Human cytomegalovirus vaccine

Abstract

Aspects of the present disclosure relate to methods for generating an antigen-specific immune response against human cytomegalovirus (hCMV) in a subject by administering an mRNA vaccine.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 070,134, filed Aug. 25, 2020 (title “Human Cytomegalovirus Vaccine”), U.S. Provisional Patent Application No. 63 / 079,421, filed Sep. 16, 2020 (title “Human Cytomegalovirus Vaccine”), and U.S. Provisional Patent Application No. 63 / 136,117, filed Jan. 11, 2021 (title “Human Cytomegalovirus Vaccine”), under 35 U.S.C. § 119(e), the disclosures of each of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Cytomegalovirus (CMV) is a virus of the Herpesviridae family. CMV is mainly acquired through contact with infectious mucosal secretions or in utero and remains latent after primary infection. The seroprevalence of CMV in the United States is 50.4% overall, but has been reported at rates of 60% - 100% in resource-poor regions.

[0003] CMV is the most common congenital viral infection, affecting 30,000 to 40,000 infants (0.6% - 2% of births) per year in the United States. Congenital CMV infection in the first trimester of pregnancy is associated with the most adverse pregnancy outcomes, but symptomatic congenital CMV can result from infection at any time during pregnancy. Approximately 30% - 35% of mothers who have a primary CMV infection during pregnancy transmit the virus to the fetus, and 12% of such newborns have symptomatic disease, with approximately 4% dying within the first year of life. In addition, approximately half of CMV-infected infants who are symptomatic at birth develop late complications such as intellectual disability, sensorineural hearing loss, and developmental delay. Due to the significant impact of congenital CMV infection on pediatric health, the 2017 Institute of Medicine Report ranked the development of a CMV vaccine to prevent congenital CMV infection as a top priority category.

[0004] In patients receiving chronic immunosuppressive therapy after solid organ or hematopoietic stem cell transplantation, CMV infection, which causes graft rejection or end-organ disease, is linked to a high mortality rate. In the United States, approximately 30,000 adults receive solid organ transplants and 22,000 receive hematopoietic stem cell transplants annually. Overall, 8%–40% of solid organ transplant patients and 3%–6% of hematopoietic stem cell transplant patients receiving antiviral prophylactic therapy develop post-transplant complications due to CMV. The main complications of CMV infection in transplant patients are acute or chronic rejection of the transplanted tissue, as well as invasive diseases (e.g., colitis, hepatitis, and encephalitis). [Overview of the project]

[0005] There is a very high unmet medical need for safe and effective methods to prevent congenital CMV infection. Another unmet medical need is the prevention of CMV infection in patients receiving long-term immunosuppressive therapy after solid organ or hematopoietic stem cell transplantation.

[0006] Messenger ribonucleic acid (mRNA)-based vaccine platforms were developed based on the principle and knowledge that targeted viral proteins or antigens are produced in vivo by the delivery and cellular uptake of corresponding synthetic viral mRNA via the delivery of an immunogenic composition formulated within lipid nanoparticles. The mRNA then undergoes ribosomal translation within the cell, endogenously expressing the viral protein antigen encoded by the vaccine immunogenic composition containing the synthetic viral mRNA. Such mRNA-based vaccines are transiently expressed without entering the cell nucleus, interacting with the human genome, or replicating. This allows mRNA vaccines and immunogenic compositions to provide a mechanism that stimulates the endogenous production of structurally intact and properly folded human glycosylated viral glycoproteins and protein antigens in a manner that accurately mimics wild-type viral infection, thereby inducing a highly targeted immune response against infectious pathogens such as CMV.

[0007] Aspects of this disclosure include (a) messenger ribonucleic acid (mRNA) polynucleotides comprising an open reading frame encoding hCMV gH polypeptide, (b) mRNA polynucleotides comprising an open reading frame encoding hCMV gL polypeptide, (c) mRNA polynucleotides comprising an open reading frame encoding hCMV UL128 polypeptide, (d) mRNA polynucleotides comprising an open reading frame encoding hCMV UL130 polypeptide, (e) mRNA polynucleotides comprising an open reading frame encoding hCMV UL131A polypeptide, and (f) hCMV The present invention relates to an hCMV immunogenic composition comprising mRNA polynucleotides containing an open reading frame encoding a gB polypeptide, wherein the molar ratio of (a):(f) in the immunogenic composition is approximately 1:1, the molar ratio of (b):(c):(d):(e) in the immunogenic composition is approximately 1:1:1:1, and the molar ratio of each of (a) and (f) to any one of (b), (c), (d), or (e) in the immunogenic composition is approximately 1.5:1 to 2:1.

