Transamniotic nucleic acid therapy for perinatal immunization

Abstract

The present disclosure provides compositions and methods for inducing an immune response in a fetus or newborn. In particular, the disclosure provides compositions and methods for transamniotic administration of a polynucleotide encoding a pathogen antigen.

Description

[0001] TRANSAMNIOTIC NUCLEIC ACID THERAPY FOR PERINATAL IMMUNIZATION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and benefit of U.S. Provisional Application No. 63 / 642,499, filed on May 3, 2024, the content of which is incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on May 1, 2025, is named 167705-034901PCT_SL.xml and is 4,568 bytes in size.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] Perinatal infections are among the leading causes of neonatal morbidity and mortality, responsible for over 500,000 deaths worldwide every year, and may be transmitted from the mother to fetus or newborn via maternal transplacental transfer, blood, genital secretions, and breastfeeding. However, vaccination remains challenging as human newborns do not typically mount an immune response to a vaccine administered after birth until 6-8 months of age. are Polynucleotide, e.g., messenger ribonucleic acid (mRNA), vaccines exhibit higher potency, safety, and efficacy, and are also more easily manufactured than traditional vaccines. However, transport of exogenous encapsulated mRNA from a mother’s bloodstream through the placenta to the fetus is typically very limited. Therefore, there is a need for improved, minimally invasive methods of delivering mRNA immunogenic compositions to a fetus that induce an immune response against a pathogen antigen, thereby reducing the propensity of the perinatal subject to become infected with the pathogen and / or reducing severity of a perinatal infection with the antigen.

[0008] SUMMARY OF THE DISCLOSURE

[0009] Provided herein are polynucleotide, e.g., mRNA, immunogenic compositions, vaccines, and methods for the delivery (e.g., intra-amniotic delivery) thereof to a fetus to induce an immune response against a pathogen, to reduce the infection of a subject with a pathogen wherein the subject has a propensity to acquire an infection, and / or to reduce the severity of a perinatal infection in a subject.

[0010] In one aspect, the disclosure provides a method of inducing an immune response against a pathogen in a fetus or neonate, the method involving administering to the amniotic fluid surrounding the fetus a polynucleotide encoding a pathogen antigen , thereby inducing an immune response in the fetus or neonate against the pathogen.

[0011] In another aspect, the disclosure provides a method for reducing the propensity of a fetus or neonate to become infected with a pathogen, the method involving administering to the amniotic fluid surrounding the fetus a messenger RNA encoding an antigen of the pathogen, where the antigen induces an immune response in the fetus or neonate, thereby reducing the infection or risk thereof from the pathogen.

[0012] In another aspect, the disclosure provides a composition for inducing an immune response in a fetus or neonate to a cytomegalovirus containing one or more polynucleotides containing SEQ ID NO: 1. In one embodiment, the one or more polynucleotides comprises SEQ ID NOS: 1 a 5’ cap and / or a polyA tail. In another embodiment, the one or more polynucleotides is packaged in a vector. In another embodiment, the vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle. In another embodiment, the vector is a lipopolyplex. In another embodiment, the vector is a lipid nanoparticle.

[0013] In another aspect, the disclosure provides a kit containing the composition of any of the above aspects, formulated as a pharmaceutical composition, and instructions for use thereof.

[0014] In various embodiments of the above aspects or any other aspect delineated herein, the messenger RNA reaches the placenta. In various embodiments of the above aspects or any other aspect delineated herein, the messenger RNA reaches the fetal circulation and therefore also reaches multiple fetal anatomical sites. In various embodiments of the above aspects, the pathogen is a virus (e.g., CMV) or a bacteria. In various embodiments of the above aspects, the antigen is an envelope glycoprotein, a CMV pentameric complex protein, a phosphoprotein, an immediate early antigen protein, or an early membrane antigen or a late membrane antigen protein. In various embodiments of the above aspects, the envelope glycoprotein is glycoprotein B. In various embodiments of the above aspects, the polynucleotide comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In various embodiments of the above aspects, the RNA comprises a messenger RNA (mRNA). In various embodiments of the above aspects, the mRNA comprises a sequence of SEQ ID NO: 1. In various embodiments of the above aspects, the mRNA comprises a sequence of SEQ ID NO: 1 and a 5’ cap and / or a polyA tail. In various embodiments of the above aspects, the polynucleotide is packaged in a vector. In various embodiments of the above aspects, the vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle. In various embodiments of the above aspects, the vector is a lipopolyplex. In various embodiments of the above aspects, the subject is a mammal. In various embodiments of the above aspects, the mammal is a human. In various embodiments of the above aspects, the method reduces horizontal transfer of the infection from an infected subject to a susceptible host. In various embodiments of the above aspects, the polynucleotide is formulated as a pharmaceutical composition. In various embodiments of the above aspects, the pharmaceutical composition is administered prior to 10 weeks of pregnancy. In various embodiments of the above aspects, the pharmaceutical composition is administered after 10 weeks of pregnancy. In various embodiments of the above aspects, where the immune response comprises the production of antibodies to the pathogen. In various embodiments of the above aspects, the immune response comprises a cellular immune response. In various embodiments of the above aspects, one or more cytokines are produced. In various embodiments of the above aspects, the one or more cytokines are selected from IFN-y, IL-2, TNF-a, GM-CSF, and IL-6.

[0015] In some aspects, provided herein are methods for reducing an infection or risk thereof from a pathogen in a fetus or neonate, comprising administering to the amniotic fluid surrounding the fetus a polynucleotide encoding an antigen of the pathogen, wherein the antigen induces an immune response in the fetus or neonate against the pathogen antigen, thereby reducing propensity of the neonate from acquiring an infection with the pathogen. In some aspects, provided herein are methods of inducing an immune response in a fetus or neonate, comprising administering to the amniotic fluid surrounding the fetus a polynucleotide encoding an antigen to a pathogen, wherein the antigen induces an immune response in the fetus or neonate.

[0016] In some embodiments of the disclosure, the pathogen is a virus, e.g, a cytomegalovirus (CMV) or a bacteria. In some embodiments, the polynucleotide comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA). In some embodiments, the mRNA comprises SEQ ID NO: 1, optionally comprising a 5’cap and a polyA tail. In some embodiments, the polynucleotide is packaged in a delivery vector, e.g., a vector comprising one or more of a lipid, a protein, a plasmid, or a nanoparticle. In some embodiments, the delivery vector is a lipopolyplex.

[0017] In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the method treats or prevents the onset or progression of an infection from the pathogen or reduces horizontal transfer of the infection from an infected subject to a susceptible host. In some embodiments, the pharmaceutical composition is administered prior to 10 weeks of pregnancy, prior to 5 weeks of pregnancy, or after 10 weeks of pregnancy.

[0018] In some aspects, the immune response induced by the composition and methods described herein comprises the production of antibodies to the pathogen. In some aspects, the immune response comprises a cellular immune response, e.g., comprising the production of one or more cytokines. In some embodiments, the or more cytokines are selected from IFN-y, IL-2, TNF-a, GM-CSF, and IL-6.

[0019] In some aspects, provided herein is a composition for inducing an immune response in a fetus or neonate comprising a polynucleotide encoding a cytolomegalovirus (CMV) antigen, e.g., an envelope glycoprotein, e.g., glycoprotein B. In some embodiments, provided herein are polynucleotides encoding a CMV pentameric complex protein, a phosphoprotein, an immediate early antigen protein, and / or an early membrane antigen or a late membrane antigen protein.

[0020] In some embodiments, the polynucleotides provided herein comprise a sequence selected of SEQ ID NO: 1, optionally comprising a 5’cap and a polyA tail. In some embodiments, the polynucleotide is packaged in a delivery vector. In some embodiments, the delivery vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle. In some embodiments, the delivery vector is a lipopolyplex.

[0021] In some aspects, provided herein is a kit comprising an mRNA vaccine to be administered transamniotically, and instructions for use thereof.

[0022] Definitions

[0023] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. By “administer” is meant giving, supplying, dispensing, delivering, or applying a composition, agent, therapeutic and the like to a subject, or applying or bringing the composition and the like into contact with the subject. Administering or administration may be accomplished by any of a number of routes, such as, for example, without limitation, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV), (injection), intrathecal, intramuscular, dermal, intradermal, intracranial, inhalation, rectal, intravaginal, or intraocular By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In some embodiments, the agent is an mRNA molecule (e.g., an mRNA molecule encoding an antigen derived from a pathogen). By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0024] By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

[0025] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0026] By “antibody” is meant an immunoglobulin (Ig) molecule produced by B lymphoid cells and having a specific amino acid sequence. Antibodies are evoked or elicited in subjects (humans or other animals or mammals) following exposure to a specific antigen (immunogen). A subject capable of generating antibodies / immunoglobulin (i.e., an immune response) directed against a specific antigen / immunogen is said to be immunocompetent. Antibodies are characterized by reacting specifically with (e.g., binding to) an antigen or immunogen in some demonstrable way, antibody and antigen / immunogen each being defined in terms of the other.

