WO2021211343A1

WO2021211343A1 - Zika virus mrna vaccines

Info

Publication number: WO2021211343A1
Application number: PCT/US2021/026256
Authority: WO
Inventors: Tal ZAKS; Brooke BOLLMAN; Hamilton BENNETT
Original assignee: Modernatx, Inc.
Priority date: 2020-04-13
Filing date: 2021-04-07
Publication date: 2021-10-21

Abstract

Provided herein are vaccines comprising a chemically-modified messenger ribonucleic acid encoding a Zika virus prME protein formulated in a cationic lipid nanoparticle formulation, and related methods for inducing an antigen- specific immune response to Zika virus.

Description

ZIKA VIRUS MRNA VACCINES

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/009,123, filed April 13, 2020, which is incorporated by reference herein in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Contract No. HHS0100201600029C awarded by Biomedical Advanced Research and Development (BARDA). The government has certain rights in the invention.

BACKGROUND

Zika vims has rapidly emerged in recent years as a pandemic with potential long-term public health implications. Zika is primarily transmitted by mosquitos but can also be transmitted sexually. Children bom to mothers infected with Zika can develop microcephaly, a severe disease characterized by small, not fully developed heads and severe disabilities. In adults, outbreaks in Latin American and Caribbean countries have been associated with Guillain-Barre syndrome, a rare but serious autoimmune disorder in which the immune system attacks part of the nervous system. There is no approved vaccine for Zika.

SUMMARY

The Zika vims (ZIKV) vaccine provided herein comprises an mRNA that encodes the structural proteins of ZIKV and is designed to cause cells to secrete virus-like particles, mimicking the response of the cell after natural infection. Preclinical data have shown that vaccination with protected against transmission of ZIKV during pregnancy in mice. Provided herein are data from a randomized, observer-blind, placebo-controlled, dose-ranging study designed to evaluate the safety, tolerability and immunogenicity of the ZIKV vaccine described herein in healthy flavivims seropositive and seronegative adults ages 18 to 49 years. Primary outcome measures include frequency and grade of adverse events; and secondary outcome measures include geometric mean titers of neutralizing antibodies against Zika vims.

Some aspects of the present disclosure provide a method comprising administering to a subject a vaccine comprising 10 qg - 250 qg of a messenger ribonucleic acid (mRNA) comprising: (a) an open reading frame (ORF) that encodes a ZIKV prME protein; and (b) a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid, wherein a neutralizing antibody response to ZIKV is produced in the subject.

In some embodiments, the ZIKV prME protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the ZIKV prME protein comprise the amino acid sequence of SEQ ID NO:

7.

In some embodiments, the ORF comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ORF comprise the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the mRNA comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the mixture of lipids comprises 45-55 mol% ionizable cationic lipid, 15-20 mol% non-cationic lipid, 35-45 mol% sterol, and 0.5-5 mol% PEG-modified lipid.

In some embodiments, the mixture of lipids comprises 50 mol% ionizable cationic lipid, 10 mol% non-cationic lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid. In some embodiments, the ionizable cationic lipid is a Compound I ionizable cationic lipid, the non- cationic lipid is DSPC, the sterol is cholesterol, and the PEG-modified lipid is PEG-DMG.

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

In some embodiments, the composition further comprises Tris buffer, propylene glycol, and diethylenetriamine pentaacetic acid (DTPA). In some embodiments, the composition further comprises 100 mM Tris buffer, 7% propylene glycol, and 1 mM DTPA.

In some embodiments, the composition comprises 10 pg, 30 pg, 100 pg, or 250 pg pg of the mRNA. In some embodiments, the subject is 18 to 49 years of age.

In some embodiments, a second dose of the vaccine is administered to the subject following a first dose, optionally 28 days following the first dose. In some embodiments, the vaccine is administered intramuscularly.

In some embodiments, the composition comprises 10 pg of the mRNA. In some embodiments, a second dose of the vaccine is administered to the subject at least 28 days following the first dose.

In some embodiments, the subject is flavivims seronegative. In some embodiments, the plaque reduction neutralization test 50 (PRNT50) geometric mean titer (GMT) of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 180 - 210, 190 - 200, or 195. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 20-50, 30-40, or 38.

In some embodiments, the microneutralization assay 50 (MN50) GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 40 - 70, 50 - 60, or 57. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 1180 - 1210, 1190 - 1200, or 1195. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 130-160, 140- 150, or 141.

In some embodiments, the subject flavivims seropositive. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 135 - 155, 140-150, or 148. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 215-235, 220-230, or 224. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 50-80, 60-70, or 68. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 360-390, 370-380, or 375. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 630-660, 640-650, or 646. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 250-280, 260-270, or 263.

In some embodiments, any one of the methods described herein further comprise administering to subjects of a population a second dose of the vaccine at least 28 days following administration of a first dose of the vaccine.

In some embodiments, at least 70%, at least 75%, or at least 80% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below a lower limit of quantification (LLOQ) to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

In some embodiments, at least 55%, at least 60%, or at least 65% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below a lower limit of quantification (LLOQ) to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

In some embodiments, at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in microneutralization (MN) from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

In some embodiments, at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in microneutralization (MN) from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

In some embodiments, the subjects are flavivirus seronegative. In some embodiments, at least 80%, at least 85%, or at least 90% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16. In some embodiments, at least 55%, at least 60%, or at least 65% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16. In some embodiments, at least 60%, at least 65%, or at least 70% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28. In some embodiments, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28. In some embodiments, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

In some embodiments, the subjects are flavivirus seropositive. In some embodiments, at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by PRNT. In some embodiments, at least 28 days following a first dose of the vaccine, and at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT. In some embodiments, at least 28 days following a first dose of the vaccine, and at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT. In some embodiments, at least 28 days following a first dose of the vaccine, and at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

In some embodiments, at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN. In some embodiments, at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN. In some embodiments, at least 45%, at least 50%, or at least 55% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by MN. In some embodiments, at least 90%, at least 95%, or at least 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by MN.

In some embodiments, the composition comprises 30 pg of the mRNA. In some embodiments, a second dose of the vaccine is administered to the subject at least 28 days following the first dose.

In some embodiments, the subject is flavivims seronegative. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose and the second dose of the vaccine, is 5-30, 10-20, or 14. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 285-320, 295-310, or 303. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 115-145, 125-135 or 130. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 1455-1495, 1465-1485, or 1478.

In some embodiments, the subject flavivims seropositive. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 70-105, 80-95, or 88. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 135-165, 145-155, or 151.

In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 215-245, 225-235, or 227. In some embodiments, the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 565-595, 575-585, or 579.

In some embodiments, any one of the methods described herein may further comprise administering to subjects of a population a second dose of the vaccine at least 28 days following administration of a first dose of the vaccine.

In some embodiments, at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below a LLOQ to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

In some embodiments, at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

In some embodiments, the subjects are flavivims seronegative.

In some embodiments, at least 30%, at least 35%, or at least 40% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16. In some embodiments, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16.

In some embodiments, at least 75%, at least 80%, or at least 85% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28. In some embodiments, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

In some embodiments, the subjects are flavivims seropositive.

In some embodiments, at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by PRNT. In some embodiments, at least 28 days following a first dose of the vaccine, and at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

In some embodiments, at least 28 days following a first dose of the vaccine, and at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

In some embodiments, at least 85%, at least 70%, or 75% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN. In some embodiments, at least 85%, at least 70%, or 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN.

Other aspects of the present disclosure provide a vaccine comprising 10 mg - 250 mg, 10 mg - 100 mg, or 10 mg - 30 mg of a messenger ribonucleic acid (mRNA) comprising: (a) ORF that encodes a ZIKV prME protein, wherein the ORF comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1; and (b) a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

In some embodiments, the ZIKV prME protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the ZIKV prME protein comprise the amino acid sequence of SEQ ID NO: 7.

In some embodiments, any one of the vaccines described herein may further comprise Tris buffer, propylene glycol, and diethylenetriamine pentaacetic acid (DTPA). In some embodiments, the vaccine comprises 100 mM Tris buffer, 7% propylene glycol, and 1 mM DTPA.

In some embodiments, the vaccine comprises 10 pg of the mRNA. In some embodiments, the vaccine comprises 30 pg of the mRNA. In some embodiments, the vaccine comprises 100 pg of the mRNA. In some embodiments, the vaccine comprises 250 pg of the mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Overview of the dosing scheme by cohort.

FIG. 2: Anti-ZIKV neutralizing antibodies by PRNT, 10 mg dose level seronegative (left) and seropositive participants (right), Per-Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination.

FIG. 3: Anti-ZIKV neutralizing antibodies by MN, 10 mg dose level seronegative (left) and seropositive participants (right), Per-Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination.

FIG. 4: Anti-ZIKV neutralizing antibodies by PRNT, 10 mg dose level and 30 mg does level seronegative (left) and seropositive participants (right), Per-Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination.

FIG. 5: Anti-ZIKV neutralizing antibodies by MN, 10 mg and 30 mg dose level seronegative (left) and seropositive participants (right), Per-Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination.

FIG. 6: Anti-ZIKV neutralizing antibodies by PRNT, 10 mg, 30 mg, 100 mg, and 250 mg dose level up to Day 57 and 10 mg dose level up to Month 7 seronegative (left) and seropositive participants (right), Per-Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination; Month 7 = 6 months after the 2nd vaccination. FIG. 7: Anti-ZIKV neutralizing antibodies by MN, 10 qg, 30 qg, 100 qg, and 250 qg dose level up to Day 57 and 10 qg dose level up to Month 7 seronegative (left) and seropositive participants (right), Per- Protocol Set. Day 1 = 1st vaccination; Day 29 = 1 month after the 1st vaccination; Day 57 = 1 month after the 2nd vaccination; Month 7 = 6 months after the 2nd vaccination.

DETAILED DESCRIPTION

Zika virus (ZIKV), first discovered in 1947, is a single-stranded RNA flavivirus, which is transmitted to humans by a mosquito vector (mainly Aedes aegypti but other Aedes mosquitoes are believed to be competent vectors) or by person to person spread, mainly through sexual transmission. In the 60 years after its discovery, ZIKV remained a relatively obscure pathogen, associated with only sporadic cases of human infection that were largely asymptomatic or resulted in a mild febrile illness. In the last decade, however, there has been rapid geographic spread into the Pacific Islands and then South America, where ZIKV outbreaks have been larger, more frequent, and more severe. Most concerning are infections in pregnant women, particularly during the first and second trimesters, which have resulted in a wide range of birth defects, including microcephaly, intrauterine growth restriction, and spontaneous abortion (Brasil et. al. 2016).

The devastating consequences of ZIKV infection, including congenital Zika syndrome and pregnancy loss and complicated neurological sequelae such as Guillain-Barre syndrome, led the World Health Organization (WHO) to declare a Public Health Emergency of International Concern on 1 February 2016 and to call on the global research and development communities to prioritize the development of preventive and therapeutic solutions. Although the WHO declared an end to its global health emergency over the spread of ZIKV in November 2016, the long-term need for a ZIKV vaccine continues as a priority need under the Blueprint Plan of Action. Currently there is no approved vaccine to protect against this disease.