[0008] In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2.

[0009] In some embodiments, the hCMV immunogenic composition is maintained as a liquid formulation until it is ready for administration to a patient. In some embodiments, the hCMV immunogenic composition is maintained as a lyophilized formulation until it is ready for administration to a patient.

[0010] In some embodiments, the hCMV immunogenic composition is stable for at least 3 months when stored at temperatures above 0°C and below 10°C. In some embodiments, the hCMV immunogenic composition is stable for at least 12 to 18 months when stored at temperatures above 0°C and below 10°C. In some embodiments, the hCMV immunogenic composition is stable for at least 24 months when stored at temperatures above 0°C and below 10°C. In some embodiments, the hCMV immunogenic composition is stable for at least 3 months when stored at a temperature of about 5°C. In some embodiments, the hCMV immunogenic composition is stable for at least 12 to 18 months when stored at a temperature of about 5°C. In some embodiments, the hCMV immunogenic composition is stable for at least 24 months when stored at a temperature of about 5°C.

[0011] In some embodiments, the hCMV immunogenic composition has increased stability compared to an hCMV immunogenic composition in which (a) to (f) are present in approximately equivalent masses. In some embodiments, the hCMV immunogenic composition has increased stability compared to an hCMV immunogenic composition in which (a) to (f) are present in approximately equivalent masses when stored for at least 3 months at temperatures above 0°C and below 10°C. In some embodiments, the hCMV immunogenic composition has increased stability compared to an hCMV immunogenic composition in which (a) to (f) are present in approximately equivalent masses when stored for at least 24 months at temperatures above 0°C and below 10°C.

[0012] In some embodiments, the hCMV immunogenic composition has increased pentamer expression compared to hCMV immunogenic compositions in which (a) to (f) are present in approximately equal masses. In some embodiments, the hCMV immunogenic composition induces increased pentamer antibody levels compared to hCMV immunogenic compositions in which (a) to (f) are present in approximately equal masses. In some embodiments, the hCMV immunogenic composition has increased gB expression compared to hCMV immunogenic compositions in which (a) to (f) are present in approximately equal masses. In some embodiments, the hCMV immunogenic composition induces increased gB antibody levels compared to hCMV immunogenic compositions in which (a) to (f) are present in approximately equal masses.

[0013] In some embodiments, the mRNA polynucleotides (a) to (f) are formulated in at least one lipid nanoparticle in an amount sufficient to induce an antigen-specific immune response to hCMV or hCMV antigen in a subject. In some embodiments, the mRNA polynucleotides (a) to (f) are formulated in at least one lipid nanoparticle in an amount sufficient to induce an antigen-specific immune response to hCMV or hCMV antigen in a subject, and then lyophilized.

[0014] In some embodiments, at least one of the mRNA polynucleotides (a) to (f) includes a chemical modification. In some embodiments, at least 80% of the uracil in the open reading frames of mRNA polynucleotides (a) to (f) has a chemical modification selected from N1-methyl-psoidouridine or N1-ethyl-psoidouridine. In some embodiments, the chemical modification is located at the carbon 5 position of uracil. In some embodiments, at least one of the mRNA polynucleotides (a) to (f) further includes at least one 5' terminal cap, 7mG(5')ppp(5')N1mpNp.

[0015] In some embodiments, the lipid nanoparticles include an ionizable aminolipid and a mixture of lipids comprising cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-sn-glycerol,methoxypolyethylene glycol (DMG-PEG). In some embodiments, the ionizable aminolipid comprises compound I. [ka] In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 20-60 mol% ionizable aminolipids, 25-55 mol% cholesterol, 5-25 mol% DSPC, and 0.5-15 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 45-55 mol% ionizable aminolipids, 35-40 mol% cholesterol, 5-15 mol% DSPC, and 1-2 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 50 mol% ionizable aminolipids, 38.5 mol% cholesterol, 10 mol% DSPC, and 1.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 49 mol% ionizable aminolipids, 38.5 mol% cholesterol, 10 mol% DSPC, and 2.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 48 mol% ionizable aminolipid, 38.5 mol% cholesterol, 11 mol% DSPC, and 2.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 47 mol% ionizable aminolipid, 38.5 mol% cholesterol, 11.5 mol% DSPC, and 3 mol% DMG-PEG.