[0027] By “antigen” is meant a compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.

[0028] By “Cytomegalovirus (CMV) mRNA” is meant a polynucleotide encoding a CMV protein. In particular embodiments, the CMV mRNA comprises a sequence having at least about 85% sequence identity to the following core sequence encoding the human CMV glycoprotein B antigen or a fragment thereof that encodes an immunogenic peptide: tcatcttct actcgtggaa cttctgctac tcacagtcac cattcctctc atacgacgtc tgctgctcat tctcgatccg gttcagtctc tcaacgcgta acttcttccc aaacggtcag ccatggtgtt aacgagacca tctacaacac taccctcaag tacggagatg tggtgggggt caacaccacc aagtacccct atcgcgtgtg ttctatggca cagggtacgg atcttattcg ctttgaacgt aatatcgtct gcacctcgat gaagcccatc aatgaagacc tggacgaggg catcatggtg gtctacaaac gcaacatcgt cgcgcacacc tttaaggtac gagtctacca gaaggttttg acgtttcgtc gtagctacgc ttacatccac accacttatc tgctgggcag caacacggaa tacgtggcgc ctcctatgtg ggagattcat catatcaaca gtcacagtca gtgctacagt tcctacagcc gcgttatagc aggcacggtt ttcgtggctt atcataggga cagctatgaa aacaaaacca tgcaattaat gcccgacgat tattccaaca cccacagtac ccgttacgtg acggtcaagg atcaatggca cagccgcggc agcacctggc tctatcgtga gacctgtaat ctgaattgta tggtgaccat cactactgcg cgctccaagt atccctatca ttttttcgca acttccacgg gtgatgtggt tgacatttct cctttctaca acggaactaa tcgcaatgcc agctattttg gagaaaacgc cgacaagttt ttcatttttc cgaactacac tatcgtctcc gactttggaa gaccgaattc tgcgttagag acccacaggt tggtggcttt tcttgaacgt gcggactcag tgatctcctg ggatatacag gacgagaaga atgttacttg tcaactcact ttctgggaag cctcggaacg caccattcgt tccgaagccg aggactcgta tcacttttct tctgccaaaa tgaccgccac tttcttatct aagaagcaag aggtgaacat gtccgactct gcgctggact gtgtacgtga tgaggccata aataagttac agcagatttt caatacttca tacaatcaaa catatgaaaa atatggaaac gtgtccgtct ttgaaaccac tggtggtttg gtggtgttct ggcaaggtat caagcaaaaa tctctggtgg aactcgaacg tttggccaac cgctccagtc tgaatcttac tcataataga accaaaagaa gtacagatgg caacaatgca actcatttat ccaacatgga gtcggtgcac aatctggtct acgcccagct gcagttcacc tatgacacgt tgcgcggtta catcaaccgg gcgctggcgc aaatcgcaga agcctggtgt gtggatcaac ggcgcaccct agaggtcttc aaggaactta gcaagatcaa cccgtcagct attctctcgg ccatctacaa caaaccgatt gccgcgcgtt tcatgggtga tgtcctgggt ctggccagct gcgtgaccat taaccaaacc agcgtcaagg tgctgcgtga tatgaatgtg aaggaatcgc caggacgctg ctactcacga ccagtggtca tctttaattt cgccaacagc tcgtacgtgc agtacggtca actgggcgag gataacgaaa tcctgttggg caaccaccgc actgaggaat gtcagcttcc cagcctcaag atcttcatcg ccggcaactc ggcctacgag tacgtggact acctcttcaa acgcatgatt gacctcagca gcatctccac cgtcgacagc atgatcgccc tagacatcga cccgctggaa aacaccgact tcagggtact ggaactttac tcgcagaaag aattgcgttc cagcaacgtt tttgatctcg aggagatcat gcgcgagttc aattcgtata agcagcgggt aaagtacgtg gaggacaagg tagtcgaccc gctgccgccc tacctcaagg gtctggacga cctcatgagc ggcctgggcg ccgcgggaaa ggccgttggc gtagccattg gggccgtggg tggcgcggtg gcctccgtgg tcgaaggcgt tgccaccttc ctcaaaaacc ccttcggagc cttcaccatc atcctcgtgg ccatagccgt cgtcattatc atttatttga tctatactcg acagcggcgt ctctgcatgc agccgctgca gaacctcttt ccctatctgg tgtccgccga cgggaccacc gtgacgtcgg gcaacaccaa agacacgtcg ttacaggctc cgccttccta cgaggaaagt gtttataatt ctggtcgcaa aggaccggga ccaccgtcgt ctgatgcatc cacggcggct ccgccttaca ccaacgagca ggcttaccag atgcttctgg ccctggtccg tctggacgca gagcagcgag cgcagcagaa cggtacagat tctttggacg gacagactgg cacgcaggac aagggacaga agcccaacct gctagaccga ctgcgacacc gcaaaaacgg ctaccgacac ttgaaagact ccgacgaaga agagaacgtc (SEQ ID NO. 1)

[0029] In particular embodiments, the mRNA comprises a Cap 1 on the 5 ’-end and an elongation of the 3 ’-end comprising about 50, 100, or 150 adenosine monophosphates, which provides a poly (A) tail.

[0030] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0031] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In an embodiment, the analyte is a marker of an immune response. In some embodiments, the analyte is an antibody that specifically binds an antigen derived from a pathogen (e.g., CMV).

[0032] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0033] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In some embodiments the disease is caused by a virus, a bacteria, or other pathogen. In some embodiments, the disease is a CMV infection.

[0034] By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, the agent is an mRNA encoding a pathogen antigen. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0035] By “fetus” is mean an unborn child.

[0036] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0037] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0038] The term “immune response” is meant any response mediated by an immunoresponsive cell. In one example of an immune response, leukocytes are recruited to carry out a variety of different specific functions in response to exposure to an antigen (e.g., a foreign entity). Immune responses are multifactorial processes that differ depending on the types of cells involved. Immune responses include cell-mediated responses (e.g., T cell responses), humoral responses (B cell / antibody responses), innate responses and combinations thereof.

[0039] By “immunogen” is meant a compound, composition, or substance which, under appropriate conditions, can elicit or stimulate an immune response, such as the production of antibodies, and / or a T-cell response, in an animal, including compositions that are injected into or otherwise delivered to an animal.

[0040] As used herein, an "immunogenic composition" is a composition comprising an immunogen or a polynucleotide encoding such immunogen or a vaccine comprising an pathogen polypeptide or peptide (e.g., a full length or a soluble form of an pathogen polypeptide) or a polynucleotide encoding such immunogen). As will be appreciated by the skilled person in the art, if administered to a subject in need prior to the subject’s contracting disease or experiencing full-blown disease, an immunogenic composition can be prophylactic and result in the subject’s eliciting an immune response, e.g., a neutralizing antibody and / or cellular immune response, to protect against disease, or to prevent more severe disease or condition, and / or the symptoms thereof. If administered to a subject in need following the subject’s contracting disease, an immunogenic composition can be therapeutic and result in the subject’s eliciting an immune response, e.g., a neutralizing antibody and / or cellular immune response, to treat the disease, e.g., by reducing, diminishing, abrogating, ameliorating, abating, alleviating, or eliminating the disease, and / or the symptoms thereof. In an embodiment, the immune response is a B cell response, which results in the production of antibodies, e.g., neutralizing antibodies, directed against the immunogen or immunogenic composition comprising the antigen or antigen sequence. In a manner similar to the foregoing, in some embodiments, an immunogen, immunogenic composition, or vaccine can be prophylactic. In some embodiments, an immunogen, immunogenic composition, or vaccine can be therapeutic.

[0041] By “immunogenic composition” is meant a composition comprising an antigen, antigen sequence, or immunogen, wherein the composition elicits an immune response in an immunized subject.

[0042] The term “immunize” (or immunization) refers to rendering a subject protected from, or immunologically responsive to, a disease or pathology caused by a pathogenic agent, e.g., an infectious disease caused by a virus, e.g., CMV, such as by vaccination. In some cases herein, the terms “immunization” and “vaccination” may be used interchangeably (e.g., immunization / vaccination). The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0043] By "isolated polynucleotide" is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0044] By “lipopolyplex” is meant a structure for encapsulation and delivery of an agent into an organism and comprises a nucleic acid molecule, cationic lipids, and cationic polymers. Nonlimiting examples of lipopolyplex compositions include those described in To et al., Expert Opinion on Drug Discovery 16, 1307-1313, 2021, the disclosure of which is incorporated herein in its entirety for all purposes.

[0045] By “lipid nanoparticle” is meant a small particle comprising lipids that is suitable for delivering an agent to a cell. In some embodiments, the cell is in a subject. In some embodiments, the lipid nanoparticle comprises (e.g., encapsulates) the agent. In various embodiments, the agent is an mRNA molecule. As used herein, the term lipid encompasses triglycerides (e.g. tristearin), diglycerides (e.g. glycerol bahenate), monoglycerides (e.g. glycerol monostearate), fatty acids (e.g. stearic acid), steroids (e.g. cholesterol), and waxes (e.g. cetyl palmitate).