Provided herein is a vaccine based on a messenger RNA (mRNA) vaccine platform. This platform is based on the principle and observations that antigens can be produced in vivo by delivery and uptake of the corresponding mRNA by cells. The mRNA then undergoes intracellular ribosomal translation to endogenously express the protein antigen(s) encoded by the vaccine mRNA. This mRNA-based vaccine does not enter the cellular nucleus or interact with the genome, is non-replicating, and expression is transient. mRNA vaccines thereby offer a mechanism to stimulate endogenous production of structurally intact protein antigens in a way that mimics wild type viral infection and is able to induce highly targeted immune responses against infectious pathogens such as ZIKV. The ZIKV mRNA vaccine of the present disclosure is a lipid nanoparticle (LNP)- encapsulated mRNA-based vaccine directed against the pre-membrane and envelope (prME) structural protein of ZIKV. This vaccine, in some embodiments, includes mRNA formulated with LNPs composed of 4 lipids: heptadecan 9 yl 8 ((2 hydroxyethyl)(6 oxo 6 (undecyloxy)hexyl)amino) octanoate (Compound I); cholesterol; 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); and 1 monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG). This vaccine, in some embodiments, is provided as a sterile liquid for injection at a concentration of 0.5 mg/mL in 100 mM Tris buffer, 7% propylene glycol, and 1 mM diethylenetriamine-pentaacetic acid (DTPA).

Antigens

Antigens, as used herein, 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 proteins and immunogenic fragments (an immunogenic fragment that induces (or is capable of inducing) an immune response to ZIKV), unless otherwise stated. It should be understood that the term “protein’ encompasses peptides and the term “antigen” encompasses antigenic fragments. Other molecules may be antigenic, such as bacterial polysaccharides or combinations of protein and polysaccharide structures, but the antigens provided herein include viral proteins, fragments of viral proteins, and variant proteins (e.g., designed and/or mutated proteins) derived from ZIKV.

The ZIKV prME protein sequence of the mRNA vaccine of the present disclosure is provided in the Sequence Listing elsewhere herein. In some embodiments, the ZIKV vaccine comprises an mRNA encoding a prME protein that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the ZIKV vaccine comprises an mRNA encoding a prME protein that comprises the amino acid sequence of SEQ ID NO: 7.

Nucleic Acids

The ZIKV mRNA vaccine of the present disclosure comprise a (at least one) ribonucleic acid (RNA) having an open reading frame encoding a ZIKV antigen. In some embodiments, the RNA is a messenger RNA (mRNA) having an open reading frame encoding a ZIKV antigen. In some embodiments, the mRNA further comprises a 5' UTR, 3' UTR, a polyA tail and/or a 5' cap.

Nucleic acids comprise a polymer of nucleotides (nucleotide monomers), 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'-amino- 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 ribonucleic acid 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 RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U ”

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 a vaccine of the present disclosure.

In some embodiments, the ZIKV vaccine comprises an mRNA that comprises an open reading frame that comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ZIKV vaccine comprises an mRNA that comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ZIKV vaccine comprises an mRNA that comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ZIKV vaccine comprises an mRNA that comprises the nucleotide of SEQ ID NO: 2. In some embodiments, the ZIKV mRNA may further comprise a 5’ cap (e.g., 7mG(5’)ppp(5’)NlmpNp), a polyA tail (e.g., -100 nucleotides), or a 5’ cap and a poly A tail.

It should also be understood that the ZIKV 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; however, other UTR sequences (e.g., of the prior art) may be used or exchanged for any of the UTR sequences described herein. UTRs may also be omitted from the vaccine constructs provided herein.

Variants

In some embodiments, the ZIKV mRNA vaccine of the present disclosure encodes a ZIKV antigen variant. Antigen 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 ZIKV mRNA vaccine comprises an mRNA ORF comprising a nucleotide sequence of SEQ ID NO: 1, or comprising 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 SEQ ID NO: 1.

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.” J. 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 ZIKV 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 ZIKV pathogen. In addition to variants that are identical to the reference protein but are truncated, 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.

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, the ZIKV mRNA vaccine includes at least one RNA polynucleotide 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 vitro- 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 ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England Bio Labs, Ipswich, MA). 5 '-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England Bio Labs, 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, the ZIKV mRNA vaccine includes one or more stabilizing elements. 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 RNA 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, the ZIKV mRNA vaccine 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, 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, the ZIKV mRNA vaccine does not comprise a histone downstream element (HDE), which 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.

The ZIKV mRNA vaccine 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 composition 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, the ZIKV mRNA vaccine 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 RNA vaccines. Alternatively the AURES may remain in the RNA vaccine.

Signal Peptides

The ZIKV mRNA vaccines provided herein comprises a mRNA having an ORF that encodes a JEV signal peptide fused to the ZIKV 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 ZIKV 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 comprises the amino acid sequence MWLVSLAIVTACAGA (SEQ ID NO: 8).

Fusion Proteins

In some embodiments, the ZIKV mRNA vaccine of the present disclosure includes a 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 ZIKV prME protein fused to a JEV signal peptide (e.g., MWLVSLAIVTACAGA (SEQ ID NO: 8)). 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 ZIKV 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-infectious (Lopez-Sagaseta, J. et. ah, 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 ZIKV antigen is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the ZIKV antigen.

In another embodiment, 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. ah, J Mol Biol. 2009;390:83-98). Several high- resolution structures 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. ah. J Biol Inorg Chem. 2003;8:105-111; Fawson D.M. et. ah, 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.

Fumazine synthase (FS) is also well-suited as a nanoparticle platform for antigen display. FS, 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 FS 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 FS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 A diameter. Even FS cages of more than 100 subunits have been described (Zhang X. et. ah, 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 2 A 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., WO2017/127750). The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will likewise appreciate that other polycistronic constructs (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 structures; 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 a ZIKV 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 RNA 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 RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA 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 RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.

Chemically Unmodified Nucleotides

In some embodiments, at least one mRNA of the ZIKV mRNA vaccine of the present disclosure 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 RNA (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 ZIKV mRNA vaccine of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one ZIKV 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/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB 2017/051367 all of which are incorporated 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 RNA nucleic acid (e.g., a modified mRNA nucleic acid), 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 RNA nucleic acid (e.g., a modified mRNA nucleic acid), 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 (m 1 y), 1 -ethyl-pseudouridine (e l \|/), 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-methoxymethyl 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 ( 1 \_|/) 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 (m 1 \_|/) 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 polyA 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 (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5' UTR does not encode a protein (is non-coding). Natural 5 TRs 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: 9), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5UTR 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 Xcnopus or human derived a-globin or b- globin (US 8278063; US 9012219), human cytochrome b-245 a polypeptide, and hydroxy steroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219). CMV immediate-early 1 (IE1) gene (US20140206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 10) (WO2014/144196) 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., W02015/101414, W02015/101415, WO/2015/062738, WO2015/024667, WO2015/024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (W02015/101414, W02015/101415, WO2015/062738), 5' UTR element derived from the 5'UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015/024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015/024667) 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 (3') 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. ah, 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: 11) 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., RNA) 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 hour, 12 hour, 24 hour, 48 hour, and 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 (US8278063, US9012219, US2011/0086907). A modified b-globin construct 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, al-globin, UTRs and mutants thereof are also known in the art (W02015/101415, WO2015/024667). Other 3 UTRs described in the mRNA constructs in the non-patent literature include CYBA (Ferizi et. ah, 2015) and albumin (Thess et. ah, 2015). Other exemplary 3 UTRs include that of bovine or human growth hormone (wild type or modified) (WO2013/185069, US2014/0206753,

WO2014/152774), rabbit b globin and hepatitis B virus (HBV), a-globin 3' UTR and Viral VEEV 3’ UTR sequences are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014/144196) is used. In some embodiments, 3 UTRs of human and mouse ribosomal protein are used. Other examples include rps9 3’UTR (W02015/101414), FIG4 (W02015/101415), and human albumin 7 (W02015/101415).

In some embodiments, a 3' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6.

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.2010/0293625 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.2009/0226470, herein incorporated by reference in its entirety, and those known in the art. In vitro Transcription of RNA cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO2014/ 152027, which is incorporated by reference herein in its entirety.

In some embodiments, the RNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA 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 RNA polynucleotide, for example, but not limited to ZIKV 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 a 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 polyA tail. The particular nucleic acid sequence composition 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 RNA 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 “polyA 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 polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA 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 polyA 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 RNA transcript is capped via enzymatic capping. In some embodiments, the RNA 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 interm olecular 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, LNATM 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 (CSL), 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 construct 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, immunoabsorbent 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 ZIKV mRNA vaccine of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable cationic 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 nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable 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 20-60 mole percent (mol%) ionizable cationic lipid (e.g., Compound I). For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid (e.g., DSPC). For example, the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non- cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol (e.g., cholesterol). For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.

In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid (e.g., PEG-DMG). For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable cationic lipid (e.g., Compound I), 5-25 mol% non-cationic lipid (e.g., DSPC), 25-55 mol% sterol (e.g., cholesterol), and 0.5-15 mol% PEG-modified lipid (e.g., PEG-DMG).

In some embodiments, an ionizable cationic 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 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, 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 selected from the group consisting of a C_3-6 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(=NR₉)N(R)2, -N(R)C(=CHR₉)N(R)₂, -0C(0)N(R)₂, -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(0R)C(=NR₉)N(R)₂, -N(0R)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, 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’)-, -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, C_2-3 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, 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 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.

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, 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, -(CFhl_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 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)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 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’)-,

-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;

Rs is selected from the group consisting of C_3-6 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 C_3-14 alkyl and C_3-14 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, or salts or isomers thereof.

R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CFh)_nQ, -(CFhl_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted Ci-₆ alkyl, where Q is selected from a C_3-6 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)_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(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)R, -C(0)N(R)0R, 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 -(CH₂)_nQ in which n is 1 or 2, or (ii) R4 is -(CH₂)_nCHQR 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 heterocyclo alkyl ; 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 C3-6 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 C1-3 alkyl, C₂-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-is 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-i₂ alkyl and C_2-i2 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.

Ri is selected from the group consisting of C5-30 alkyl, Cs-₂o 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, -(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)0R, -0C(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)0R, -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)0R, -N(0R)C(0)R, -N(0R)S(0)₂R, -N(0R)C(0)0R, -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)0R, and -C(=NR₉)N(R)₂, 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 C_1-3 alkyl, C2-3 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 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 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, C2-14 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 -(CFh)nQ or -(CFh)nCF[QR, where Q is -N(R)2, and n is selected from 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; 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 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.

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 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 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)(OR’)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; 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 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 -(CH2)_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)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 C_2-i4 alkenyl.

(II):

(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)₂, -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 C_2-i4 alkenyl.

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

, or

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

(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 cationic 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, 1 ,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 cationic 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 mol% ionizable cationic lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable cationic lipid.

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

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

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

In some embodiments, the lipid nanoparticle comprises 50 mol% ionizable cationic lipid (e.g., Compound I), 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% 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 cationic lipid component to the RNA 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 cationic lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA 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.

Multivalent Vaccines

The ZIKV vaccines, as provided herein, may include mRNA or multiple mRNAs encoding two or more antigens of the same or different ZIKV species. In some embodiments, the ZIKV vaccine includes an RNA or multiple RNAs encoding two or more antigens. In some embodiments, the mRNA of a ZIKV vaccine may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more antigens.

In some embodiments, two or more different mRNA encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNA encoding antigens may be formulated in separate lipid nanoparticles (each RNA formulated in a single lipid nanoparticle). The lipid nanoparticles may then be combined and administered as a single vaccine (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.

Combination Vaccines

The ZIKV vaccines, as provided herein, may include an RNA or multiple RNAs encoding two or more antigens of the same or different viral strains. Also provided herein are combination vaccines that include RNA encoding one or more ZIKV and one or more antigen(s) of a different organism. 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 ZIKV infection is high or organisms to which an individual is likely to be exposed to when exposed to a ZIKV. Pharmaceutical Formulations

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention or treatment of ZIKV in humans and other mammals, for example. ZIKV mRNA vaccines can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat infectious disease.

In some embodiments, the ZIKV vaccine containing mRNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen). In some embodiments, a subject is flavivims seropositive. In some embodiments, a subject is flavivims seronegative.