[0016] In some embodiments, the molar ratio of mRNA(a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2, resulting in 10%, 20%, 30%, 40%, or 50% less lipids administered to the patient compared to administering equal amounts of mRNA(a):(b):(c):(d):(e):(f). In some embodiments, the molar ratio of mRNA(a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2, resulting in 30% less lipids administered to the patient compared to administering equal amounts of mRNA(a):(b):(c):(d):(e):(f). In some embodiments, the molar ratio of mRNA(a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2, resulting in 40% less lipids administered to the patient compared to administering equal amounts of mRNA(a):(b):(c):(d):(e):(f). In some embodiments, the molar ratio of mRNA(a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2, resulting in 50% less lipids administered to the patient compared to administering equal amounts of mRNA(a):(b):(c):(d):(e):(f).

[0017] In some embodiments, the mRNA encoding the hCMV gH protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 5, the mRNA encoding the hCMV gL protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 6, the mRNA encoding the hCMV UL128 protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 2, the mRNA encoding the hCMV UL130 protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 3, the mRNA encoding the hCMV UL131A protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 3, and / or the mRNA encoding the hCMV gB protein includes a nucleotide sequence having at least 90% identity to the nucleotide sequence of Sequence ID No. 1.

[0018] In some embodiments, the mRNA encoding the hCMV gH protein comprises the nucleotide sequence of SEQ ID NO: 5, the mRNA encoding the hCMV gL protein comprises the nucleotide sequence of SEQ ID NO: 6, the mRNA encoding the hCMV UL128 protein comprises the nucleotide sequence of SEQ ID NO: 2, the mRNA encoding the hCMV UL130 protein comprises the nucleotide sequence of SEQ ID NO: 3, the mRNA encoding the hCMV UL131A protein comprises the nucleotide sequence of SEQ ID NO: 4, and / or the mRNA encoding the hCMV gB protein comprises the nucleotide sequence of SEQ ID NO: 1.

[0019] In some embodiments, an open reading frame encoding the hCMV gH polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 11; an open reading frame encoding the hCMV gL polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 12; an open reading frame encoding the hCMV UL128 polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 8; an open reading frame encoding the hCMV UL130 polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 9; an open reading frame encoding the hCMV UL131A polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 10; and / or an open reading frame encoding the hCMV gB polypeptide includes a sequence having at least 90% identity to the sequence of SEQ ID NO: 7.

[0020] In some embodiments, the open reading frame encoding the hCMV gH polypeptide includes SEQ ID NO: 11, the open reading frame encoding the hCMV gL polypeptide includes SEQ ID NO: 12, the open reading frame encoding the hCMV UL128 polypeptide includes SEQ ID NO: 8, the open reading frame encoding the hCMV UL130 polypeptide includes SEQ ID NO: 9, the open reading frame encoding the hCMV UL131A polypeptide includes SEQ ID NO: 10, and / or the open reading frame encoding the hCMV gB polypeptide includes SEQ ID NO: 7.

[0021] In some embodiments, each of the mRNA polynucleotides (a) to (f) further comprises a poly-A tail. In some embodiments, the poly-A tail is 100 nucleotides long.

[0022] In some embodiments, the hCMV gH polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19, the hCMV gL polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20, the hCMV UL128 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16, the hCMV UL130 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17, the hCMV UL131A polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18, and / or the hCMV gB polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15.

[0023] In some embodiments, the hCMV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 19, the hCMV gL polypeptide comprises the amino acid sequence of SEQ ID NO: 20, the hCMV UL128 polypeptide comprises the amino acid sequence of SEQ ID NO: 16, the hCMV UL130 polypeptide comprises the amino acid sequence of SEQ ID NO: 17, the hCMV UL131A polypeptide comprises the amino acid sequence of SEQ ID NO: 18, and / or the hCMV gB polypeptide comprises the amino acid sequence of SEQ ID NO: 15.

[0024] Aspects of the present disclosure relate to a method for generating an antigen-specific immune response against hCMV in a subject, comprising administering to a human subject an effective amount of the hCMV immunogenic composition described herein, thereby inducing an antigen-specific immune response against hCMV or hCMV antigen in the human subject.

[0025] In some embodiments, the hCMV immunogenic composition is administered via intramuscular injection. In some embodiments, the human subject is CMV serologically positive prior to administration of the hCMV mRNA vaccine. In some embodiments, the human subject is CMV serologically negative prior to administration of the hCMV mRNA vaccine. In some embodiments, the hCMV immunogenic composition is administered in doses of 25 μg to 300 μg mRNA. In some embodiments, the hCMV immunogenic composition is administered in doses of 50 μg to 150 μg mRNA. In some embodiments, the hCMV immunogenic composition is administered in doses of 50 μg. In some embodiments, the hCMV immunogenic composition is administered in doses of 100 μg. In some embodiments, the hCMV immunogenic composition is administered in doses of 150 μg.