[0046] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0047] As used herein, a “pathogen” is any biologically based agent that causes an infection, e.g, a virus, a bacteria, a fungi, or other. A “pathogen antigen” may include an antigenic protein, peptide, or fragment thereof, of the pathogen which induces an immune response.

[0048] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0049] By “reference” is meant a standard or control condition. In some cases, a reference may be a healthy, uninfected subject or cell, e.g., a subject or cell not infected with virus (e.g., CMV).

[0050] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0051] A compound or antibody that "specifically binds" refers to one that recognizes and binds to a polypeptide, such as a virus polypeptide, peptide, or vaccine product, but which does not substantially recognize and bind to other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide, such as a virus polypeptide or peptide.

[0052] Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule (e.g., DNA or RNA) that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. In some instances, the nucleic acid molecule is an mRNA molecule. In embodiments, the mRNA molecule comprises a 5’ cap, a poly(A) tail, a 3’ untranslated region, and / or a 5’ untranslated region. Components of mRNA molecules suitable for use in the methods of the present disclosure include those described in Sahin, etal., “mRNA-based therapeutics - developing a new class of drugs,” Nature Reviews Drug Discovery, 13:759-780 (2014), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0053] In some embodiments, the polynucleotides provided herein contain one or more modifications or nucleotide analogs. For example, in some embodiments a polynucleotide contains one or more analogs (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.

[0054] In some embodiments, the polynucleotide contains a modified backbone and / or linkages (e.g., between adjacent nucleosides). Non-limiting examples of modified backbones include those that contain a phosphorus atom in the backbone and those that do not contain a phosphorus atom in the backbone. Non-limiting examples of modified backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3' -alkylene phosphonates, 5 '-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', a 5' to 5' or a 2' to 2' linkage.

[0055] In some embodiments, a polynucleotide contains short chain alkyl or cycloalkyl linkages (e.g., between adjacent nucleosides), mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. In embodiments, a polynucleotide includes one or more of the following: morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. In embodiments, a polynucleotide contains a nucleic acid mimetic. The term “mimetic” can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar- backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In embodiments, the backbone in PNA compounds contains two or more linked aminoethylglycine units that give PNA an amide containing backbone. Heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0056] In some embodiments, a polynucleotide contains a morpholino backbone structure. For example, a nucleic acid can contain a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace a phosphodiester linkage.

[0057] A polynucleotide can contain linked morpholino units having heterocyclic bases attached to the morpholino ring. Linking groups can link morpholino monomeric units. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). In some instances, the furanose ring normally present in a nucleic acid molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. In some cases, incorporation of CeNA monomers into a nucleic acid chain increases the stability of a DNA / RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. In embodiments, a polynucleotide contains Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C, 4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene ( — CH2), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +10 ° C ), stability towards 3'- exonucleolytic degradation and good solubility properties.

[0058] In some embodiments, a polynucleotide contains nucleobase modifications (often referred to simply as “base modifications”) or substitutions. In embodiments, unmodified nucleobases include one or more of the purine bases, (e.g., adenine (A) and guanine (G)), and / or the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)). Non-limiting examples of modified nucleobases include nucleobases such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine. Further non-limiting examples of modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin- 2(3H)-one), G-clamps such as a substituted phenoxazine cytidine e.g., 9-(2-aminoethoxy)-H- pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4, -b)indol-2- one), pyridoindole cytidine (H-pyrido(3',2':4, 5)pyrrolo[2,3-d]pyrimidin-2-one).

[0059] By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0060] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In an embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0061] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0062] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0063] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0064] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0065] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0066] A delivery “vector” as used herein refers to an agent that facilitates the delivery and / or cell entry of a polynucleotide to a cell. In some embodiments, a delivery vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle. In some embodiments the delivery vector comprises a lipid nanoparticle (LNP). In some embodiments a delivery vector comprises a viral- like particle (VLP). In some embodiments a delivery vector comprises a plasmid comprising the polynucleotide. In some embodiments a delivery vector comprises a lipopolyplex, z.e., a vector comprising a protein and a lipid.

[0067] By “virus-like particles (VLPs)” is meant virus particles made up of one of more viral structural proteins, but lacking the viral genome. Most VLPs comprise at least a viral core protein that drives budding and release of particles from a host cell.

[0068] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to preventing, reducing the risk of, or reducing or ameliorating the symptoms associated with a disease or disorder. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. By “vaccine” is meant a preparation of immunogenic material (e.g., protein or nucleic acid), such as a protein or peptide antigen, capable of stimulating (eliciting) an immune response, administered to a subject to treat a disease, condition, or pathology, or to prevent or protect against a disease, condition, or pathology, such as an infectious disease, e.g., a virus infection. The immunogenic material may include, for example, DNA, or RNA derived from a pathogen. Vaccines may elicit a prophylactic (preventative) immune response in the subject; they may also elicit a therapeutic response immune response in a subject. Vaccines may be administered to human subjects, non-human subjects, or veterinary subjects. The terms vaccine and immunogen are used interchangeably herein.

[0069] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0070] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0071] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0072] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 is a schematic overview of the experimental protocol in which control and hCMV mRNA lipopolyplexes were administered to rats via intraamniotic injection to elicit an immune response at day 17 (El 7) of gestation. At the indicated time points, human cytomegalovirus glycoprotein B (hCMV gB) production was measured in the placenta at term via protein immunoblotting, and IgG antibody production was measured in fetal and neonatal serum via ELISA. E=gestational day (term=E21-22). P=post term days.

[0074] FIGs. 2A-2B show the expression of human cytomegalovirus (hCMV) glycoprotein B in term rat placentas from hCMV mRNA and control groups. FIG. 2A shows a protein immunoblot of hCMV glycoprotein B in term (E21) rat fetuses. FIG. 2B is a bar graph showing the quantification of hCMV glycoprotein B in term rat placentas. Data presented as median with interquartile range. * p<0.05 for mRNA vs. control

[0075] FIG. 3 is a bar chart showing human cytomegalovirus (hCMV) glycoprotein B antigen IgG antibody levels in rat serum at term (E21), as well as day 7 (P7) and day 14 (P 14) after birth. Data presented as mean ± SEM, * p<0.05 for mRNA vs. Control.

[0076] FIG. 4 is a schematic overview of the experimental protocol in which control and human cytomegalovirus (hCMV) mRNA lipopolyplexes were administered to rats at day 17 (El 7) of gestation via intraamniotic injection to elicit an immune response. After term, spleen lymphocytes from the rat neonates were isolated and assessed for T-cell response in response to antigen challenge. At the indicated time points, IgG antibody production was measured in neonatal and maternal serum via ELISA. E=gestational day; term=E21-22.

[0077] FIG. 5 is a bar graph of the antigen-specific serum IgG antibody levels at 1 month, 1.5 months, and 3 months of age after transamniotic fetal mRNA administration as described in FIG. 4, against an hCMV antigen in rats. Data presented in relative units (RU) as median (interquartile range). Mo = months. ***p=0.029, 1 vs. 3 months.

[0078] FIGS. 6A-6E show flow cytometry data of cytokines relevant to cellular immune response after incubation of spleen lymphocytes with or without a human cytomegalovirus glycoprotein B (hCMV-gB) recombinant envelope protein antigen at three different postnatal time points. FIG. 6A shows the production of interferon-gamma (IFN-y) in control and hCMV- gB challenged rat neonates. FIG. 6B shows the production of tumor necrosis factor alpha (TNF- a) in control and hCMV-gB challenged rat neonates. FIG. 6C shows the production of interleukin-2 (IL-2) in control and hCMV-gB challenged rat neonates. FIG. 6D shows the production of interleukin-6 (IL-6) in control and hCMV-gB challenged rat neonates. FIG. 6E shows the production of granulocyte-macrophage colony stimulating factor (GM-CSF) in control and hCMV-gB challenged rat neonates.

[0079] DETAILED DESCRIPTION OF THE DISCLOSURE

[0080] Provided herein are polynucleotide, e.g., messenger RNA (mRNA) immunogenic compositions and methods for the delivery thereof to a fetus to induce an immune response against a pathogen antigen, thereby reducing the propensity of the perinatal subject to become infected with the pathogen, and / or reducing the severity of perinatal infections. Perinatal infections are among the leading causes of neonatal morbidity and mortality, responsible for over 500,000 deaths worldwide every year, and may be transmitted from the mother to fetus or newborn via maternal transfer, blood, genital secretions, and breastfeeding. However, vaccination remains challenging as human newborns do not typically mount an immune response to a vaccine administered after birth until 6-8 months of age. Polynucleotide, e.g., mRNA, vaccines exhibit higher potency, safety, and efficacy, and are also more easily manufactured than traditional vaccines. However, transport of exogenous encapsulated mRNA from a mother’s bloodstream through the placenta to the fetus is typically very limited. Therefore, there is a need for improved, minimally invasive methods of delivering mRNA immunogenic compositions to a fetus that reduce the risk and / or severity of perinatal infections.