An “effective amount” of a ZIKV vaccine is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (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 ZIKV mRNA 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 ZIKV mRNA vaccine containing RNA polynucleotides having at least one chemical modifications are more efficient than a composition 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 RNA 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 mRNA in a ZIKV vaccine of the present disclosure, in some embodiments, is 10 mg to 250 mg. For example, an effective amount of mRNA in a ZIKV vaccine may be 10-100 mg, 10-30 mg, 30-250 mg, 30-100 mg, or 100-250 mg. In some embodiments, an effective amount of mRNA in a ZIKV vaccine is 10 mg. In some embodiments, an effective amount of mRNA in a ZIKV vaccine is 30 mg. In some embodiments, an effective amount of mRNA in a ZIKV vaccine is 100 mg. In some embodiments, an effective amount of mRNA in a ZIKV vaccine is 250 mg.

The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

In some embodiments, RNA vaccines (including polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment or prevention of ZIKV. The ZIKV mRNA 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 RNA vaccines of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

The ZIKV mRNA vaccine may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster.

In some embodiments, an initial dose of a vaccine is administered followed by a booster dose. A booster dose is a dose that is given at a certain interval after completion of the primary dose or series of doses that is/are intended to boost immunity to, and therefore prolong protection against, the disease (e.g., ZIKV) that is to be prevented. A booster dose may be given after an earlier administration of an immunizing composition. The time of administration between the initial administration of an immunizing composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month (e.g., 28 days, 29 days, 30 days, or 31 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, or more. In some embodiments, the time of administration between the initial administration of the vaccine and the booster may be, but is not limited to, 28 days. In some embodiments, the ZIKV mRNA vaccine may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art.

The ZIKV mRNA vaccine may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and/or prevent a variety of infectious disease. RNA 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.

Provided herein are pharmaceutical compositions including the ZIKV mRNA vaccine and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

The ZIKV mRNA vaccine may be formulated or administered alone or in conjunction with one or more other components. For instance, the ZIKV mRNA vaccine may comprise other components including, but not limited to, adjuvants.

In some embodiments, the ZIKV mRNA vaccine does not include an adjuvant (they are adjuvant free).

The ZIKV mRNA vaccine may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccines comprise at least one additional active substances, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. 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).

In some embodiments, the ZIKV mRNA vaccine are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example,

RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens.

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 polynucleotide) into association with an excipient and/or one or more other accessory ingredients, 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, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition 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 composition is to be administered. By way of example, the composition 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) active ingredient.

In some embodiments, the ZIKV mRNA vaccine 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 ZIKV mRNA vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

Dosing/Administration

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of ZIKV in humans and other mammals. The ZIKV vaccine can be used as therapeutic or prophylactic agents. In some aspects, the RNA vaccines of the disclosure are used to provide prophylactic protection from ZIKV. In some aspects, the RNA vaccines of the disclosure are used to treat a ZIKV infection. In some embodiments, the ZIKV mRNA vaccine of the present disclosure is used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.

In some embodiments, the ZIKV 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 immune response. The RNA encoding the ZIKV antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.

Prophylactic protection from ZIKV can be achieved following administration of the ZIKV mRNA vaccine of the present disclosure. Vaccines can be administered 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 possible, although less desirable, to administer the vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

A method of eliciting an immune response in a subject against ZIKV is provided in aspects of the present disclosure. The method involves administering to the subject a ZIKV mRNA vaccine comprising at least one mRNA having an open reading frame encoding at least one ZIKV antigen, thereby inducing in the subject an immune response specific to a ZIKV antigen, 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 ZIKV. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.

A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. 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 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 ZIKV 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 ZIKV or an unvaccinated subject.

A method of eliciting an immune response in a subject against ZIKV is provided in other aspects of the disclosure. The method involves administering to the subject the ZIKV mRNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigen, thereby inducing in the subject an immune response specific to ZIKV antigen, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the ZIKV at 2 times to 100 times the dosage level relative to the RNA 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 the ZIKV mRNA 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 three times the dosage level relative to the ZIKV mRNA 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 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to the ZIKV mRNA 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 10 times to 1000 times the dosage level relative to the ZIKV mRNA 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 100 times to 1000 times the dosage level relative to the ZIKV mRNA vaccine.

In other 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 ZIKV transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.

Other aspects the disclosure provide methods of eliciting an immune response in a subject against ZIKV by administering to the subject the ZIKV mRNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigen, thereby inducing in the subject an immune response specific to ZIKV antigen, wherein 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 prophylactically effective dose of a traditional vaccine against ZIKV. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.

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.

Also provided herein are methods of eliciting an immune response in a subject against a ZIKV by administering to the subject the ZIKV mRNA vaccine having an open reading frame encoding a first antigen, wherein the RNA polynucleotide does not include a stabili ation element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

The ZIKV mRNA vaccine may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The ZIKV mRNA vaccine is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the ZIKV mRNA vaccine may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The vaccine of the present disclosure are administered to a subject in an effective amount to induce an immune response, such as a neutralizing antibody response, in the subject. In some embodiments, the effective amount of mRNA is 10 pg to 250 pg, or 20 pg to 500 pg. For example, an effective amount of mRNA may be 10-100 pg, 10-30 pg, 30-250 pg, 30-100 pg, or 100-250 pg administered as a single dose or as two (or more) doses. In some embodiments, an effective amount of mRNA is a single dose of 10 pg. In some embodiments, an effective amount of mRNA is a first dose of 10 pg and a second dose of 10 pg. In some embodiments, an effective amount of mRNA is a single dose of 30 pg. In some embodiments, an effective amount of mRNA is a first dose of 30 pg and a second dose of 30 pg. In some embodiments, an effective amount of mRNA is a single dose of 100 pg. In some embodiments, an effective amount of mRNA is a first dose of 100 pg and a second dose of 100 pg. In some embodiments, an effective amount of mRNA is a single dose of 250 pg. In some embodiments, an effective amount of mRNA is a first dose of 250 pg and a second dose of 250 pg. The ZIKV mRNA vaccine 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).

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of the ZIKV mRNA vaccine, wherein the ZIKV mRNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an anti-ZIKV antigen). “An effective amount” is a dose of the ZIKV mRNA vaccine 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) ZIKV 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-ZIKV antigen antibody titer produced in a subject administered the ZIKV mRNA 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-ZIKV antigen) 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 the ZIKV mRNA vaccine.

In some embodiments, an anti-ZIKV antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-ZIKV 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-ZIKV 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- ZIKV antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-ZIKV 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-ZIKV antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-ZIKV antigen 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-ZIKV 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-ZIKV antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the anti-ZIKV 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, antibody-mediated immunogenicity in a subject is assessed at one or more time points. Methods of assessing antibody-mediated immunogenicity are known and include geometric mean concentration (GMC) of antibody to antigen, geometric mean fold rise (GMFR) in serum antibody, geometric mean titer (GMT), median, minimum, maximum, 95% confidence interval (Cl), geometric mean ratio (GMR) of post-baseline/baseline titers, and seroconversion rate.

The GMC is the average antibody concentration for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data. GMT is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data. In some embodiments, antibody-mediated immunogenicity in a subject is assessed using the plaque reduction neutralization test (PRNT). In some embodiments, antibody-mediated immunogenicity in a subject is assessed using the microneutralization (MN) assay. Seroconversion, in some embodiments, is assessed using the PRNT and/or the MN assay.

In some embodiments, seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below the lower limit of quantification (LLOQ) to a PRNT equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. In other embodiments, seroconversion is defined as a change in microneutraliztion (MN) from below the lower limit of quantification (LLOQ) to a MN equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. For both the PRNT and the MN assay, values lower than the LLOQ are assigned a value of 50% of the LLOQ.

In some embodiments, a subject is administered a ZIKV vaccine comprising 10 pg of mRNA. In some embodiments, a second dose of the vaccine is administered to the subject at least 28 days following a first dose.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 150-250, e.g., 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or 250. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 195.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Month 7, following the first dose and the second dose of the vaccine, is 10-110, e.g., 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75,

80, 85, 90, 95, 100, 105, or 110. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Month 7, following the first dose and the second dose of the vaccine, is 38.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 29, following the first dose of the vaccine, is 25-125, e.g., 25, 30, 35,

40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,

120, 125, 130, 134, 140, 145, or 150. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 29, following the first dose of the vaccine is, 57. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 1150-1250, e.g., 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1191, 1192, 1193, 1194,

1195, 1196, 1197, 1198, 1199, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, or 1250. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 1195.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Month 7, following the first dose and the second dose of the vaccine, is 100-200, e.g., 100, 105, 110, 115, 120, 125, 130, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seronegative subject at Month 7, following the first dose and the second dose of the vaccine, is 141.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seropositive subject at Day 29, following the first dose of the vaccine, is 100-200, e.g., 100, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims positive subject at Day 29, following the first dose of the vaccine, is 148.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 150-250, e.g., 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 121, 222, 223, 224, 225, 226, 227, 228, 229, 230, 235, 240, 245, 250. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims positive subject at Day 57, following the first dose and the second dose of the vaccine, is 224.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims seropositive subject at Month 7, following the first dose and the second dose of the vaccine, is 25-125, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a flavivims positive subject at Month 7, following the first dose and the second dose of the vaccine, is 68.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivims seropositive subject at Day 29, following the first dose of the vaccine, is 350-450, e.g., 355, 360, 365, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, or 450. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivirus positive subject at Day 29, following the first dose of the vaccine, is 375.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivirus seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 600-700, e.g., 605, 610, 615, 620, 625, 630, 635, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 655, 670, 675, 680, 685, 690, or 700. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivirus positive subject at Day 57, following the first dose and the second dose of the vaccine, is 646.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivirus seropositive subject at Month 7, following the first dose and the second dose of the vaccine, is 200-300, e.g., 200, 205, 210, 220, 225, 230, 235, 240, 245, 250, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 275, 280, 285, 290, 295, or 300. In some embodiments, the MN50 GMT of neutralizing antibody induced in a flavivirus positive subject at Month 7, following the first dose and the second dose of the vaccine, is 263.

In some embodiments, a subject is administered a ZIKV vaccine comprising 30 pg of mRNA. In some embodiments, a second dose of the vaccine is administered to the subject at least 28 days following a first dose.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seronegative subject at Day 29, following the first dose and the second dose of the vaccine, is 5- 30, e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seronegative subject at Day 29, following the first dose and the second dose of the vaccine, is 5 14.

In some embodiments, the PRNT50 GMTof neutralizing antibody induced in a seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 250-350, e.g., 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 301, 302, 303, 304305,

306, 307, 308, 309, 310, 315, 320, 325, 330, 335, 340, 345, or 350. In some embodiments, the PRNT50 GMTof neutralizing antibody induced in a seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 303.

In some embodiments, the MN50 GMTof neutralizing antibody induced in a seronegative subject at Day 29, following the first dose of the vaccine, is 100-200, e.g., 105, 110, 115, 120, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200. In some embodiments, the MN50 GMTof neutralizing antibody induced in a seronegative subject at Day 29, following the first dose of the vaccine, is 130. In some embodiments, the MN50 GMT of neutralizing antibody induced in a seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 1400-1500, e.g., 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455,

1460, 1465, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1485, 1490, 1495, or 1500. In some embodiments, the MN50 GMT of neutralizing antibody induced in a seronegative subject at Day 57, following the first dose and the second dose of the vaccine, is 1478.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seropositive subject at Day 29, following the first dose of the vaccine, is 50-150, e.g., 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seropositive subject at Day 29, following the first dose of the vaccine, is 88.

In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 100-200, e.g., 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200. In some embodiments, the PRNT50 GMT of neutralizing antibody induced in a seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 151.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a seropositive subject at Day 29, following the first dose of the vaccine, is 150-250, e.g., 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 240, 245, or 250. In some embodiments, the MN50 GMT of neutralizing antibody induced in a seropositive subject at Day 29, following the first dose of the vaccine, is 227.

