results are presented as GMT (titer expressed in arbitrary units per milliliter [AU/mL]) and GMFR. All immunogenicity analyses were performed on the Per Protocol Set (defined in the protocol). These analyses were conducted with samples collected at prespecified study time points.

Neutralizing antibodies against RSV-A and RSV-B were confirmed to be present at baseline in all treatment groups. At baseline for the placebo group and the 12.5 pg, 25 pg, and 50 pg 1 dose mRNA 1345 groups, the GMTs were 1209.7, 847.5, 1041.5, and 1042.6 for RSV-A and 1366.9, 1327.0, 2539.8 and 1839.1 for RSV-B; while the GMT IU/mLs were 1279.6, 896.5, 1101.7, and 1103.0 for RSV-A and 948.1, 920.3, 1761.7, and 1275.7 for RSV-B, respectively.

Vaccination was found to boost neutralization antibody titers against RSV (both A and B lineages) at all 3 dose levels evaluated (FIGs. 11-12). The GMT at Month 1 was lowest with the 12.5 pg dose, and similar with 25 pg and 50 pg doses of vaccine. Specifically, at Month 1, the 12.5 pg, 25 pg, and 50 pg vaccine (single dose) groups had the following results: the GMTs were 8209.1, 11686.6, and 11633.7 for RSV-A and 10440.8, 16456.7, and 16487.3 for RSV-B; the GMT IU/mL values were 8683.5, 12362.2, and 12305.9 for RSV-A and 7241.6, 11414.5, and 11435.2 for RSV-B; and the GMFRs were 9.69, 11.22, and 11.16 for RSV-A and 7.87, 6.48, and 8.96 for RSV-B. There was minimal change in RSV-A and RSV-B neutralizing antibody titers in the placebo group from baseline to Month 1 , represented by GMFRs of approximately 1.

With respect to baseline binding antibodies, binding antibodies against PreF and PostF were confirmed to be present at baseline in all treatment groups. At baseline for the placebo group and the 12.5 pg, 25 pg, and 50 pg dose vaccine (single dose) groups: the GMTs were 7108.7, 5925.0, 7094.5, and 7045.1 for PreF and 9033.1, 7515.1, 8031.5, and 9464.6 for PostF, respectively.

Vaccination boosted PreF- and PostF-binding antibodies at all 3 dose levels evaluated (FIGs. 13-14). The preF GMT at Month 1 was the lowest with the 12.5 pg dose, and similar with 25 pg and 50 pg doses. The postF GMT at Month 1 was similar between the 12.5 pg and 25 pg groups, and highest in the 50 pg group. The PreF GMFR was greater than the PostF GMFR for each dose level, suggesting that the vaccine preferentially boosts antibodies to the PreF conformation. Specifically, at Month 1 for the 12.5 pg, 25 pg, and 50 pg (single dose) groups, the GMTs were 50908.5, 67953.4, and 69289.7 for PreF binding antibody and 58957.9, 58023.9, and 72615.6 for PostF binding antibody, respectively; and the GMFRs were 8.59, 9.58, and 9.84 for PreF binding antibody and 7.85, 7.22, and 7.67 for PostF binding antibody, respectively. There was minimal change in PreF or PostF binding antibody titer in the placebo group from baseline through Month 1 , represented by GMFRs of approximately 1.

Pediatric Cohorts

Two pediatric cohorts of RSV seropositive children (12 to 59 months of age) will be enrolled and randomized via IRT. In the pediatric cohorts (Cohorts 5 and 6), 2 escalating dose levels of the mRNA vaccine (30 pg and 100 pg) or placebo administered in a 3-dose series will be evaluated.

SEQUENCES

Wild-type RSV F Glycoprotein

MELLIHRS S AIFLTLAINTL YLT S S QNITEEF Y QS TCS A V S RGYFS ALRTGW YT S VITIELS N IKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINT TKNLN V S IS KKRKRRFLGFLLGV GS AI AS GI A V S KVLHLEGE VNKIKN ALLSTNKA V V S L SNG V S VLTS KVLDLKN YINN QLLPIVN QQS CRIS NIET VIEFQQKN S RLLErrREFS VN AG VTTPLS T YMLTN SELLS LINDMPITNDQKKLMS S NV QIVRQQS YSIMS IIKEE VL A Y V V QL PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSN RVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVF PS DEFD AS IS Q VNEKIN QSLAFIRRS DELLHN VNTGKS TTNIMIT AIII VIIV VLLS LIAIGLLL Y CKAKNTPVTLS KDQLSGINNIAFS K (SEQ ID NO: 1)

It should be understood that any of the mRNA sequences described herein may include a 5' UTR and/or a 3' UTR. The UTR sequences may be selected from the following sequences, or other known UTR sequences may be used. It should also be understood that any of the mRNAs described herein may further comprise a poly(A) tail and/or cap ( e.g ., 7mG(5')ppp(5')NhnpNp). Further, while many of the mRNAs and encoded antigen sequences described herein include a signal peptide and/or a peptide tag (e.g., C-terminal His tag), it should be understood that the indicated signal peptide and/or peptide tag may be substituted for a different signal peptide and/or peptide tag, or the signal peptide and/or peptide tag may be omitted.