[0081] As far as is known, the present disclosure demonstrates the first therapeutic transamniotic delivery of a composition comprising a polynucleotide, e.g. an mRNA, encoding a pathogen antigen, i.e., an antigen from a virus, bacteria, or other pathogen. As provided herein, the transamniotic delivery of an mRNA encoded pathogen antigen induces an immune response in a fetal subject, thereby reducing the propensity of the subject to become infected with the pathogen, and / or reducing the severity of a perinatal infection from the pathogen (e.g., virus (e.g., CMV), bacteria, or other pathogen).

[0082] The present disclosure describes the discovery that transamniotic fetal mRNA delivery of an example human viral antigen, human cytomegalovirus envelope glycoprotein-B (hCMV-gB), induces a lasting adaptive cell-mediated immune response as well as postnatal and early neonatal antigen-specific immunoglobulin production in a healthy rat model. Thus, fetal mRNA vaccination via the minimally invasive transamniotic route is a practical strategy for inducing an immune response in the fetal subject, thereby reducing the propensity of the subject to acquire a perinatal infection. Methods of Inducing an Immune Response

[0083] The disclosure provides methods for delivering, via the transamniotic fluid, polynucleotides, e.g., mRNAs, encoding a pathogen antigen to induce an immune response in a fetus, and / or to reduce the risk and / or severity of prenatal or perinatal infections. The methods and compositions provided herein may be used to reduce the risk of and / or severity of a perinatal infection from a virus, a bacteria, or other pathogen. In some embodiments, the method reduces horizontal transfer of the infection from an infected subject, e.g., the mother to a susceptible host, e.g., the fetus or neonate.

[0084] As provided herein, a polynucleotide, e.g., an mRNA, encoding a pathogen antigen is administered by intra-amniotic injection. In some embodiments, provided herein is a method for reducing an infection or risk thereof from a pathogen in a fetus or neonate comprising administering to the amniotic fluid surrounding the fetus a polynucleotide encoding an antigen of the pathogen, wherein the antigen induces an immune response in the fetus or neonate. In some embodiments, provided herein is a method of inducing an immune response in a fetus or neonate, comprising administering to the amniotic fluid surrounding the fetus a polynucleotide encoding an antigen to a pathogen, wherein the antigen induces an immune response in the fetus or neonate.

[0085] In some embodiments, an mRNA is formulated with a lipid, a protein, a plasmid, or a nanoparticle as part of a delivery vector. In some embodiments, the method of delivery can include the use of lipopolyplexes such as, for example, those described in: Bofinger et al., “Development of Lipopolyplexes for Gene Delivery: A Comparison of the Effects of Differing Modes of Targeting Peptide Display on the Structure and Transfection Activities of Lipopolyplexes,” Journal of Peptide Science, 2018, Vol. 24, No. 12, p. e3131 ” The mRNA can be delivered using common microencapsulation methods utilizing lipid nanoparticle delivery such as, for example, those described in: Hou et al., “Lipid Nanoparticles for MRNA Delivery,” Nature Reviews Materials, December 2021, Vol. 6, No. 12, pp. 1078-1094.” In some embodiments, the mRNA is encoded by a plasmid. In some embodiments, the mRNA is associated with a viral like particle, e.g., as described in “Ikwuagwu et al., “Virus-like particles for drug delivery: a review of methods and applications.” Current Opinions Biotechnology, 2022, Vo. 78, p. 102785.

[0086] The therapeutic methods of the disclosure (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of a polynucleotide, e.g., an mRNA encoding a pathogen antigen to the amniotic fluid surrounding a fetus. In embodiments, the fetus is a human fetus. Such treatment will be suitably administered to fetuses at risk for a disease, infection, and / or disorder, or symptom thereof, before and / or after birth. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

[0087] In some embodiments, a polynucleotide, e.g., an mRNA encoding a pathogen antigen is administered to the amniotic fluid in an amount of about or at least about 0.01 pg, 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1,000 mg. In some embodiments, a polynucleotide, e.g., an mRNA encoding a pathogen antigen is administered to the amniotic fluid in an amount of no more than about 0.01 pg, 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1,000 mg.

[0088] In some embodiments, doses of a polynucleotide, e.g., an mRNA encoding a pathogen antigen of the disclosure are from about 0.0001 mg / kg fetal weight per day to about 1000 mg / kg fetal weight per administration. In some embodiments, doses ranging from about 1 to about 50 mg / kg will be suitable. In an embodiment, a polynucleotide, e.g., an mRNA encoding a pathogen antigen is administered to the amniotic fluid in amounts sufficient to deliver about or at least about 0.1 mg / kg fetal weight, 0.0001 mg / kg fetal weight, 0.001 mg / kg fetal weight, 0.01 mg / kg fetal weight, 0.1 mg / kg fetal weight, 0.5 mg / kg fetal weight, 1 mg / kg fetal weight, 2 mg / kg fetal weight, 3 mg / kg fetal weight, 4 mg / kg fetal weight, 5 mg / kg fetal weight, 6 mg / kg fetal weight 7 mg / kg fetal weight, 8 mg / kg fetal weight, 9 mg / kg fetal weight, or 10 mg / kg fetal weight of the polynucleotide to the fetus. In an embodiment, a polynucleotide, e.g., an mRNA is administered to the amniotic fluid in amounts sufficient to deliver no more than about 0.0001 mg / kg fetal weight, 0.001 mg / kg fetal weight, 0.01 mg / kg fetal weight, 0.1 mg / kg fetal weight, 05 mg / kg fetal weight, 1 mg / kg fetal weight, 2 mg / kg fetal weight, 3 mg / kg fetal weight, 4 mg / kg fetal weight, 5 mg / kg fetal weight, 6 mg / kg fetal weight 7 mg / kg fetal weight, 8 mg / kg fetal weight, 9 mg / kg fetal weight, or 10 mg / kg fetal weight of the polynucleotide to the fetus. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses) may be employed to the extent that subject tolerance permits.

[0089] In some embodiments, administration of a polynucleotide, e.g., an mRNA encoding a pathogen antigen of the disclosure to the amniotic fluid is associated with a reduction in the risk of infection or the intensity, severity, or frequency, or delays the onset of a disorder, disease, and / or deficiency in the fetus before and / or after birth (z.e., prenatal or perinatal). In some embodiments, administration of a polynucleotide, e.g.,an mRNA encoding a pathogen antigen of the disclosure reduces horizontal transfer of the infection from an infected subject to a susceptible host.

[0090] In some embodiments, a polynucleotide, e.g.,an mRNA encoding a pathogen antigen is administered at a particular time during the development of the fetus. In some embodiments, the mRNA is administered prior to, at, after, and / or until about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, or 40 weeks of pregnancy. In some embodiments, it is advantageous to administer the mRNA prior to 20 weeks of pregnancy.

[0091] In embodiments, the transamniotic delivery of the polynucleotide to the amniotic fluid results in the presence of and / or an increase in levels of the polynucleotide, e.g, an mRNA in the fetal blood plasma. In embodiments, the transamniotic delivery of a polynucleotide to the amniotic fluid results in an increase in levels of the polynucleotide in fetal tissues (e.g., thymus, spleen, brain, and / or bone marrow, and / or others, given the presence of the polynucleotide in the fetal blood plasma). Further non-limiting examples of tissues include liver, stomach, intestines, pancreas, spleen, thymus, lymph nodes, brain, meninges, heart, blood vessels, lungs, airways, kidneys, ureters, urethra, ovaries, testicles, genitalia, skin, skin annexes, muscle, bone, cartilage, bone marrow, eyes, ears, mouth, nose, pharynx, larynx, vitreous body, cerebrospinal fluid, and peripheral blood.

[0092] A skilled practitioner will readily be able to identify suitable methods for detecting and measuring levels of a polynucleotide in the amniotic fluid or in any samples taken from a fetus or from a neonate that received such treatment before birth. For example, quantitative real-time polymerase chain reaction (qRT-PCR) is one example of many techniques available for measuring polynucleotide levels in biological samples such as, to provide non-limiting examples, serum, bone marrow, spleen tissue, thymus tissue, and brain tissue. The biological samples can be collected from the fetus and / or the mother.

[0093] Cytomegalovirus

[0094] In some embodiments, the methods and compositions provided herein may be used to induce an immune response against a cytomegalovirus (“CMV”), thereby reducing the propensity of the subject to acquire a perinatal infection, and / or thereby reducing the severity of a perinatal infection from a cytomegalovirus. As provided herein, a polynucleotide, e.g., an mRNA of the disclosure may encode a pathogen antigen, e.g., an envelope protein, e.g., a glycoprotein B envelope antigen of human CMV. In some embodiments, provided herein are polynucleotides, e.g., mRNAs encoding other CMV antigens, e.g., a CMV pentameric complex protein, a phosphoprotein, an immediate early antigen protein, and / or an early membrane antigen or a late membrane antigen protein.