In some embodiments, the MN50 GMT of neutralizing antibody induced in a seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 550-650, e.g., 550, 555, 560, 565, 570, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, or 650. In some embodiments, the MN50 GMT of neutralizing antibody induced in a seropositive subject at Day 57, following the first dose and the second dose of the vaccine, is 579.

In some embodiments, subjects of a population are administered a ZIKV vaccine comprising 10 pg of mRNA. In some embodiments, subjects of a population are administered a ZIKV vaccine comprising 30 pg of mRNA. In some embodiments, subjects of a population are administered a ZIKV vaccine comprising 100 pg of mRNA. In some embodiments, subjects of a population are administered a ZIKV vaccine comprising 150 pg of mRNA. In some embodiments, a second dose of the vaccine is administered to the subjects at least 28 days following a first dose. In some embodiments, the subjects are seropositive. In some embodiments, the subjects are seronegative.

In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 29 following the first dose and the second dose of the vaccine, as assessed by the PRNT or MN assay.

In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, as assessed by the PRNT or MN assay.

In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, as assessed by the PRNT or MN assay.

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 by Day 29 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay.

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 by Day 29 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay.

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 by Day 57 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay.

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 by Day 57 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay. 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 by Month 7 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay.

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 by Month 7 following vaccination, relative to baseline, as assessed by the PRNT or the MN assay.

Neutralizing antibody response to ZIKV, in some embodiments, is assessed on Day 29, Day 57, Month 7, and/or Month 12 post vaccination (e.g., initial dose of vaccine).

A control, in some embodiments, is the anti-ZIKV antigen antibody titer produced in a subject who has not been administered the ZIKV mRNA vaccine. In some embodiments, a control is an anti-ZIKV antigen antibody titer produced in a subject administered a recombinant or purified ZIKV 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 the ZIKV mRNA vaccine to be effective is measured in a murine model. For example, the ZIKV mRNA 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, the ZIKV mRNA vaccine may be administered to a murine model, the murine model challenged with ZIKV, 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 the ZIKV mRNA vaccine is a dose that is reduced compared to the standard of care dose of a recombinant ZIKV 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 ZIKV protein vaccine, or a live attenuated or inactivated ZIKV vaccine, or a ZIKV VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent ZIKV, or a ZIKV- related condition, while following the standard of care guideline for treating or preventing ZIKV, or a ZIKV -related condition.

In some embodiments, the anti-ZIKV antigen antibody titer produced in a subject administered an effective amount of the ZIKV mRNA vaccine is equivalent to an anti-ZIKV antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified ZIKV protein vaccine, or a live attenuated or inactivated ZIKV vaccine, or a ZIKV 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 Jun 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 ZIKV mRNA vaccine is at least 60% relative to unvaccinated control subjects. For example, efficacy of the ZIKV mRNA 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 ZIKV mRNA 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 the ZIKV mRNA 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 the ZIKV mRNA 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 the ZIKV mRNA vaccine of the present disclosure is sufficient to produce detectable levels of ZIKV 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-ZIKV 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 the ZIKV mRNA vaccine of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the ZIKV 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 ZIKV 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 ZIKV 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-ZIKV antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, an anti-ZIKV 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-ZIKV antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, an anti-ZIKV 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 ZIKV vaccine, an inactivated ZIKV vaccine, or a protein subunit ZIKV vaccine.

Additional Embodiments

1. A method comprising administering to a subject a vaccine comprising 10 pg-250 pig of a messenger ribonucleic acid (mRNA) comprising: (a) an open reading frame (ORF) that encodes a Zika virus (ZIKV) prME protein; and (b) a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid, wherein a neutralizing antibody response to ZIKV is produced in the subject.

2. The method of paragraph 1, wherein the ZIKV prME protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

3. The method of paragraph 2, wherein the ZIKV prME protein comprise the amino acid sequence of SEQ ID NO: 7.

4. The method of any one of the preceding paragraphs, wherein the ORF comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 1.

5. The method of paragraph 4, wherein the ORF comprise the nucleotide sequence of SEQ ID NO: 1.

6. The method of any one of the preceding paragraphs, wherein the mRNA comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the nucleotide sequence of SEQ ID NO: 2.

7. The method of paragraph 6, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2. 8. The method of any one of the preceding paragraphs, wherein the mixture of lipids comprises 45-55 mol% ionizable cationic lipid, 15-20 mol% non-cationic lipid, 35-45 mol% sterol, and 0.5-5 mol% PEG-modified lipid.

9. The method of paragraph 8, wherein the mixture of lipids comprises 50 mol% ionizable cationic lipid, 10 mol% non-cationic lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid.

10. The method of any one of the preceding paragraphs, wherein ionizable cationic lipid is a Compound I ionizable cationic lipid, the non-cationic lipid is DSPC, the sterol is cholesterol, and the PEG-modified lipid is PEG-DMG.

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

12. The method of any one of the preceding paragraphs, wherein the composition further comprises Tris buffer, propylene glycol, and diethylenetriamine pentaacetic acid (DTPA).

13. The method of paragraph 12, wherein the composition further comprises 100 mM Tris buffer, 7% propylene glycol, and 1 mM DTPA.

14. The method of any one of paragraphs 1-13, wherein the composition comprises 10 pg, 30 pg, 100 pg, or 250 pg of the mRNA.

15. The method of any one of the preceding paragraphs, wherein the subject is 18 to 49 years of age.

16. The method of any one of the preceding paragraphs, wherein a second dose of the vaccine is administered to the subject following a first dose, optionally 28 days following the first dose.

17. The method of any one of the preceding paragraphs, wherein the vaccine is administered intramuscularly.

18. The method of any one of paragraphs 14-17, wherein the composition comprises 10 pg of the mRNA.

19. The method of paragraph 18, wherein a second dose of the vaccine is administered to the subject at least 28 days following the first dose.

20. The method of paragraph 18 or 19, wherein the subject is flavivims seronegative.

21. The method of paragraph 20, wherein the plaque reduction neutralization test 50 (PRNT50) geometric mean titer (GMT) of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 180 - 210, 190 - 200, or 195.

22. The method of paragraph 20, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 20-50, 30-40, or 38. 23. The method of any one of paragraphs 20-22, wherein the microneutralization assay 50 (MN50) GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 40 - 70, 50 - 60, or 57.

24. The method of any one of paragraphs 20-23, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 1180 - 1210, 1190 - 1200, or 1195.

25. The method of any one of paragraphs 20-23, wherein the MN50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 130-160, 140-150, or 141.

26. The method of paragraph 18 or 19, wherein the subject flavivims seropositive.

27. The method of paragraph 26, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 135 - 155, 140-150, or 148.

28. The method of paragraph 26 or 27, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 215-235, 220-230, or 224.

29. The method of any one of paragraphs 26-28, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 50-80, 60-70, or 68.

30. The method of any one of paragraphs 26-29, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 360-390, 370-380, or 375.

31. The method of any one of paragraphs 26-30, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 630-660, 640-650, or 646.

32. The method of any one of paragraphs 26-31, wherein the MN50 GMT of neutralizing antibody induced in the subject at Month 7, following the first dose and the second dose of the vaccine, is 250-280, 260-270, or 263.

33. The method of paragraph 18, further comprising administering to subjects of a population a second dose of the vaccine at least 28 days following administration of a first dose of the vaccine.

34. The method of paragraph 33, wherein at least 70%, at least 75%, or at least 80% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below a lower limit of quantification (LLOQ) to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

35. The method of paragraph 33 or 34, wherein at least 55%, at least 60%, or at least 65% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below a lower limit of quantification (LLOQ) to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

36. The method of any one of paragraphs 33-35, wherein at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in microneutralization (MN) from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

37. The method of any one of paragraphs 33-36, wherein at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in microneutralization (MN) from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

38. The method of any one of paragraphs 33-36, wherein the subjects are flavivirus seronegative.

39. The method of paragraph 32, wherein at least 80%, at least 85%, or at least 90% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16.

40. The method of paragraph 38 or 39, wherein at least 55%, at least 60%, or at least 65% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16.

41. The method of any one of paragraphs 38-40, wherein at least 60%, at least 65%, or at least 70% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

42. The method of any one of paragraphs 38-41, wherein at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28. 43. The method of any one of paragraphs 38-42, wherein at least 90%, at least 95%, or 100% of the subjects have seroconverted by Month 7 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

44. The method of any one of paragraphs 33-36, wherein the subjects are flavivims seropositive.

45. The method of paragraph 44, wherein at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by PRNT.

46. The method of paragraph 44 or 45, wherein at least 28 days following a first dose of the vaccine, and at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

47. The method of any one of paragraphs 44-46, wherein at least 28 days following a first dose of the vaccine, and at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

48. The method of any one of paragraphs 44-47, wherein at least 28 days following a first dose of the vaccine, and at least 40%, at least 45%, or at least 50% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

49. The method of any one of paragraphs 44-48, wherein at least 90%, at least 95%, or 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN.

50. The method of any one of paragraphs 44-49, wherein at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN.

51. The method of any one of paragraphs 44-50, wherein at least 45%, at least 50%, or at least 55% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by MN.

52. The method of any one of paragraphs 44-51, wherein at least 90%, at least 95%, or at least 100% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Month 7 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by

MN.

53. The method of any one of paragraphs 14-17, wherein the composition comprises 30 pg of the mRNA.

54. The method of paragraph 53, wherein a second dose of the vaccine is administered to the subject at least 28 days following the first dose.

55. The method of paragraph 53 or 54, wherein the subject is flavivirus seronegative.

56. The method of paragraph 55, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose and the second dose of the vaccine, is 5-30, 10-20, or 14.

57. The method of paragraph 55 or 56, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 285-320, 295-310, or 303.

58. The method of any one of paragraphs 55-57, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 115-145, 125-135 or 130.

59. The method of any one of paragraphs 55-58, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 1455-1495, 1465-1485, or 1478.

60. The method of paragraph 53 or 54, wherein the subject flavivirus seropositive.

61. The method of paragraph 60, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 70-105, 80-95, or 88.

62. The method of paragraph 60 or 61, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is

135-165, 145-155, or 151.

63. The method of any one of paragraphs 60-62, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 29, following the first dose of the vaccine, is 215-245, 225-235, or 227.

64. The method of any one of paragraphs 60-63, wherein the MN50 GMT of neutralizing antibody induced in the subject at Day 57, following the first dose and the second dose of the vaccine, is 565-595, 575-585, or 579.

65. The method of paragraph 53, further comprising administering to subjects of a population a second dose of the vaccine at least 28 days following administration of a first dose of the vaccine. 66. The method of paragraph 65, wherein at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below a LLOQ to a PRNT equal to or above the LLOQ, and the LLOQ for the PRNT is 16.

67. The method of paragraph 65 or 66, wherein at least 85%, at least 90%, or at least 95% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN is 28.

68. The method of any one of paragraphs 65-67, wherein the subjects are flavivims seronegative.

69. The method of paragraph 68, wherein at least 30%, at least 35%, or at least 40% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16.

70. The method of paragraph 68 or 69, wherein at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, wherein seroconversion is defined as a change in PRNT from below the LLOQ to a PRNT equal to or above LLOQ, and the LLOQ for the PRNT is 16.

71. The method of any one of paragraphs 68-70, wherein at least 75%, at least 80%, or at least 85% of the subjects have seroconverted by Day 29 following the first dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

72. The method of any one of paragraphs 68-71, wherein at least 90%, at least 95%, or 100% of the subjects have seroconverted by Day 57 following the first dose and the second dose of the vaccine, is defined as a change in MN from below the LLOQ to a MN equal to or above LLOQ, and the LLOQ for the MN assay is 28.