It should be further understood that any of the mRNA sequences described herein may be fully or partially chemically modified (e.g., by Nl-methylpseudouridine). In Table 1 below, the sequences numbers are given as unmodified/fully modified by Nl-methylpseudouridine).

5' UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 2; fully modified by Nl-methylpseudouridine, SEQ ID NO: 33)

5' UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC (SEQ ID NO: 3; fully modified by Nl-methylpseudouridine, SEQ ID NO: 40)

3' UTR:

UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 4; fully modified by Nl-methylpseudouridine, SEQ ID NO: 35)

3' UTR:

UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 5; fully modified by Nl-methylpseudouridine, SEQ ID NO: 41)

Table 1. Vaccine Sequences

* It should be understood that any one of the open reading frames and/or corresponding amino acid sequences described in Table 1 may include or exclude a signal sequence. It should also be understood that the signal sequence may be replaced by a different signal sequence, for example, any one of SEQ ID NOs: 18-34.

EQUIVALENTS All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,”

“composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

The entire contents of International Application Nos. PCT/US2015/002740, PCT/US2016/043348, PCT/US2016/043332, PCT/US2016/058327, PCT/US2016/058324,

PCT/US2016/058314, PCT/US2016/058310, PCT/US2016/058321, PCT/US2016/058297, PCT/US2016/058319, and PCT/US2016/058314 are incorporated herein by reference.

Claims

CLAIMS What is claimed is:

1. A method comprising: administering to a human subject a dose of a vaccine comprising a lipid nanoparticle that comprises a messenger ribonucleic acid (mRNA), wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human respiratory syncytial vims (RSV) F glycoprotein that comprises a deletion of a cytoplasmic tail domain, the dose of the vaccine comprises 12.5 pg to 200 pg or 50 pg to 100 or 50 pg to 200 pg of the mRNA, and the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 3-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

2. The method of claim 1, wherein the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject are at least 4-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

3. The method of any one of the preceding claims, wherein the geometric mean titer (GMT) of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 29 post administration of the dose is at least 15,000.

4. The method of any one of the preceding claims, wherein the geometric mean (GM) of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 15,000 IU/mL.

5. The method of any one of the preceding claims, wherein the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 11,000 IU/mL.

6. The method of any one of the preceding claims, wherein the geometric mean fold rise (GMFR) of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 20.

7. The method of any one of the preceding claims, wherein the GMFR of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 10.

8. The method of any one of claims 1-5, wherein the dose comprises 50 pg of the mRNA.

9. The method of any one of claims 1-5, wherein the dose comprises 100 pg of the mRNA.

10. The method of any one of claims 1-5, wherein the dose comprises 200 pg of the mRNA.

11. The method of any one of claims 1-5, wherein the dose comprises 12.5 pg of the mRNA.

12. The method of any one of claims 1-5, wherein the dose comprises 25 pg of the mRNA.

13. The method of any one of claims 1-9, further comprising administering to the subject a second dose of the vaccine.

14. The method of claim 13, wherein the second dose of the vaccine is administered at least 2 months after the first dose is administered.

15. The method of claim 13 or 14, wherein the GMT of neutralizing antibody titers induced against RSV-A in the subject at Day 141 post administration of the first dose is at least 5,000.

16. The method of claim 13 or 14, wherein the GMT of neutralizing antibody titers induced against RSV-B in the subject at Day 141 post administration of the first dose is at least 11,000.

17. The method of any one of claims 13-16, wherein the GM of neutralizing antibody titers induced against RSV-A in the subject at Day 141 post administration of the first dose is at least 5,000 IU/mL.

18. The method of any one of claims 13-16, wherein the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 141 post administration of the first dose is at least 8,000 IU/mL.

19. The method of any one of claims 13-18 wherein the GMFR of neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 141 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 7.

20. The method of any one of claims 13-19, wherein the neutralizing antibody titers induced against RSV-A and/or RSV-B in the subject at Day 141 post administration of the first dose are at least 4-fold higher than the neutralizing antibody titers against RSV-A and/or RSV-B in the subject at baseline.

21. The method of any one of claims 13-20, further comprising administering to the subject a third dose of the vaccine.

22. The method of claim 21, wherein the third dose of the vaccine is administered at least 2 months after the second dose is administered.