[0095] The data presented herein show that the postnatal humoral immune response elicited by the transamniotic delivery of an mRNA vaccine encoding for the human cytomegalovirus glycoprotein B (hCMV-gB) envelope antigen lasts for at least 3 months postnatally in a rat model. Of note, whereas the hCMV-gB-specific antibody was detected in 53.9% of prenatally vaccinated pups by day 7, it was detected in 97.2% of the pups at 1-3 months after birth.

[0096] In addition, a cell-based immune activation pathway was observed based on the pattern of cytokine production by the spleen lymphocytes. Cells challenged with recombinant hCMV-gB protein showed significantly increased production of TNF-a and IL-6 compared to nonchallenged cells; these cytokines are typically seen early in the cellular immune response, being produced by APCs as well as by CD8+ T-cells (which can be directly activated by APCs). Production of these cytokines is a prominent early signal to naive CD4+ T-cells. Accordingly, the levels of these cytokines peaked by 1.5 months. In addition, challenged spleen lymphocytes showed significantly increased production of cytokines, i.e., IFN-y, GM-CSF, and IL-2 compared to non-challenged cells. IFN-y is typically produced by Thl cells, which coordinate a response against intracellular pathogens, activating macrophages and cytotoxic T-cells, while GM-CSF has a known immunomodulatory role, enhancing antigen presentation to T-cells. IL-2 is secreted by T-cells and promotes T-cell proliferation, survival, and effector functions, indicating that the specific T-cells were indeed activated during repeat challenge in vitro. The production of these cytokines increased over time postnatally, indicating a maturing Thl- predominant response.

[0097] Nucleotide sequences and compositions

[0098] The methods of the disclosure comprise administering a polynucleotide, e.g., an mRNA encoding a pathogen antigen to the amniotic fluid surrounding a fetus. In some embodiments, the disclosure may include modified nucleobases in a messenger RNA. Such modifications provide a number of advantages, including, for example, increasing the stability of the mRNA, masking the nucleic acid payload from the immune system, and / or decreasing the chances of an unintended immune response or triggering anaphylaxis. In some embodiments, such modifications can include, but are not limited to those modifications described herein, such as pseudouridine, thiouridine, and 5-methylcytidine. In some embodiments, the mRNA is singlestranded. In further embodiments, the mRNA can include untranslated regions (UTRs) at the 5’ and 3’ end to enhance stability. In some embodiments, the mRNA includes one or more of a 3’ poly(A) tail, a 5’ cap, UTRs, and a protein-encoding region. In embodiments, the mRNA molecule comprises a 5’ cap, a poly(A) tail, a 3’ untranslated region, and / or a 5’ untranslated region. Components of mRNA molecules suitable for use in the methods of the present disclosure include those described in Sahin, etal., “mRNA-based therapeutics - developing a new class of drugs,” Nature Reviews Drug Discovery, 13:759-780 (2014), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0099] In some embodiments, a DNA template can be used to produce synthetic mRNA. In further embodiments, in vitro transcription technology is used to transcribe the sequence of interest into mRNA, which can be purified for delivery into a target or fetus. In some embodiments, mRNA synthesis takes place in a cell-free system. In other embodiments, cells engineered to express an mRNA sequence of interest can be cultivated and purified. In some embodiments, the engineered cells have been transfected with a plasmid containing a DNA template for the mRNA sequence of interest.

[0100] In some embodiments, the mRNA encoding a pathogen antigen to be administered to a subject has been modified to improve translation efficiency in a target cell or has been modified to augment expression, which can include optimization of stop and start codons for efficient elongation and termination of translation. This optimization can include the replacement of similar codons with a major codon that is preferred by highly expressed genes or a codon that is decoded by more tRNA than similar codons. Methods for codon optimization are well known and include those methods described, for example, in Mauro and Chappel, “A critical analysis of codon optimization in human therapeutics,” Trends Mol Med., 20:604-613 (2014), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0101] In some embodiments, the polynucleotide is encoded by a DNA vector and is administered in a lentiviral or adenovirus viral delivery vector.

[0102] Pharmaceutical Compositions

[0103] Provided herein are compositions and methods for delivery of polynucleotide, e.g., mRNA immunogenic compositions comprising a pathogen antigen to a fetus that induce an immune response against the pathogen antigen, thereby reducing the propensity of the subject to become infected with the pathogen perinatally, and / or reducing the severity of a perinatal infection with the antigen. In some embodiments, the compositions provided herein contain a therapeutically effective amount of a polynucleotide, e.g.,an mRNA encoding an antigen in a unit of weight or volume suitable for administration to a subject. Agents (e.g., mRNA) of the disclosure may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. In some embodiments, the agents of the disclosure may be administered through injection of lipid nanoparticles or lipoplexes containing the a polynucleotide, e.g., mRNA, of interest.

[0104] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, viscous paraffin, fatty acid esters, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and / or coloring substances and the like) which do not deleteriously react with the active compounds or interfere with their activity. In an embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0105] A suitable pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A composition can be a liquid solution.

[0106] Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful delivery systems for agents of the disclosure include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

[0107] The formulations can be administered to human patients in therapeutically effective amounts. The preferred dosage of a polynucleotide the disclosure is likely to depend on such variables as the volume of the amniotic cavity, or the nature of a disorder being treated. In an embodiment, the polynucleotide is administered more than once in a given pregnancy.

[0108] A pharmaceutical composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0109] Kits

[0110] Provided herein are kits for delivering a polynucleotide, e.g.,an mRNA encoding a pathogen antigen to a fetal subject to induce an immune response against the antigen, thereby reducing the propensity of the perinatal subject to become infected with the antigen, and / or reducing the severity of a perinatal infection with the antigen. In some embodiments, the kit contains equipment (e.g., hypodermic needles and syringes) to aid in administration of compositions of the disclosure to amniotic fluid surrounding a fetus.

[0111] Optionally, the kit includes directions for administering the pharmaceutical composition to amniotic fluid. In other embodiments, the kit comprises a sterile container which contains the pharmaceutical composition. Such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding compositions containing mRNA. The instructions will generally include information about the pharmaceutical composition (e.g., safety information, recommended doses, and the like) and how to administer the composition to the amniotic fluid surrounding a fetus. In other embodiments, the instructions include at least one of the following: description of the mRNA; methods for using the enclosed materials; precautions; warnings; indications; clinical or research studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0112] The practice of the disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for specific embodiments will be discussed in the sections that follow.

[0113] The following are example embodiments of the disclosure and are not intended to be limiting in any way.

[0114] Enumerated Embodiments:

[0115] Embodiment 1. A method for vaccinating a fetal subject, the method comprising administering a polynucleotide encoding a cytomegalovirus (CMV) antigen to an amniotic fluid of the subject.

[0116] Embodiment 2. The method of Embodiment 1, wherein the CMV antigen is a component of an envelope of CMV.

[0117] Embodiment 3. The method of Embodiment 1 or Embodiment 2, wherein the CMV antigen is a glycoprotein.

[0118] Embodiment 4. The method of Embodiment 3, wherein the glycoprotein is glycoprotein B (gB).

[0119] Embodiment 5. The method of any one of Embodiments 1-4, wherein the polynucleotide is an mRNA.

[0120] Embodiment. The method of any one of Embodiments 1-5, wherein the polynucleotide is encapsulated within a vector.

[0121] Embodiment 7. The method of Embodiment 6, wherein the vector is a lipopolyplex.

[0122] Embodiment 8. The method of any one of Embodiments 1-7, wherein the subject is a human subject.

[0123] Embodiment 9. The method of any one of Embodiments 1-8, wherein the polynucleotide is administered through intra-amniotic injection.

[0124] Embodiment 10. The method of any one of Embodiments 1-9, wherein the CMV is human CMV (hCMV).

[0125] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

[0126] EXAMPLES

[0127] Example 1: Transamniotic administration of an mRNA encoding a human cytomegalovirus (hCMV) antigen induced neonate antibody production to hCMV

[0128] Pregnant Sprague Dawley dams underwent volume-matched intraamniotic injections in all their fetuses (n=103) of either human CMV (hCMV) envelope glycoprotein B (hCMV-gB) factor VIII antigen mRNA encapsulated in lipopolyplex (mRNA group; n=56), or of the same lipopolyplex without mRNA (controls; n=47) on gestational day 17 (E17; term=E21-22). Term placentas were screened for host production of hCMV-gB by protein immunoblotting. Serum hCMV-gB IgG antibodies were measured at term, and 7 (P7) and 14 (P 14) days after birth by ELISA. The encapsulation efficiency of the hCMV-gB mRNA into the lipopolyplex was 98.2%. The experimental design is shown in FIG. 1.