73. The method of any one of paragraphs 65-67, wherein the subjects are flavivims seropositive.

74. The method of paragraph 73, wherein at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by PRNT.

75. The method of paragraph 73 or 74, wherein at least 28 days following a first dose of the vaccine, and at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

76. The method of any one of paragraphs 73-75, wherein at least 28 days following a first dose of the vaccine, and at least 65%, at least 70%, or at least 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 57 following the first dose and the second dose of the vaccine, relative to baseline, as assessed by PRNT.

77. The method of any one of paragraphs 73-76, wherein at least 85%, at least 70%, or 75% of the subjects achieve an at least 2-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN.

78. The method of any one of paragraphs 73-77, wherein at least 85%, at least 70%, or 75% of the subjects achieve an at least 4-fold increase in neutralizing antibody titer Day 29 following the first dose of the vaccine, relative to baseline, as assessed by MN.

79. A vaccine comprising 10 pg - 250 pig of a messenger ribonucleic acid (mRNA) comprising: (a) an open reading frame (ORF) that encodes a Zika vims (ZIKV) prME protein, wherein the ORF comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1; and (b) a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

80. A vaccine comprising 10 pg - 30 pg of a messenger ribonucleic acid (mRNA) comprising: (a) an open reading frame (ORF) that encodes a Zika virus (ZIKV) prME protein, wherein the ORF comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1; and (b) a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

81. The vaccine of any one of paragraphs 79-80, wherein the ZIKV prME protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

82. The vaccine of paragraph 81, wherein the ZIKV prME protein comprise the amino acid sequence of SEQ ID NO: 7.

83. The vaccine of any one of paragraphs 79-82, wherein the ORF comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the amino acid sequence of SEQ ID NO: 1.

84. The vaccine of paragraph 83, wherein the ORF comprise the nucleotide sequence of SEQ ID NO: 1. 85. The vaccine of any one of paragraphs 79-84, wherein the mRNA comprises a nucleotide sequence having at least 90%, at least 95%, at least 98% identity to the nucleotide sequence of SEQ ID NO: 2.

86. The vaccine of paragraph 85, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2.

87. The vaccine of any one of paragraphs 79-86, wherein the mixture of lipids comprises 45- 55 mol% ionizable cationic lipid, 15-20 mol% non-cationic lipid, 35-45 mol% sterol, and 0.5-5 mol% PEG-modified lipid.

88. The vaccine of paragraph 87, wherein the mixture of lipids comprises 50 mol% ionizable cationic lipid, 10 mol% non-cationic lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid.

89. The vaccine of any one of paragraphs 79-88, wherein ionizable cationic lipid is a Compound I ionizable cationic lipid, the non-cationic lipid is DSPC, the sterol is cholesterol, and the PEG-modified lipid is PEG-DMG.

90. The vaccine of any one of paragraphs 79-89, wherein the mRNA comprises a 1- methylpseudourine chemical modification.

91. The vaccine of any one of paragraphs 79-90, further comprising Tris buffer, propylene glycol, and diethylenetriamine pentaacetic acid (DTP A).

92. The vaccine of paragraph 91, comprising 100 mM Tris buffer, 7% propylene glycol, and 1 mM DTPA.

93. The vaccine of any one of paragraphs 79-92, comprising 10 pg of the mRNA.

94. The vaccine of any one of paragraphs 79-93, comprising 30 pg of the mRNA.

95. The vaccine of any one of paragraphs 79-93, comprising 100 pg of the mRNA.

96. The vaccine of any one of paragraphs 79-93, comprising 250 pg of the mRNA.

EXAMPLES

Phase 1 Study

This is a Phase 1, first-in-human (FIH), randomized, observer-blind, placebo-controlled, dose-ranging study to evaluate the safety, tolerability and immunogenicity of a Zika virus (ZIKV) mRNA vaccine (SEQ ID NO: 2), administered intramuscularly (IM) on a 2-dose vaccination schedule 28 days apart in healthy adults (18 through 49 years of age). A total of 120 participants (30 participants per cohort) are enrolled into 1 of 4 vaccine dose cohorts (10, 30, 100 or 250 meg).

The study comprises a Screening Phase (up to 28 days), a Vaccination Phase (up to 57 days), and a Long-Term Follow-up Phase (up to 12 months after last vaccination). Participants are screened and stratified for their baseline flavivirus serological status prior to vaccine administration. Study vaccine dosing began with Cohort 1 (10 pg vaccine or placebo), followed sequentially by Cohort 2 (30 pg vaccine or placebo), Cohort 3 (100 pg vaccine or placebo), and Cohort 4 (250 pg vaccine or placebo).

For all participants receiving the ZIKV mRNA vaccine, independent of serostatus at baseline, the seroconversion rate was 86.4% (65.1, 97.1%) for the 10 pg cohort and at 95.5% (77.2, 99.9%) for the 30 pg. MN data were consistent with PRNT50 data.

Objectives and Endpoints

Primary Objective

The primary objective of the study is to evaluate the safety, tolerability, and reactogenicity of a 2-dose vaccination schedule of the ZIKV mRNA vaccine, given 28 days apart, across a range of dose levels in flavivirus- seronegative and flavivirus-seropositive participants compared with placebo.

Primary Safety Endpoints

• Frequency and grade of each solicited local and systemic reactogenicity AE during a7- day follow-up period after each vaccination

• Frequency and grade of any unsolicited AEs during the 28 day follow up period after each vaccination

• Frequency of any MAAE, SAE and AESI from Day 1 to the EOS Visit at Month 13

Secondary Objective

To evaluate the immunogenicity of a 2-dose vaccination schedule of the ZIKV mRNA vaccine, given 28 days apart as measured by ZIKV-specific neutralization assay (Plaque Reduction Neutralization Test, [PRNT]).

Exploratory Objectives

To evaluate the immunogenicity of a 2-dose vaccination schedule of ZIKV mRNA vaccine, given 28 days apart, as measured by enzyme-linked immunosorbent assay (ELISA) and additional ZIKV-specific neutralization assays, including microneutralization (MN) and the reporter viral particle neutralization (RVP) assays.

Study Design and Methodology

This is a Phase 1, randomized, observer-blind, placebo-controlled, dose-ranging study to evaluate the safety, tolerability, and immunogenicity of the ZIKV mRNA vaccine administered to healthy flavivirus-seropositive and seronegative adult participants (18 to 49 years of age, inclusive). The study will comprise a Screening Phase (up to 28 days), a Vaccination Phase (up to 57 days), and a Long-Term Follow-up Phase (up to 12 months after last vaccination). Participants will have approximately 8 clinic visits with an additional 13 safety telephone calls. Study duration will be approximately 13 months for each participant. Participants will provide written informed consent before any study- specific procedures are performed.

A total of 120 participants (30 participant per cohort) will be enrolled into 1 of 4 vaccine dose cohorts (10, 30, 100, or 250 pg). Within each cohort, eligible participants will be randomly assigned to vaccine or placebo (4:1 vaccine to placebo) and administered the study vaccine as a 0.5-mL intramuscular (IM) injection on a 2-dose vaccination schedule, 28 days apart (Day 1 and Day 29). Participants will be stratified by baseline flavivirus serostatus (seropositive and seronegative).

For each dose cohort (Cohorts 1 through 4), Safety Oversight will be performed 7 days after the first 5 participants (all seronegative) are randomly assigned and receive their first study vaccination (Day 1). Once safety is confirmed, the remaining 25 participants in the dose level cohort will be randomly assigned to dosing. Seven days after all 30 participants in the cohort have received the first study vaccination, a blinded 1ST that is not directly involved in the day-to- day activities of the study, will review all available safety data for the currently dosed cohort and any cumulative safety data of all cohorts as the trial advances to determine the acceptability to escalate to the next vaccine dose level. The 30 participants in the first cohort will continue to receive their second study vaccine dose on Day 29 (+7 days).

The blinded 1ST will oversee the safety of the trial and will review safety data to ensure adherence to the protocol, will monitor safety laboratory test results and reactogenicity, and may request input from the SMC should the study meet pause rules or for any other study events that could potentially affect participant safety. The 1ST will approve escalation to the next higher dosing cohort after review of blinded safety data of the currently dosed cohort through 7 days after the first vaccination and any cumulative safety data of all cohorts as the trial advances.

At each vaccination visit (Day 1 and Day 29), a diary card will be provided to the participant and study staff will provide training on its proper use. Participants will record daily body temperature, any solicited local (injection site) and systemic AEs (solicited AEs), any unsolicited AEs, and any concomitant medications and vaccinations on the day of each vaccination and on 7 subsequent days. Participants will be instructed to return the completed diary card to the Investigator at the subsequent planned study visit (Day 8 and Day 36). Participants will record on the same diary card daily any unsolicited AEs experienced, daily body temperature, and concomitant medications and vaccinations (excluding vitamins and minerals) received from 7 through 28 days after each vaccination. Participants will be instructed to return the completed diary card to the Investigator at Day 29 and Day 57. All concomitant medications and vaccinations (excluding vitamins and minerals) received and SAEs, MAAEs, AESIs, and AEs leading to withdrawal from vaccine dosing or from the study will be collected from Day 1 until Month 13 (EOS Visit). Other safety assessments will include clinical laboratory test results (hematology, serum chemistry, and coagulation); vital sign measurements; and physical examination findings. Blood samples for immunogenicity assessments will be collected the day of each vaccination (Days 1 and 29 before vaccination), 28 (+7) days after each vaccination, and during the Long-Term Follow-up Phase at Month 7 (± 14 days) and Month 13 (± 14 days).

Once all subjects from a cohort have completed the Vaccination Phase through Day 57 (±7 days), the database will be locked for that cohort and safety and immunogenicity data will be analyzed through 28 days following the second vaccination by an unblinded statistician. As dose escalation occurs, cumulative analyses will be included for each subsequent data lock to allow for all prior dosing cohorts to be analyzed by dose assignment, and in aggregate for vaccine exposure. Immunogenicity and safety data, including mean group analyses of change from baseline, where applicable, will be summarized for each dose group. These data are required to inform decisions on dose selection for this and other development programs using the same messenger RNA (mRNA) platform.

When all participants have completed their final contact (approximately 12 months after the last vaccination), immunogenicity testing is completed, and all queries resolved, the database will be locked and analyzed with a final clinical study report.

Study Population

Participants (males and females 18 to 49 years of age, inclusive) will be included in the study if they are in good health as determined by medical history, clinical laboratory assessments, vital sign measurements, a physical examination at screening and per investigator judgement. Negative pregnancy tests will be required at screening and before vaccine administration for female participants of childbearing potential. Flavivirus serostatus (positive or negative) will be determined by ELISA or other commercially available serological assay.

Safety Assessments

Safety assessments will include monitoring and recording of solicited AEs (local and systemic reactogenicity events) and unsolicited AEs, serious AEs (SAEs), AEs of special interest (AESIs), AEs leading to study withdrawal, medically attended AEs (MAAEs); clinical laboratory test results (hematology, serum chemistry, and coagulation); vital sign measurements; and physical examination findings. Immunogenicity Assessments

Immunogenicity assessments will include the following:

• Serum neutralizing antibodies (nAb) against ZIKV

• Serum binding antibodies (bAb) against ZIKV

Vaccine, Dosage, and Route of Administration

The ZIKV mRNA vaccine is a novel lipid nanoparticle (LNP)-encapsulated mRNA- based vaccine encoding the full pre-membrane and envelope (prME) structural polyproteins of ZIKV. The ZIKV mRNA vaccine includes an mRNA (SEQ ID NO: 2) formulated with LNPs.

The ZIKV mRNA vaccine is provided as a sterile liquid for injection at a concentration of 0.5 mg/mL in 100 mM Tris buffer, 7% propylene glycol, and 1 mM diethylenetriamine- pentaacetic acid (DTPA).