23. The method of any one of claims 13-22, wherein the second dose comprises 50 pg of the mRNA.

24. The method of any one of claims 13-22, wherein the second dose comprises 100 pg of the mRNA.

25. The method of claim 22, wherein each of the second dose and the third dose comprises 50 pg of the mRNA.

26. The method of claim 22, wherein each of the second dose and the third dose comprises 100 pg of the mRNA.

27. The method of any one of the preceding claims, wherein the subject is an 18-49 year old subject.

28. The method of any one of claims 8-13, wherein the subject is a 65-79 year old subject.

29. The method of claim 28, wherein the GMT of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 12,000.

30. The method of claim 28 or claim 29, wherein the GMT of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 11,000.

31. The method of any one of claims 28-30, wherein the GM of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the dose is at least 12,500 IU/mL.

32. The method of any one of claims 28-31, wherein the GM of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the dose is at least 7,500 IU/mL.

33. The method of any one of claims 28-32, wherein the GMFR of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 9.5.

34. The method of any one of claims 28-33, wherein the GMFR of neutralizing antibody titers induced against RSV-B in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 5.

35. The method of any one of claims 28-34, wherein the GMT of binding antibody titers induced against RSV prefusion F protein (preF) in the subject at Day 29 post administration of the dose is at least 55,500.

36. The method of any one of claims 28-35, wherein the GMT of binding antibody titers induced against RSV postfusion F protein (postF) in the subject at Day 29 post administration of the dose is at least 48,000.

37. The method of any one of claims 28-36, wherein the GMFR of binding antibody titers induced against RSV preF in the subject at Day 29 post administration of the dose is at least 7.0.

38. The method of any one of claims 28-37, wherein the GMFR of binding antibody titers induced against RSV postF in the subject at Day 29 post administration of the dose is at least 4.5.

39. The method of any one of the preceding claims, wherein the F glycoprotein encoded by the ORF of the mRNA is membrane anchored.

40. The method of any one of the preceding claims, wherein the F glycoprotein encoded by the ORF of the mRNA comprises interprotomer disulfide stabilizing mutations.

41. The method of any one of the preceding claims, wherein the F glycoprotein encoded by the ORF of the mRNA has at least 95% identity to the amino acid sequence of SEQ ID NO: 8.

42. The method of claim 41, wherein the F glycoprotein encoded by the open reading frame of the mRNA comprises the amino acid sequence of SEQ ID NO: 8.

43. The method of claim 42, wherein the F glycoprotein encoded by the open reading frame of the mRNA consists of the amino acid sequence of SEQ ID NO: 8.

44. The method of any one of the preceding claims, wherein the mRNA comprises a 5’ 7mG(5’)ppp(5’)NlmpNp cap and a 3’ polyA tail.

45. The method of any one of the preceding claims, wherein the mRNA comprises a 1- methylpseudourine chemical modification.

46. The method of any one of the preceding claims, wherein the lipid nanoparticle comprises 45-55 mol% ionizable amino lipid, 15-20 mol% neutral lipid, 35-45 mol% cholesterol, and 0.5-5 mol% PEG-modified lipid.

47. The method of any one of the preceding claims, wherein the ionizable amino lipid is Compound I:

(Compound I).

48. The method of any one of claims 1-47, wherein the lipid nanoparticle comprises 50 mol% ionizable amino lipid.

49. The method of any one of claims 1-47, wherein the lipid nanoparticle comprises 49 mol% ionizable amino lipid.

50. The method of any one of claims 1-47, wherein the lipid nanoparticle comprises 48 mol% ionizable amino lipid.

51. The method of any one of the preceding claims, wherein the vaccine is administered intramuscularly.

52. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

53. A method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and each of the three doses comprises 50 pg of the mRNA.

54. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and the dose comprises 100 pg of the mRNA.

55. A method comprising: administering to a human subject intramuscularly three doses of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

56. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 18-49 years old, and the dose comprises 200 pg of the mRNA.

57. The method of any one of claims 1-5, wherein the GMFR of neutralizing antibody titers induced against RSV-A in the subject at Day 29 post administration of the first dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 10.

58. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 65-79 years old, and the dose comprises 50 pg of the mRNA.

59. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I: (Compound I), the human subject is 65-79 years old, and the dose comprises 100 pg of the mRNA.

60. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

the human subject is 65-79 years old, and the dose comprises 200 pg of the mRNA.

61. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

(Compound I), the human subject is 65-79 years old, and the dose comprises 12.5 pg of the mRNA.

62. A method comprising: administering to a human subject intramuscularly a dose of vaccine comprising lipid nanoparticle that comprises a chemically-modified mRNA, wherein the mRNA comprises an open reading frame encoding a stabilized trimeric prefusion form of a human RSV F glycoprotein that comprises a deletion of a cytoplasmic tail domain and has at least 95% identity to the amino acid sequence of SEQ ID NO: 8, the lipid nanoparticle comprises 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable amino lipid of Compound I:

the human subject is 65-79 years old, and the dose comprises 25 pg of the mRNA.

63. The method of any one of the preceding claims, wherein administration of any one of the vaccines described herein produces an immune response with an RSV preF bias.

64. Use of the vaccine of any one of claims 1-63 in the manufacture of a medicament for inducing an immune response in a subject.

65. A composition for use in the method of any one of claims 1-63.