[0129] Materials and methods used to carry out the experiments described in Example 1 follow. mRNA formulation and encapsulation

[0130] A custom-designed hCMV envelope gB antigen (hCMV-gB) mRNA was commercially obtained (GenBank:M22343.1; Ribo Pro, The Netherlands). A modification of the mRNA was the addition of Cap 1 on the 5’-end and the elongation of the 3’-end by adding 150 adenosine monophosphates to create a longer poly (A) tail. Next, a proprietary sequence optimization method which uses only canonical nucleotides and relies on swapping synonymous codons was implemented by the supplier Ribo Pro to create the final mRNA product consisting of 2,800 nucleotides. The above modifications were used to enhance translational efficiency and mRNA stability. The final mRNA product was then kept in RNAse-free water at the 1 pg / pL at -80 °C.

[0131] Just prior to in vivo delivery, the mRNA was encapsulated into self-assembling semisynthetic particles using the commercially available TransIT mRNA transfection kit (Minis Bio, Madison, WI). TransIT consists of 2 components: a proprietary synthetic polymer and a lipid. This combination encapsulates mRNA and therefore enables both high transfection efficiency and low cellular toxicity. The interaction of the polymer, lipid, and mRNA forms a semisynthetic lipid-polymer nanoparticle complex referred to as a lipopolyplex, which enhances mRNA delivery in vivo. First Ipg of mRNA (1 pg / pL stock) was suspended in 45pL of phosphate-buffered saline (PBS). Then 2pL of mRNA Boost Reagent and 2pL of TransIT Reagent proper (the two components of the TransIT kit) were added to the mixture, carefully pipetted and incubated for 5-10 minutes at room temperature to allow for self-assembling mRNA encapsulation prior to injection in vivo. mRNA lipopolyplex encapsulation efficiency

[0132] TransIT encapsulation efficiency of the hCMV-gB mRNA into the lipopolyplex particles was measured using the ultrasensitive fluorescent nucleic acid stain-based Quant-iT RiboGreen assay (Thermo Fisher Scientific, Waltham, MA). Briefly, following the manufacturer’s instructions, samples of naked hCMV-gB mRNA in PBS (lpg / 50pL stock), of hCMV-gB mRNA lipopolyplexes (lpg / 50pL stock), and of mRNA standards were plated on 96-well plate, mixed first with TE buffer (lOmM Tris-HCl, ImM EDTA, pH 7.5; Thermo Fisher Scientific, Waltham, MA), then fluorescent RiboGreen reagent (Thermo Fisher Scientific) was added. After 5 minutes of incubation at room temperature and protected from light, fluorescence intensity was measured on a microplate reader (BMG Labtech, Cary, NC) to quantify the amount of free mRNA in solution. Encapsulation efficiency was calculated by subtracting the amount of free mRNA from the total originally used to generate the mRNA lipopolyplexes and expressed as a percentage.

[0133] Intra-amniotic injections

[0134] An overview of the experimental design is shown in FIG. 1. Nine time-dated Sprague Dawley dams (Charles River Laboratories, Wilmington, MA) underwent surgical intra-amniotic injections in all their viable fetuses on gestational day 17 (El 7, term=E21-22). Briefly, under general anesthesia with 2-4% of inhaled isoflurane (Patterson Veterinary, Greeley, CO), a midline laparotomy was performed and the bicornuate uterus was eviscerated. All viable fetuses (n=103) received a volume-matched (50pL) intra-amniotic injection of either a suspension of the custom-made hCMV-gB mRNA lipopolyplex (mRNA group; n=56) or of the same lipopolyplex without mRNA (control group; n=47) to control for possible inter-species homology of the glycoprotein B. Injections were performed using a 33G non-coring needle fitted on a lOOpL syringe (both from the Hamilton Company, Reno, NV). Injections were carried under direct vision with care not to disturb the fetus, placenta, or umbilical cord. The incision was then closed in two layers followed by application of powdered metronidazole (Unichem Pharmaceuticals, Hasbrouck Heights, NJ) to the wound. Extended-release buprenorphine Ethiqa XR (Fidelis Animal Health, North Brunswick, NJ) was administered subcutaneously for post-operative analgesia. Specimen procurement

[0135] Dams in both groups were either euthanized at term (~21 days aka E21) or allowed to deliver spontaneously and had their pups survive after birth for either 7 days (post-term day P7; P7 mRNA and P7 control) or 14 days (P14; P14 mRNA and P14 control). At P7 and P14, pups were euthanized by CO2 chamber followed by rapid cervical dislocation. Mothers were euthanized by CO2 chamber only. For the E21 groups, following euthanasia, the laparotomy was re-opened, and the uterus was screened for gross signs of fetal hydrops or fetal resorption. Only fetuses exhibiting signs of full viability at the time of euthanasia were presumed survivors and underwent further tissue procurement. First, umbilical cord structures were milked toward the fetus to allow for additional blood return. Then, an incision in the subclavian area was performed to pool and collect fetal blood from both the subclavian artery and vein, which was procured. This was followed by procurement of the placenta. A midline laparotomy was then performed on the fetus and any eventual gross abnormalities of the liver and spleen such as splenomegaly or hepatomegaly were noted. For the P7 and P14 pups, following euthanasia, a left lateral thoracotomy was performed, and the great vessels were transected just above diaphragm to allow for mixed arterio-venous blood to pool in the thoracic cavity and be procured. A midline laparotomy was then performed to evaluate any gross abnormalities of the liver and spleen. Maternal blood samples were obtained from all dams of the E21 or P14 groups via direct cardiac puncture. All blood samples were centrifuged at 5,000 relative centrifugal force for 8 minutes to allow for separation of the serum. All samples were then rapidly frozen in dry ice-ethanol bathsand stored at -80° C until further processing. hCMV-gB protein immunoblotting

[0136] Term placentas were screened for mRNA incorporation and translation by the host via detection of hCMV-gB antigen production through protein immunoblotting. Briefly, placental samples from each dam at E21 (n=8 for E21 mRNA and n=4 for E21 control) were randomly selected for further analysis. Protein was extracted from approximately 30 mg of placental tissue using Tissue Protein Extraction Reagent (T-PERTM) with added protease and phosphatase inhibitors (all from ThermoFisher Scientific, Waltham, MA) and protein concentration was determined using the Bradford assay (BioRad, Hercules, CA). Samples were then separated on an SDS 4-12% Criterion XT Bis-Tris gel (BioRad) and transferred to polyvinyl difluoride (PVDF) membranes (MilliporeSigma, Burlington, MA). The EveryBlot block buffer (BioRad) was used for membrane blocking. Membranes were incubated overnight at 4° C in 1 :4,000 dilution of anti-hCMVgB antibody (Abeam, Waltham, MA) in the EveryBlot buffer. Membranes were then washed with lx Tris-Buffered Saline with 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody (R&D Systems, Minneapolis, MN) for 1 hour at room temperature (1 :2,000 dilution). Protein targets were normalized using HRP-conjugated P-actin antibody (Sigma-Aldrich, St. Louis, MO). Immunoblots were developed with enhanced chemiluminescence reagents (Bio-Rad) on a ChemiDoc Touch System Imager (Bio-Rad) and quantified with Image Lab Software v6.1.0 (Bio-Rad). hCMV-gB antigen-specific IgG antibody ELISA

[0137] Fetal and maternal serum samples were standardized by volume and used at 1 :3 dilution for hCMV-gB antigen-specific IgG antibody detection using a commercially available ELISA kit (Novatein Biosciences, Woburn, MA) according to the manufacturer’s instructions. Serum samples, negative controls and standards were all run in duplicates on 96 well pre-coated plates. The final amount of hCMV-gB antigen-specific IgG antibodies was calculated using a standard curve and expressed in relative units (RU).

[0138] Statistical analyses

[0139] Fetal and neonatal survival comparisons between the groups was calculated by the Fisher’s exact test. Protein immunoblotting and ELISA data were compared by the nonparametric Wilcoxon rank sum test and Fisher’s exact test. Statistical significances were defined as p<0.05. The data were presented either as mean ± SEM, as absolute numbers (for survival analysis), or as median with interquartile range (IQR) for the protein immunoblotting analysis.

[0140] Overall fetal / neonatal survival was 86% (89 / 103) with no significant differences between the groups either at term [E21 : 100% (23 / 23) for mRNA and 91% (10 / 11) for control; p=0.324] or postnatally [P7+P14 combined: 91% (30 / 33) for mRNA and 72% (26 / 36) for control; p=0.066]. There was no maternal mortality and no premature labor in any group. Spontaneous delivery in the postnatal groups occurred either at E22 or E23. No gross hepatic or splenic abnormalities were observed in any term fetus or pup.

[0141] Immunoblots of hCMV-gB protein antigen in term placental tissue confirmed successful protein translation following intra-amniotic mRNA delivery (FIGs. 2A-2B). Presence of hCMV- gB was significantly higher in the E21 mRNA group [1.77 (1.37, 1.98) fold] compared to E21 controls [0.99 (0.86, 1.11) fold; p=0.008].