The ZIKV mRNA vaccine (10, 30, 100, or 250 pg) and placebo will be prepared as outlined in the pharmacy manual and administered via IM injection (0.5 mL) into the deltoid muscle on designated vaccination days. The second dose of vaccine or placebo will be administered preferably in the same arm used for the first dose.

The placebo is 0.9% sodium chloride injection, United States Pharmacopeia (USP) or British Pharmacopeia (BP).

Sample Size

A total of 120 participants (30 participant per cohort) are planned for enrollment in the study and random assignment to study dosing. The sample size is considered sufficient to meet the study objective of identifying a dose and establishing initial safety results in a population of healthy adults in both endemic and nonendemic Zika regions. Formal sample size calculations were not performed.

Statistical Methods:

Safety: Reactogenicity will be summarized by dosing group (10, 30, 100, or 250 pg vaccine or placebo), vaccination (first or second dose), duration, and severity. Adverse events will be coded by preferred term and system organ class using MedDRA and summarized by dose group, vaccination (first or second dose), and overall. Adverse events will also be summarized by severity and relationship to the study vaccine. Descriptive statistics will be presented, and the difference in the proportion of participants with AEs will be provided, comparing each dose level with placebo pooled across all cohorts. Individual participant listings will be provided for all AEs, AEs leading to study withdrawal, AES Is, MAAEs, and SAEs.

Safety data from clinical laboratory test results and vital sign measurements will be graded by severity scoring and analyzed by dose group and vaccination (first or second). Absolute and change from baseline values will be provided according to the toxicity table, along with mean, median, and standard deviation. Results of serology, urine drug screening, physical examinations, and pregnancy tests will be listed for all participants randomly assigned to receive study vaccine.

Medical history data for all participants randomly assigned to receive study vaccine will be presented by participant in a listing. Baseline demographic and background variables will be summarized by dosing group for all randomly assigned participants. The number of participants who enroll in the study and the number and percentage of participants who complete the study will be presented. Frequency and percentage of participants who withdraw or discontinue from the study and the reason for withdrawal or discontinuation will also be summarized.

Prior and concomitant medications will be listed (with start and stop dates) for each participant and summarized by common medical dictionary coding. Any vaccinations that occur during the trial will also be captured and summarized.

Immunogenicity: The following secondary immunogenicity outcome measures and the 95% confidence intervals, where appropriate, will be summarized by dose group and by visit:

• Serum neutralizing antibodies (nAb titers) against ZIKV by PRNT:

• Geometric mean titer (GMT) of nAb against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by PRNT.

• GMT of nAb in initially seronegative participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by PRNT.

• GMT of nAb in initially seropositive participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by PRNT.

• Percentage of participants who seroconverted from Day 1 (baseline) to Day 29, from Day 1 to Day 57, from Day 1 to Month 7, and from Day 1 to Month 13. A seroconversion is defined as a change of plaque reduction neutralization test (PRNT) from below the limit of quantification (LOQ) to a PRNT equal to or above 1:10, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers.

• Proportion of initially seronegative participants with a seroresponse at Day 29, Day 57, Month 7, and Month 13 as measured by PRNT.

• Proportion of initially seropositive participants with a 2-fold or 4-fold increase in nAb titers as compared with baseline as measured by PRNT.

• The following exploratory immunogenicity outcome measures and the 95% confidence intervals, where appropriate, will be summarized by dosing group and by visit: • GMT of nAb against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by additional neutralization assay.

• GMT of nAb in initially seronegative participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by additional neutralization assay.

• GMT of nAb in initially seropositive participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by additional neutralization assay.

• Percentage of participants who seroconverted from Day 1 (baseline) to Day 29, from Day 1 to Day 57, from Day 1 to Month 7, and from Day 1 to Month 13. A seroconversion is defined as change of nAb titer (by additional neutralization assay) from below the LOQ to a titer equal to or above 1:10, or a multiplication by at least 4 in pre-existing neutralizing titer.

• Proportion of initially seronegative participants with a seroresponse at Day 29, Day 57, Month 7, and Month 13 as measured by additional neutralization assay.

• Proportion of initially seropositive participants with a 2-fold or 4-fold increase in nAb as compared with baseline as measured by additional neutralization assay.

• GMT of bAb against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by ELISA binding assay.

• GMT of bAb in initially seronegative participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by ELISA binding assay.

• GMT of bAb in initially seropositive participants against ZIKV at Day 1, Day 29, Day 57, Month 7, and Month 13 as measured by ELISA binding assay.

• Percentage of participants that seroconverted from Day 1 (baseline) to Day 29, from Day 1 to Day 57, from Day 1 to Month 7, and from Day 1 to Month 13. A seroconversion is defined as a change of binding antibody titer from below the LOQ to a binding antibody titer equal or above the LOQ, or a multiplication by at least 4 in pre-existing bAb titers.

• Proportion of initially seronegative participants with a seroresponse at Day 29, Day 57, Month 7, and Month 13 as measured by ELISA binding assay.

• Proportion of initially seropositive participants with a 2-fold or 4-fold increase in bAb as compared with baseline as measured by ELISA binding assay.

• IgG and IgM antibodies against envelope- and NS1- based antigens present in serum collected at baseline and at end of study measured as antibody binding by ELISA or equivalent methodology to flavivimses. Summary of Results - Cohort 1: 10 mg Dose Level

This summary presents the interim safety and immunogenicity data for Cohort 1 (10 pg) of the ZIKV mRNA vaccine study through Month 7, approximately 6 months post-second vaccine administration.

The ZIKV mRNA vaccine 10 pg dose level has an acceptable safety profile. No severe adverse events and no adverse events of special interest were reported during the study period. Neither a second dose, administered 28 days post first vaccine administration, nor a flavivirus positive baseline serostatus seem to affect this profile, although the number of the initially seropositive participants is limited.

The ZIKV mRNA vaccine 10 pg dose level induces a strong neutralizing antibody response both in flavivirus infection naive participants and in participants with pre-existing flavivirus infection. Of all participants, 81.8% seroconverted (PRNT) after completing the vaccination series (Day 57). The ZIKV-specific neutralizing antibody response persisted at Month 7 in flavivirus baseline seronegative and seropositive participants. Overall, 61.9% (39.4, 81.9%) and 95.2% (76.2, 99.9%) of participants remained seroconverted compared to baseline as measured by PRNT and MN assays, respectively.

Demographics and Population set

Demographic and baseline characteristics were generally balanced across the cohort and showed a mean age of 36.4 years (range: 21-49) in the vaccine treatment arm. More females than males were included in this cohort (sex ratio: 24/6). All five baseline flavivirus seropositive participants were enrolled at the Puerto Rico investigational site. The other demographic parameters (height, weight, BMI) were quite homogenous across the cohort.

Thirty participants were enrolled and received one vaccine administration; 29 of these participants (23 in the vaccine treatment arm corresponding to 19 baseline flavivirus seronegative and 4 baseline flavivirus seropositive; 6 in placebo arm corresponding to 5 baseline flavivirus seronegative and 1 baseline flavivirus seropositive) received two vaccine doses. One participant (baseline flavivirus seronegative) declined to receive the second dose, however agreed to continue in the trial as planned through their 12-month follow-up. No participants have yet been lost to follow up.

Safety

Solicited safety data were collected through 7 days after each vaccination and are based on the Solicited Safety Set. Unsolicited events were collected through 28 days after each vaccination and are based on the Exposed Set. The solicited safety set following the first vaccination includes 24 participants in the vaccine treatment and 6 in the saline Placebo treatment arm. The second vaccination solicited safety set includes 23 participants in the vaccine treatment and 6 in the saline placebo treatment arm.

Solicited Local Adverse Events

The most frequently reported adverse reaction was local pain at injection site which was reported by 12 participants (50%) in the vaccine treatment arm after the first administration and 8 participants (34.8%) after the second vaccine administration, all were of Grade 1 or 2. No local erythema and no local induration were reported. Although it is difficult to conclude given the limited number of flavivirus seropositive participants, it seemed that the solicited local reactions were slightly more frequent in the seronegative participants.

Solicited Systemic Adverse Events

Following the first vaccine administration, fatigue (8 participants, 33.3%), headache (7 participants, 29.2%) and myalgia (5 participants, 20.8%) were the most common solicited systemic AEs. After the second vaccine administration the most frequent solicited systemic AEs were myalgia (7 participants, 30.4%), fatigue (6 participants, 26.1%) and headache (5 participants, 21.7%). Fever was reported once each following the first and second administration. No Grade 3 solicited AEs were reported. No events were reported in the diary cards for rash as a solicited adverse reaction. Solicited systemic adverse events were observed in a larger proportion of seronegative than seropositive participants.

Unsolicited Adverse Events

Nineteen Unsolicited Treatment-Emergent Adverse Events (TEAEs) attributed to 9 participants (37.5%) were reported in the vaccine treatment arm compared to 3 events in 2 participants (33.3%) in the placebo arm. Among those, one case of a generalized rash was reported. All were Grade 1 or Grade 2 and none were considered related to the vaccine by the PI. Two MAAEs were reported in the vaccine treatment arm and 3 in the placebo arm; none were considered treatment related. No SAEs and no AESI were reported.

Laboratory Abnormalities

Inclusion criteria for the study required that all participants have Grade 0 lab tests at enrollment. No Grade 2 or higher laboratory abnormalities were observed through 7 days post vaccine administration. In total 14 Grade 1 events occurred 7 days post-vaccine administration, however those modifications were considered not clinically relevant.

Immunogenicity

All immunogenicity analysis is based on the Per Protocol (PP) immunogenicity set.

At the 10-pg dose level, the PP immunogenicity set following the first administration (Day 29) included 24 participants in the vaccine treatment and 6 in the saline Placebo treatment arm. The second vaccination immunogenicity set (Day 57) included 22 participants in the vaccine treatment arm (91.7%) and 6 in the saline placebo treatment arm. The Month 7 immunogenicity set included 21 participants in the vaccine treatment arm (87.5%) and 6 in the saline placebo treatment arm.

Immunogenicity evaluation is performed Day 1, at Day 29, Day 57, Month 7 and Month 13 and measured by the neutralization activity against ZIKV with a Plaque Neutralization assay (PRNT) as secondary endpoint, and by a Microneutralization assay (MN), Reporter Virus Particle neutralization assay (RVP) and enzyme-linked immunosorbent assay (ELISA) as exploratory endpoints. PRNT provides a gold standard in measuring ZIKV- specific neutralizing antibodies and has served to define protective titers for some other flavivirus vaccines such as Yellow Fever and Japanese Encephalitis, however it is labor-intensive and requires biosafety equipment. MN is a high-throughput neutralization assay, modified from a qualified dengue virus micro neutralization assay. To date only the PRNT and MN results are available at Day 1, Day 29, Day 57 and Month 7 and are presented in this summary.

Flavivirus serostatus at enrollment was determined using a commercially available WNV IgG/IgM ELISA. While all participants in the flavivirus seropositive group were enrolled at the Puerto Rico site, the exact etiology of their flavivirus priming is unknown.

ZIKV mRNA Vaccine Neutralizing Antibody Responses by PRNT

Seroconversion is defined as a change in plaque reduction neutralization test (PRNT) from below the lower limit of quantification (LLOQ) to a PRNT equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. The LLOQ for the PRNT assay is 16; values lower than the LLOQ are assigned a value of 50% of the LLOQ. The GMTs for placebo participants were below the LLOQ at all time points.