[0142] No hCMV-gB antigen-specific IgG was detected in the serum of any of the term fetuses (0.0±0.0 RU), i.e., 4 days following intra-amniotic injection of hCMV-gB mRNA. However, significantly increased serum levels of hCMV-gB antigen-specific IgG were present in mRNA pups at P7 (0.017±0.001 RU; p=0.008) and P14 (0.016±0.01 RU; p=0.006) when controlled by mRNA-free injections (0.0001±0.00015 RU and 0.0±0.0 RU, respectively), i.e., 12-20 days after transamniotic administration (FIG. 3). Antigen-specific IgG antibodies were significantly increased at P7 in the mRNA group when compared to mRNA fetuses at term (p<0.001) and persisted significantly elevated until P14 (E21 mRNA vs. P14 mRNA, p<0.001), with no statistically significant difference between P7 and P14 pups in the mRNA group (p=0.639).

[0143] In the P7 mRNA group, 53.9% (7 / 13) of pups mounted a hCMV-gB antigen-specific IgG antibody response versus 0% (0 / 23) in the E21 mRNA group (p<0.001). At P14, 46.7% (7 / 15) of pups showed persistence of said immune response (p=0.999 vs. P7 mRNA).

[0144] Maternal serum samples showed no antibodies except for a single dam from the P 14 control group (0.075 RU), possibly due to a partially hemolyzed serum sample, as hemolysis is known to cause false positivity on ELISA due to the peroxidase activity of hemoglobin.

[0145] Example 2: Transamniotic administration of an mRNA encoding a human cytomegalovirus (hCMV) antigen induced neonate humoral and cellular immune responses to hCMV

[0146] The rat humoral and cellular response to transamniotic administration of an mRNA encoding a human cytomegalovirus envelope glycoprotein-B antigen (hCMV-gB) were next measured up to 3 months post term to determine whether there was a sustained humoral (antibody) immune response, and whether a cellular immune response was also induced. An overview of the experiments is shown in FIG. 4.

[0147] Materials and methods used to carry out the experiments described in Example 1 follow.

[0148] Seven pregnant Sprague Dawley dams underwent volume-matched intra-amniotic injections in all their fetuses (n=82) of a custom-made mRNA encoding for hCMV envelope glycoprotein-B (hCMV-gB) antigen encapsulated by a lipid-polymer composite on gestational day 17 (E17; term=E21-22) (see FIG. 4). At three time points between 1 and 3 months after birth, serum levels of antigen-specific hCMV-gB IgG antibodies were measured by ELISA. Host spleen lymphocytes were incubated with or without challenge with the hCMV-gB antigen, followed by flow cytometry of culture supernatants to assess T-cell response. mRNA formulation and encapsulation

[0149] A custom-designed hCMV-gB mRNA with a modification of the mRNA consisting of the addition of Cap 1 on the 5 ’-end and the elongation of the 3 ’-end by adding 150 adenosine monophosphates to create a longer poly (A) tail was commercially obtained (GenBank:M22343.1; Ribo Pro, The Netherlands). A proprietary sequence optimization method utilizing only canonical nucleotides and relying on swapping synonymous codons was implemented to create the final mRNA product consisting of 2,800 nucleotides, as used in Example 1. These modifications were used to enhance translational efficiency and mRNA stability. The final mRNA product was then kept in RNAse-free water at Ipg / pL at -80°C.

[0150] Just prior to delivery in vivo, the mRNA was encapsulated into self-assembling semisynthetic particles using the commercially available TransIT mRNA transfection kit (Minis Bio, Madison, WI) which consists of two components: a proprietary synthetic polymer and a lipid combining to encapsulate mRNA. The interaction of the polymer, lipid, and mRNA forms a semisynthetic lipid-polymer nanoparticle complex referred to as a lipopolyplex, which increases mRNA transfection efficiency and lowers cellular toxicity, thus enhancing mRNA delivery in vivo. First Ipg of mRNA (Ipg / pL stock) was suspended in 45pL of phosphate-buffered saline (PBS). Then 2pL of mRNA Boost Reagent and 2pL of TransIT Reagent proper (the two components of the TransIT kit) were carefully pipetted, added to the mixture sequentially, and incubated for 5-10 minutes at room temperature to allow for self-assembling mRNA encapsulation prior to injection in vivo. mRNA lipopolyplex encapsulation efficiency

[0151] Encapsulation efficiency of the hCMV-gB mRNA into the lipopolyplex particles was measured using the ultrasensitive fluorescent nucleic acid stain-based Quant-iT RiboGreen assay (Thermo Fisher Scientific, Waltham, MA). Briefly, samples of naked hCMV-gB mRNA in PBS (1 pg / 50pL stock), of hCMV-gB mRNA lipopolyplexes (1 pg / 50pL stock), and of mRNA standards were plated on a 96-well plate, mixed first with TE buffer (lOmM Tris-HCl, ImM EDTA, pH 7.5; Thermo Fisher Scientific, Waltham, MA), and subsequently with the fluorescent RiboGreen reagent (Thermo Fisher Scientific). After 5 minutes of incubation at room temperature and protected from light, fluorescence intensity was measured on a microplate reader (BMG Labtech, Cary, NC) to quantify the amount of free mRNA in solution. Encapsulation efficiency was calculated by subtracting the amount of free mRNA from the total originally used to generate the mRNA lipopolyplexes and expressed as a percentage, which in this study was 98%. Intra-amniotic injections Seven pregnant Sprague Dawley dams (Charles River Laboratories, Inc., Wilmington, MA) were fed a normal diet ad libitum, and housed individually under standard dark / light cycling conditions. All of them underwent general anesthesia on gestational day 17 (E17; term = E21-22) with isoflurane (Patterson Veterinary, Greeley, CO) maintained at 2-2.5% in 100% oxygen. Using sterile technique, a midline laparotomy was made and the bicornuate uterus was eviscerated. All their fetuses (n=82) then received volume-matched (50pL) doses of a suspension of the custom- made hCMV-gB mRNA lipopolyplex injected into the amniotic fluid using a 33G non-coring needle on a lOOpL syringe (both from Hamilton Company, Reno, NV) introduced into the amniotic cavity under direct visualization by the ventral aspect of the fetus, carefully avoiding it, the placenta, and the umbilical cord. Upon conclusion of the injections, the uterus was returned to the abdomen and the laparotomy was closed in 2 layers with 3-0 Vicryl (Ethicon, Somerville, NJ) and 5-0 Monocryl (Ethicon) simple running sutures followed by application of 10% povidone-iodine (Medline, Northfield, IL) to the wound. Animals were allowed to recover, and analgesia was maintained by sustained-release buprenorphine (Zoopharm, Windsor, CO).

[0152] Specimen procurement

[0153] Dams were allowed to deliver spontaneously and had their pups survive after birth for either approximately 1 month (postnatal day 28-30: P28-30), 1.5 months (P41-43), or 3 months (P82-84). Two to three dams were randomly assigned to each time point, when they and their corresponding pups were euthanized via chamber-inhaled carbon dioxide. Under standard sterile conditions in a laminar flow hood, a median sternotomy was then performed, and the great vessels were sectioned, allowing for collection of whole blood, which was initially placed in a 1.5mL tube on ice, allowed to clot, and then centrifuged at 4000 g for 8 minutes, followed by procurement of supernatant serum. Serum samples were then rapidly frozen in a dry ice-ethanol bath and stored at -80° C until further processing. A midline laparotomy was performed for aseptic procurement of the spleen, which was initially placed in a tube with 5 mL sterile PBS on ice for immediate processing. hCMV-gB antigen-specific IgG antibody ELISA

[0154] Pup and maternal serum samples were standardized by volume and used at a 1 :2 to 1 :4 dilution (subject to volume constraints) for hCMV-gB antigen-specific IgG antibody detection using a custom ELISA kit (Novatein Biosciences, Woburn, MA). Serum samples, negative controls, and standards were all run in duplicates on 96-well pre-coated plates. The final concentration of hCMV-gB antigen-specific IgG antibodies was calculated using a standard curve and expressed in relative units (RU). Values <1.3 x the negative control were considered negative. Spleen lymphocyte plating and antigen challenge

[0155] On the same day of euthanasia, each aseptically procured spleen was minced, filtered through a sterile 70pm cell strainer (Corning, Coming, NY), and centrifuged to obtain a singlecell suspension. Splenocytes underwent red blood cell (RBC) lysis using ammonium-chloride- potassium (ACK) lysis buffer (Gibco, Waltham, MA). Briefly, the cell suspension was centrifuged at 400g for 5 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in ACK lysis buffer and incubated at room temperature for 5 minutes with occasional gentle mixing. Following incubation, the reaction was quenched by adding an excess volume of complete Roswell Park Memorial Institute (RPMI)-1640 media, and the cells were centrifuged at 400g for 5 minutes. The supernatant containing lysed RBCs was removed, and the cell pellet was resuspended in 5mL complete RPMI-1640 media. Complete RPMI-1640 media was prepared by supplementing RPMI- 1640 basal medium (Coming) with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 200pg / mL penicillin (Thermo Fisher Scientific), 200U / mL streptomycin (Thermo Fisher Scientific), ImM sodium pyruvate (Gibco), lx minimum essential medium (MEM) non-essential amino acids solution (Gibco), and 0.05mM 2-mecaptoethanol (Gibco). Extra media was stored at 4°C and warmed to 37°C in a water bath prior to cell culture.