For participants stratified to the flavivirus seronegative group receiving the ZIKV mRNA vaccine (N=17), the baseline (Day 1) PRNT50 geometric mean titer (GMT) was 9.5 (95% Cl: 6.7, 13.4). No response (GMT<16) was detected at Day 29 following a single vaccine administration. Completing the two-dose vaccination series, the GMT at Day 57 increased to 195.6 (108.4, 352.8). The proportion of initially flavivirus seronegative participants that seroconverted by Day 57 was 94.4%. At Month 7, for participants stratified to the flavivirus seronegative group (N=17) receiving the ZIKA mRNA vaccine, PRNT50 geometric mean titer (GMT) was 38.2 (20, 73) with a seroconversion rate of 64.7% (38.3, 85.8%). The GMTs for placebo participants were below the LLOQ at Day 197 and were assigned a GMT of 8.0, 50% of the LLOQ of 16. At Month 7, the proportion of participants with a result higher than baseline by a factor of 2 was 64.7% (38.3-85.8%), 11 participants out of 17, and the proportion of participants with a result higher than baseline by a factor of 4 was 47.1% (23, 72.2%), 8 participants out of 17.

For participants stratified to the flavivirus seropositive group receiving the vaccine (N=4), the baseline PRNT50 GMT was 41.5 (5, 344.3). Following a single administration of the vaccine GMT increased to 147.9 (6.2, 3507.8), with 50% of participants (2/4) achieving a 4-fold increase in neutralizing antibody titers compared with baseline. Completing the two-dose vaccination series, GMT at Day 57 increased to 224.1 (43.4, 1153.5); 100% of participants (4/4) achieved a 2-fold increase and 50% of participants achieved a 4-fold increase in neutralizing antibody titers compared with baseline. At Month 7, the PRNT50 GMT was 68.8 (3.6, 1321.4), with a seroconversion rate of 50% (6.8, 93.2%). The proportion of participants with a result higher than baseline by a factor of 2 and by a factor of 4 was 50% (6.8, 93.2%), 2 participants out of 4 participants. This demonstrates that even in participants with a prior flavivirus infection, the ZIKV mRNA vaccine is able to induce a ZIKV-specific neutralizing antibody response that persisted above the baseline pre-vaccination value.

In both baseline flavivirus seronegative and seropositive participants, the ZIKV-specific immune response persisted at Month 7. A total of 61.9% (39.4, 81.9%) of the participants seroconverted at Month 7, relative to baseline, compared with 86.4% (65.1, 97.1%) of the participants at Day 57. The GMTs were 200.5 (121.1, 331.9) at Day 57 and 42.7 (23, 79.3) at Month 7.

When using a PRNT80 as readout for the neutralizing activity instead of PRNT50 the results were slightly lower as expected, however the conclusions were the same.

ZIKV mRNA Vaccine Neutralizing Antibody Responses by MN

Seroconversion is defined as a change in microneutraliztion (MN) from below the lower limit of quantification (LLOQ) to a MN equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. The lower limit of quantification (LLOQ) for the MN assay is 28; values lower than the LLOQ are assigned a value of 50% of the LLOQ. The GMTs for placebo participants were below the LLOQ at all time points.

For participants stratified to the flavivirus seronegative group receiving the vaccine (N=17), the baseline (Day 1) MN50 geometric mean titer (GMT) was 14.0. Following a single vaccine administration GMT increased to 57.3 (32, 102.7) with a seroconversion rate of 73.7%. Completing the two-dose vaccination series, GMT at Day 57 increased to 1195.3 (949.3,

1504.9). The proportion of initially flavivirus seronegative participants with a seroresponse at Day 57 was 100%. At Month 7, the MN50 geometric mean titer (GMT) was 141.8 (81.6, 246.7), with a seroconversion rate at 100% (80.5, 100%). The GMTs for placebo participants were below the LLOQ at Month 7 and assigned a value of 14, 50% of the LLOQ. At Month 7, the proportion of participants with a result higher than baseline by a factor of 2 was 100% (80.5, 100%), and the proportion of participants with a result higher than baseline by a factor of 4 was 76.5% (50.1, 93.2%), 13 participants out of 17.

For participants stratified to the flavivirus seropositive group receiving the vaccine (N=4), the baseline MN50 GMT of was 54 (4.5, 646.6). Following a single administration of the vaccine GMTs increased to 375 (32.2, 4362.9), 100% of participants (4/4) achieved a 2-fold increase and 75% of participants achieved a 4-fold increase in neutralizing antibody titers compared with baseline. Completing the two-dose vaccination series, GMT at Day 57 increased to 645.9 (176.3, 2366.3). At Month 7, the MN50 geometric mean titer (GMT) was 263.4 (22.3, 3111.4) with a seroconversion rate at 75% (19.4, 99.4%). At Month 7, the proportion of participants with a result higher than baseline by a factor of 2 was 100% (39.8, 100%), and the proportion of participants with a result higher than baseline by a factor of 4 was 50% (6.8, 93.2%), 2 participants out of 4. Consistent with the PRNT results, the vaccine is able to induce a ZIKV-specific neutralizing antibody response by MN50 in participants with a prior flavivirus infection.

Regardless of baseline flavivirus serostatus, there was a clear benefit from a two-dose series of the vaccine. The seroconversion rates, defined as a change of MN titers from below the LLOQ to a MN level equal or above the LLOQ (i.e. 28) or a multiplication by at least four in participants with pre-existing neutralizing titers, increased from 47.8% at Day 29 to 95.5% at Day 57 and remained at 95.2% (76.2, 99.9%) at Month 7. The GMTs for all participants randomized to receive placebo remained below the LLOQ through Month 7.

Concluding Comments

These interim data corresponding to the first dose level support the following conclusions:

The ZIKV mRNA vaccine 10 pg dose level has an acceptable safety profile. Neither a second dose administered 28 days post the first dose nor a flavivirus positive baseline serostatus seem to affect this profile although the number of the initially seropositive participants is limited. No severe adverse events, adverse events of special interest, and no adverse events leading to participant withdrawal related to ZIKA mRNA vaccine administration were observed during the period from enrollment to six months post second vaccine administration.

The ZIKV mRNA vaccine 10 pg dose level induces a strong neutralizing ZIKV-specific antibody response in both flavivirus infection naive participants and in participants with pre existing flavivirus infection. Of all participants, 81.8% seroconverted (PRNT50) after completing the ZIKV mRNA vaccination series. Further, the ZIKV-specific neutralizing antibody response persisted at Month 7 in flavivirus baseline seronegative and seropositive participants. Overall, 61.9% (39.4, 81.9%) and 95.2% (76.2, 99.9%) of participants remained seroconverted at Month 7 relative to baseline as measured by PRNT and MN assay, respectively.

Summary of Results - Cohort 2: 30 pg Dose Level

This summary presents the interim safety and immunogenicity data for Cohort 2 (30 pg) of the vaccine study through Day 57, one month post-second vaccine administration.

The ZIKV mRNA vaccine 30 pg dose level has an acceptable safety profile compatible with wide use of the vaccine candidate. Neither a second dose administered 28 days post first vaccine administration nor a flavivirus positive baseline serostatus seem to negatively affect this profile.

The ZIKV mRNA vaccine 30 pg dose level induces a strong ZIKV-specific neutralizing antibody response in both flavivirus infection naive participants and in participants with pre existing flavivirus infection. Of all participants, 95.5% (21/24) seroconverted (PRNT50) after completing the ZIKV mRNA vaccine vaccination series.

When compared to a 10 pg dose, a 30 pg dose demonstrated a slightly higher neutralizing antibody response in terms of GMTs and seroconversions after two dose administrations at Day 57. Following a single administration, at Day 29, the 30 pg dose level was able to induce PRNT50 titers above the limit of detection.

Demographics and Population Set

Demographic and baseline characteristics were generally balanced across Cohort 2 and showed a mean age of 35 years (range: 20-48) in the vaccine treatment arm. A similar number of males and females were included in this Cohort 2 (sex M/F ratio: 13/11). All five baseline flavivirus seropositive participants were enrolled at the Puerto Rico investigational site. The other demographic parameters (height, weight, BMI) were homogenous across the cohort.

Thirty participants were enrolled and received one vaccine administration; 29 of these participants (23 in the vaccine treatment arm and 6 in placebo arm) received two vaccine doses. One participant belonging to the flavivirus baseline seronegative subgroup did not receive the second dose of the vaccine after a joint decision from the medical monitor and the sponsor because of a previous medical finding that was not disclosed at screening that could potentially interfere with safety assessments. He will be followed for safety and other procedures as required by the protocol procedures. No participants have yet been lost to follow up. Safety

Solicited safety data were collected through 7 days after each vaccination via diary and are based on the Solicited Safety Set. Unsolicited events were collected through 28 days after each vaccination. Data in the tables are presented by the vaccine dose level individually and grouped and by the placebo grouped. The solicited safety set following the first vaccination includes 24 participants in the ZIKV mRNA vaccine 30 pg treatment and 6 in the saline Placebo treatment arm corresponding to the dose level 30 pg. The second vaccination solicited safety set includes 23 participants in the ZIKV mRNA vaccine 30 pg treatment and 6 in the saline placebo treatment arm.

Solicited Local Adverse Events

The most frequently reported adverse reaction was local pain at injection site which was reported by 12 participants (52.2%) out of 23 who returned their diary in the vaccine treatment arm after the first administration. The intensity of the local pain was predominantly Grade 1. No local erythema and no local induration were reported after the first vaccine administration. Eleven out of 22 participants (50%) reported local pain with three observations of a Grade 2 intensity after the second vaccine administration; in terms of objective signs, one case of Grade 3 (5.1- 10cm) erythema and two cases, one Grade 1 (2.5-5cm) and one Grade 3 (greater than 10 cm) of swelling were observed post-administration. Although the number of flavivirus seropositive participants is limited, more solicited local adverse events were observed in the seronegative participants.

Solicited Systemic Adverse Events

Following the first administration of the vaccine, headache and fatigue (5 participants, 21.7% in each category) followed by myalgia (3 participants, 13%) were the most common solicited systemic AEs. Most of them were Grade 1 or 2, except one case of headache (1, 4.2%) reported as Grade 3. The participant belonged to the flavivirus baseline seronegative subgroup and reported the Grade 3 headache on Day 4 and 5 post-vaccine administration; he did not take any medication, nor did he seek medical attention. After the second vaccine administration solicited systemic AEs of fatigue (10 participants, 45.5%), headache (8 participants, 36.4%), chills (5 participants, 22.7%), arthralgia (4 participants, 18.2%) and myalgia (4 participants, 18.2%) were reported. All solicited AEs were either Grade 1 or 2. Fever was not reported after the first vaccine administration, but three cases (12.6%) were noted after the second administration - one, Grade 1 (38.0 - 38.4°C) and two, Grade 2 (38.5 - 38.9°C) all in the baseline flavivirus seronegative participants. No cases of rash were reported. Solicited systemic adverse events were observed in a larger number of seronegative than seropositive participants, with acknowledgement that sample size is limited, as previously mentioned.

Unsolicited Adverse Events

Thirty seven unsolicited Treatment-Emergent Adverse Events (TEAEs) attributed to 10 participants (41.7%) with 4 related (16.7%) were reported in the vaccine treatment arm compared to 11 events in 4 participants (33.3%) with none related in the placebo arm. None of TEAEs related to vaccine administration were of a Grade 3 or 4 intensity. Throughout the 28 days follow up post-vaccine administration 6 participants (25%) reported 20 events compared to 7 (30.4%) with 15 events, respectively, after the first and second vaccine administration of mRNA- 1893 and it did not seem that an administration of a 2nd vaccine dose had a great impact on the safety profile. Twenty four TEAEs were reported in 7 baseline seronegative participants compared to 13 TEAEs in 3 seropositive participants. No SAEs and no AESI related to the vaccine were reported.

Laboratory Abnormalities

Inclusion criteria for the study required that all participants have Grade 0 lab tests at enrollment. Laboratory abnormalities were infrequent, mostly Grade 1 or 2. One participant experienced a Prothrombin Test increase at Day 29 reported as Grade 4, however this was considered not related to the vaccine administration. The participant was part of flavivirus baseline seronegative subset. None of those modifications were considered clinically relevant.

Immunogenicity

Immunogenicity evaluation is performed at Day 1, Day 29, Day 57, Month 7 and Month 13 and measured by the neutralization activity against ZIKV with a Plaque Neutralization assay (PRNT) as secondary endpoint, and by a Microneutralization assay (MN), a Reporter Virus Particle neutralization assay (RVP) and an enzyme-linked immunosorbent assay (ELISA) as exploratory endpoints. PRNT provides a gold standard in measuring ZIKV- specific neutralizing antibodies and has served to define protective titers for some other flavivirus vaccines such as Yellow Fever and Japanese Encephalitis, however it is labor-intensive and requires biosafety equipment. MN is a high-throughput neutralization assay, modified from a qualified dengue virus microneutralization assay. To date only the PRNT and MN results are available at Day 1, Day 29 and Day 57 and are presented in this summary.

Flavivirus serostatus at enrollment was determined using a commercially available WNV IgG/IgM ELISA. While all participant in the flavivirus seropositive group were enrolled at the Puerto Rico site, the exact etiology of their flavivirus priming is unknown. ZIKV mRNA Vaccine Neutralizing Antibody Responses by PRNT

Seroconversion is defined as a change in PRNT from below the lower limit of quantification to a PRNT equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. The LLOQ for the PRNT assay is 16; values lower than the LLOQ are assigned a value of 50% of the LLOQ. The GMTs for placebo participants were below the LLOQ at all time points.

For participants stratified to the flavivirus seronegative group receiving the vaccine, the baseline (Day 1) PRNT50 geometric mean titer (GMT) was reported as 8 i.e. below the LLOQ.

A ZIKV-specific response was detected at Day 29 following a single vaccine administration with a GMT at 14 (9.8-20.1) and a seroconversion rate at 40% (19.1-63.9%). Completing the two- dose vaccination series, the GMT at Day 57 increased to 303.4 (195.5-470.9) with 100% (81.5- 100%) of participants seroconverting.

For participants stratified to the flavivirus seropositive group receiving the vaccine, the baseline PRNT50 GMT was 12.3 (3.1-48.7). Following a single administration of the vaccine GMT increased to 88.1 (3.6-2166.7) at Day 29, with 75% (19.4-99.4%) of participants (3/4) achieving a 4-fold increase in neutralizing antibody titers compared with baseline. Completing the two-dose vaccination series, GMT at Day 57 continued to increase to 150.9 (6.2-3699.1);

75% of participants (3/4) achieved a 2-fold increase and 75% of participants achieved a 4-fold increase in neutralizing antibody titers compared with baseline. This demonstrates that even in participants with a prior flavivirus infection background, the vaccine is able to mount a ZIKV- specific neutralizing antibody response.

In all participants of a two-dose series of the ZIKV mRNA vaccine 30 pg, the seroconversion rate increased from 45.8% (25.6-67.2%) at Day 29 to 95.5% (77.2-99.9%) at Day 57.

ZIKV mRNA Vaccine Neutralizing Antibody Responses by MN

Seroconversion is defined as a change in MN from below the lower limit of quantification to a MN equal to or above LLOQ, or a multiplication by at least 4 in subjects with pre-existing neutralizing titers. The lower limit of quantification (LLOQ) for the MN assay is 28; values lower than the LLOQ are assigned a value of 50% of the LLOQ. The GMTs for placebo participants were below the LLOQ at all time points.

For participants stratified to the flavivirus seronegative group receiving the vaccine, the baseline (Day 1) MN50 geometric mean titer (GMT) was 14.0 i.e. below the LLOQ. Following a single vaccine administration, GMT increased to 129.7 (71.3-236) with a seroconversion rate of 85% (62.1-96.8%). Completing the two-dose vaccination series, GMT at Day 57 increased to 1478. The proportion of initially flavivirus seronegative participants with a seroresponse at Day 57 was 100% (81.5-100%).

For participants stratified to the flavivirus seropositive group receiving the vaccine, the baseline MN50 GMT of was 39.4 (1.5-1061.7). Following a single administration of the vaccine GMTs increased to 226.7 (30.1, 1709.6), 75% of participants (3/4) achieved a 2-fold increase and 75% of participants achieved a 4-fold increase in neutralizing antibody titers compared with baseline. Completing the two-dose vaccination series, GMT at Day 57 increased to 578.5 (84, 3986.2). Consistent with the PRNT results, the vaccine was able to induce a ZIKV-specific neutralizing antibody response by MN50 in participants with a prior flavivirus infection.

There was a clear benefit from a two-dose series of the vaccine particularly for the flavivirus baseline seronegative participants. The seroconversion rates, defined as a change of MN titers from below the LLOQ to a MN level equal or above the LLOQ (i.e. 28) or a multiplication by at least a factor of four in all participants, increased from 83.3% (62.6-95.3%) at Day 29 to 95.5 (77.2, 99.9%) at Day 57. The GMTs for all participants randomized to receive placebo remained below the LLOQ through Day 57. The MN data provided higher results (titers) compared to those reported in the PRNT assay. This is consistent with known differences between the assays. Irrespective of the assay, both MN and PRNT provided equivalent guidance in terms of ZIKV-specific neutralization activity.

Concluding Comments

These interim data corresponding to the second dose level support the following conclusions:

The ZIKV mRNA vaccine 30 pg dose level has an acceptable safety profile compatible with a wide use of the vaccine candidate. Neither a second dose administered 28 days post first vaccine administration nor a flavivirus positive baseline serostatus seem to negatively affect this profile.

The ZIKV mRNA vaccine 30 pg dose level induces a strong neutralizing ZIKV-specific antibody response in both flavivirus infection naive participants and in participants with pre existing flavivirus antibodies. Of all participants, 95.5% (21/24) seroconverted (PRNT50) after completing the ZIKV mRNA vaccination series.

Notably, the 30 pg dose level is sufficient to seroconvert (PRNT) baseline flavivirus seronegative subjects following only a single vaccine administration. Comparing 10 pg and 30 pg ZIKV mRNA Vaccine Dose Levels

In terms of safety the 30 pg dose level was generally well tolerated. As expected, compared to the 10 pg dose level there was a trend to have more observations of local pain and a few cases of erythema or swelling at the injection site, in particular after the 2nd vaccine administration. Slightly more solicited systemic adverse events were also noted with 30 pg dose level. Regarding the unsolicited adverse events there was no major difference between the two dose levels. No SAE, no AESI related to mRNA were reported in any of those dose levels.

In terms of Zika- specific immune response, the vaccine induces a strong neutralizing antibody response at both 10 and 30 pg. Following a single administration, sampled on Day 29, a 30 pg dose was sufficient to seroconvert 40% (19.1-63.9%) of baseline seronegative participants, compared to a 5% (0.1, 24.9%) seroconversion rate with the 10 pg dose. At Day 57 in both dose levels the seroconversion rates were similar with 100% (81.5-100%) and 94.4% (72.7-99.9%) in 30 pg and 10 pg dose levels, respectively, however the GMTs were slightly higher in the 30 pg dose level. Similar conclusions were made with the MN data. In the flavivirus seropositive subgroup the ZIKV-specific antibody increase was compatible with a booster, comparable to the response previously observed with the 10 pg dose.

SEQUENCES

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 mRNA constructs described herein may further comprise a polyA tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp). 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.

5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 3)

5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 5)

3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC (SEQ ID NO: 4)

3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC (SEQ ID NO: 6)

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 the upper and lower ends of the range are specifically contemplated and described herein.

The entire contents of International Application Nos. PCT/US2015/02740, 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

What is claimed is: CLAIMS

1. A method comprising: administering to a human subject a first dose of a vaccine comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a Zika virus (ZIKV) prME protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 7, wherein the mRNA is in a lipid nanoparticle comprising 45-55 mol% ionizable cationic lipid, 15-20 mol% neutral lipid, 35-45 mol% cholesterol, and 0.5-5 mol% PEG-modified lipid; and administering to the human subject a second dose of the vaccine at least 28 days after administering the first dose, wherein the first dose and the second dose each comprise 10 pg to 30 pig of the mRNA.

2. The method of claim 1, wherein the first dose and the second dose each comprise 30 pg of the mRNA.

3. The method of claim 1, wherein the first dose and the second dose each comprise 10 pg of the mRNA.

4. The method of any one of claims 1-3, wherein the second dose is administered 28 days after administering the first dose.

5. The method of any one of claims 1-4, wherein the first dose and the second dose of the vaccine are administered intramuscularly.

6. The method of any one of claims 1-5, wherein the ZIKV prME protein comprises the amino acid sequence of SEQ ID NO: 7.

7. The method of any one of claims 1-6, wherein the mRNA comprises a 5’ 7mG(5’)ppp(5’)NlmpNp cap.

8. The method of any one of claims 1-7, wherein the mRNA comprises a 1- methylpseudourine chemical modification.

9. The method of any one of claims 1-8, wherein the lipid nanoparticle comprises 15-20 mol% DSPC; 35-45 mol% cholesterol; 0.5-5 mol% PEG-modified lipid; and 45-55 mol% ionizable cationic lipid of Compound I:

(Compound I).

10. The method of any one of claims 1-9, wherein the lipid nanoparticle comprises 50 mol% ionizable cationic lipid.

11. The method of any one of claims 1-9, wherein the lipid nanoparticle comprises 49 mol% ionizable cationic lipid.

12. The method of any one of claims 1-9, wherein the lipid nanoparticle comprises 48 mol% ionizable cationic lipid.

13. The method of any one of claims 1-12, wherein the human subject is 18 to 49 years of age.

14. The method of any one of claims 1-13, wherein the vaccine further comprises Tris buffer, propylene glycol, and diethylenetriamine pentaacetic acid (DTPA)

15. A method comprising: administering to a human subject intramuscularly a first dose of vaccine comprising a chemically-modified messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a Zrka virus (ZIKV) prME protein comprising the amino acid sequence of SEQ ID NO: 7, wherein the mRNA is in a lipid nanoparticle comprising 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable cationic lipid of Compound I:

(Compound I), and administering to the human subject a second dose of the vaccine 28 days after administering the first dose, wherein the first dose and the second dose each comprise 30 pg of the mRNA.

16. A method comprising: administering to a human subject intramuscularly a first dose of a vaccine comprising a chemically-modified messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a Zika virus (ZIKV) prME protein comprising the amino acid sequence of SEQ ID NO: 7, wherein the mRNA is in a lipid nanoparticle comprising 15-20 mol% neutral lipid, 35-45 mol% cholesterol, 0.5-5 mol% PEG-modified lipid, and 45-55 mol% ionizable cationic lipid of Compound I:

administering to the human subject a second dose of the vaccine 28 days after administering the first dose, wherein the first dose and the second dose each comprise 10 pg of the mRNA.

17. The method of any one of claims 1-16, wherein the plaque reduction neutralization test 50 (PRNT50) geometric mean titer (GMT) of neutralizing antibody induced in the subject at Day 57 post administration of the first dose is 180 - 210.

18. The method of any one of claims 1-17, wherein the PRNT50 GMT of neutralizing antibody induced in the subject at Month 7 post administration of the first dose is 20-50.

19. The method of any one of claims 1-18, wherein the microneutralization assay 50 (MN50) GMT of neutralizing antibody induced in the subject at Day 57 is 1180 - 1210.

20. The method of any one of claims 1-19, wherein the MN50 GMT of neutralizing antibody induced in the subject at Month 7 post administration of the first dose is 130-160.