[0156] A hemocytometer was then utilized to manually count the number of viable cells, with trypan blue stain (Gibco) utilized to distinguish nonviable cells. Cells were then resuspended in complete RPMI-1640 media to achieve a concentration of 2xl05cells / well in lOOuL / well and plated in sterile 96-well U-bottom plates (10 wells / sample). Five wells per sample were incubated with lOOuL / well of complete RPMI-1640 media, while five wells were incubated with lOOul / well complete RPMI-1640 media containing hCMV-gB recombinant protein at a concentration of lOug / mL (Abeam #43040, Cambridge, MA). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 5 days. After 5 days in culture, plates were centrifuged at 400g for 5 minutes and supernatant were collected using a multichannel pipette and transferred to 96- well flat-bottom plates. Plates were then taped closed and stored at -80°C until subsequent analyses.

[0157] Flow cytometry

[0158] At the time of analysis, cell culture supernatants were thawed, and samples were analyzed for production of interleukin- 10 (IL- 10), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin-2 (IL-2), GM-CSF using the LEGENDplex™ Rat Thl / Th2 Panel V02 (9-plex) w / V- bottom plate according to the published manufacturer’s protocol (Biolegend #741228, San Diego, CA). Samples were read on a BD LSRII flow cytometer (BD Bioscience, Franklin Lakes, NJ) on the same day the assay was performed.

[0159] Statistical analysis

[0160] Survival rates were compared using Fisher’s exact test. Differences in ELISA and flow cytometry data between time points and according to antigen challenge were assessed by the nonparametric Wilcoxon rank sum test. A two-tailed alpha level of p<0.05 was considered statistically significant. Statistical analyses were carried out using Stata version 16.0 software (StatCorp LLC, College Station, TX). The data were presented either as absolute numbers (survival analysis), or as median with interquartile range (IQR) (ELISA, flow cytometry).

[0161] Results

[0162] Overall neonatal survival was 44% (36 / 82), with no significant differences between the groups. Antigen-specific hCMV-gB antibodies were present in the serum at all time points, albeit decreasing significantly from 1 to 3 months postnatally (p=0.029). Spleen lymphocytes from vaccinated pups showed significantly increased production of IFN-y, IL-2, TNF-a, GM-CSF, and IL-6 following antigen-specific challenge (p=0.021 to <0.001 vs. non-challenged cells). Cellular response increased significantly over time (p=0.043 to <0.001), indicating a maturing Thl response.

[0163] There was no maternal mortality or premature labor in the study. Spontaneous deliveries occurred between term days E22 and E24. Overall neonatal survival was 44% (36 / 82), with no significant differences between the 1-month [P28-30: 40.9% (9 / 22)], 1.5-month [P41-43: 60% (15 / 25)], or 3-month [P82-84: 34.3% (12 / 35)] time points (p=0.067 to 0.779). No gross hepatic or splenic abnormalities were observed in any dam or pup. hCMV-gB antigen-specific IgG production

[0164] Antigen-specific hCMV-gB antibodies were detectable in the fetal serum at 1 month [0.044 RU (0.036, 0.098 IQR)], 1.5 months [0.019 RU (0.015, 0.022 IQR)], and 3 months [0.027 RU (0.023, 0.038 IQR)]. Overall, antibody levels decreased significantly from 1 to 3 months postnatally (p=0.029) (FIG. 5). Notably, all dams and pups demonstrated detectable levels of hCMV-gB-specific antibodies except for one pup in the 1.5-month group.

[0165] Maternal serum samples also showed the presence of antigen-specific antibodies at 1 month (0.054 RU), 1.5 months [0.052 RU (0.042, 0.062 IQR)], and 3 months [0.049 RU (0.042, 0.066 IQR)], without significant differences between the time point groups (p=0.999). Flow cytometry

[0166] When compared with non-challenged cells, spleen lymphocytes from vaccinated pups showed significantly increased production of IFN-y, IL-2, TNF-a, GM-CSF, and IL-6 following the antigen-specific challenge at nearly every postnatal time point (p=0.021 to <0.001), with the exception of IFN-y, IL-2, and GM-CSF at 1 month (p=0.317 to 0.947), and IL-2 at 3 months (p=0.166), as shown in Table 1 below.

[0167] Table 1

[0168] By contrast, IL- 10, IL-5, IL-4, and IL- 13 production remained minimal / unchanged when hCMV antigen was re-presented (not shown). Cytokine production following antigen challenge demonstrated a pattern of increasing production with increasing postnatal age (p=0.043 to <0.001), with IFN-y, IL-2, and GM-CSF production peaking at the 3-month time point, although TNF-a and IL-6 levels peaked at the 1 ,5-month time point (FIGS. 6A-6E). This pattern is compatible with a maturing Type-1 T-helper (Thl) cell response. Additional Embodiments

[0169] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0170] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method of inducing an immune response against a pathogen in a fetus or neonate, the method comprising administering to the amniotic fluid surrounding the fetus a polynucleotide encoding a pathogen antigen , thereby inducing an immune response in the fetus or neonate against the pathogen.

2. A method for reducing the propensity of a fetus or neonate to become infected with a pathogen, the method comprising administering to the amniotic fluid surrounding the fetus a messenger RNA encoding an antigen of the pathogen, wherein the antigen induces an immune response in the fetus or neonate, thereby reducing the infection or risk thereof from the pathogen.

3. The method of claim 1 or claim 2, wherein the messenger RNA reaches the placenta.

4. The method of claim 1 or claim 2, wherein the messenger RNA reaches the fetal circulation and therefore also reaches multiple fetal anatomical sites.

5. The method of claim 1 or claim 2, wherein the pathogen is a virus or a bacteria.

6. The method of claim 3, wherein the virus is a cytomegalovirus (CMV).

7. The method of claim 6, wherein the antigen is an envelope glycoprotein, a CMV pentameric complex protein, a phosphoprotein, an immediate early antigen protein, or an early membrane antigen or a late membrane antigen protein.

8. The method of claim 7, wherein the envelope glycoprotein is glycoprotein B.

9. The method of any one of claims 1-8, wherein the polynucleotide comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

10. The method of claim 9, wherein the RNA comprises a messenger RNA (mRNA).

11. The method of claim 10, wherein the mRNA comprises a sequence of SEQ ID NO: 1.

12. The method of claim 11, wherein the mRNA comprises a sequence of SEQ ID NO: 1 and a 5’ cap and / or a poly A tail.

13. The method of any one of claims 1-12, wherein the polynucleotide is packaged in a vector.

14. The method of claim 13, wherein the vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle.

15. The method of claim 13, wherein the vector is a lipopolyplex.

16. The method of any one of claims 1-14, wherein the subject is a mammal.

17. The method of claim 16, wherein the mammal is a human.

18. The method of any one of claims 1-17, wherein the method reduces horizontal transfer of the infection from an infected subject to a susceptible host.

19. The method of any one of claims 1-18, wherein the polynucleotide is formulated as a pharmaceutical composition.

20. The method of claim 19, wherein the pharmaceutical composition is administered prior to 10 weeks of pregnancy.

21. The method of claim 19, wherein the pharmaceutical composition is administered after 10 weeks of pregnancy.

22. The method of any one of claims 1-21, wherein the immune response comprises the production of antibodies to the pathogen.

23. The method of any one of claims 1-20, wherein the immune response comprises a cellular immune response.

24. The method of claim 21, wherein one or more cytokines are produced.

25. The method of claim 24, wherein the one or more cytokines are selected from JFN-y, IL- 2, TNF-a, GM-CSF, and IL-6.

26. A composition for inducing an immune response in a fetus or neonate to a cytomegalovirus comprising one or more polynucleotides comprising SEQ ID NO:

127. The composition of claim 26, wherein the one or more polynucleotides comprises SEQ ID NOS: 1 a 5’ cap and / or a polyA tail.

28. The composition of claim 26 or claim 27, wherein the one or more polynucleotides is packaged in a vector.

29. The composition of claim 28, wherein the vector comprises one or more of a lipid, a protein, a plasmid, or a nanoparticle.

30. The composition of claim 29, wherein the vector is a lipopolyplex.

31. The composition of claim 29, wherein the vector is a lipid nanoparticle.

32. A kit comprising the composition of any one of claims 26-30, formulated as a pharmaceutical composition, and instructions for use thereof.

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