[0026] In some embodiments, the hCMV immunogenic composition is administered at least once, at least twice, or at least three times. In some embodiments, the hCMV immunogenic composition is administered in a primary immunization followed by one booster immunization. In some embodiments, the hCMV immunogenic composition is administered in a primary immunization followed by two booster immunizations.

[0027] In some embodiments, the effective amount is sufficient to produce a serum neutralizing anti-CMV antibody titer against epithelial cell infection at any of day 29, day 56, day 84, day 168, or day 196 after administration of the hCMV immunogenic composition. In some embodiments, the effective amount is sufficient to produce a serum neutralizing anti-CMV antibody titer against fibroblast cell infection at any of day 29, day 56, day 84, day 168, or day 196 after administration of the hCMV immunogenic composition. In some embodiments, the effective amount is sufficient to produce a serum neutralizing anti-CMV antibody titer against epithelial cell infection at any of day 29, day 56, day 84, day 168, or day 196 after immunization and a related geometric mean ratio (GMR) of post-baseline / baseline titers at one or more time points after administration of the hCMV immunogenic composition. In some embodiments, the effective amount is sufficient to produce a serum neutralizing anti-CMV antibody titer against fibroblast cell infection at any of day 29, day 56, day 84, day 168, or day 196 after immunization and a related geometric mean ratio (GMR) of post-baseline / baseline titers at one or more time points after administration of the hCMV immunogenic composition. In some embodiments, the percentage of participants in which the neutralizing antibody (nAb) against epithelial cell infection increases by at least 2-fold, at least 3-fold, or at least 4-fold over baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition. In some embodiments, the percentage of participants in which the neutralizing antibody (nAb) against fibroblast cell infection increases by at least 2-fold, at least 3-fold, or at least 4-fold over baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0028] Aspects of this disclosure relate to a method for inducing an antigen-specific immune response to human cytomegalovirus (hCMV) in a subject, comprising administering an effective amount of an hCMV immunogenic composition to a human subject, the composition comprising: (a) messenger ribonucleic acid (mRNA) polynucleotide containing an open reading frame encoding hCMV gH polypeptide; (b) mRNA polynucleotide containing an open reading frame encoding hCMV gL polypeptide; (c) mRNA polynucleotide containing an open reading frame encoding hCMV UL128 polypeptide; (d) mRNA polynucleotide containing an open reading frame encoding hCMV UL130 polypeptide; (e) mRNA polynucleotide containing an open reading frame encoding hCMV UL131A polypeptide; and (f) mRNA polynucleotide containing an open reading frame encoding hCMV gB polypeptide, thereby inducing an antigen-specific immune response to hCMV or hCMV antigen in the human subject, wherein the amount of the hCMV immunogenic composition is 25 μg to 300 μg. The present invention relates to a method in which, when administered in mRNA doses, the proportion of human subjects whose neutralizing antibodies (nAbs) against epithelial cell infection increase by more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, or 13-fold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0029] Further aspects of the present disclosure relate to a method for inducing an antigen-specific immune response to human cytomegalovirus (hCMV) in a subject, comprising administering an effective amount of an hCMV immunogenic composition to a human subject, the composition comprising: (a) messenger ribonucleic acid (mRNA) polynucleotide including an open reading frame encoding hCMV gH polypeptide; (b) mRNA polynucleotide including an open reading frame encoding hCMV gL polypeptide; (c) mRNA polynucleotide including an open reading frame encoding hCMV UL128 polypeptide; (d) mRNA polynucleotide including an open reading frame encoding hCMV UL130 polypeptide; (e) mRNA polynucleotide including an open reading frame encoding hCMV UL131A polypeptide; and (f) mRNA polynucleotide including an open reading frame encoding hCMV gB polypeptide, thereby inducing an antigen-specific immune response to hCMV or hCMV antigen in the human subject, wherein the amount of the hCMV immunogenic composition is 25 μg to 300 μg The present invention relates to a method in which, when administered at mRNA doses, the proportion of human subjects whose neutralizing antibodies (nAbs) against fibroblast infection increase more than twofold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0030] Aspects of this disclosure relate to a method for inducing an antigen-specific immune response to human cytomegalovirus (hCMV) in a subject, comprising administering an effective amount of an hCMV immunogenic composition to a human subject, the composition comprising: (a) messenger ribonucleic acid (mRNA) polynucleotide containing an open reading frame encoding hCMV gH polypeptide; (b) mRNA polynucleotide containing an open reading frame encoding hCMV gL polypeptide; (c) mRNA polynucleotide containing an open reading frame encoding hCMV UL128 polypeptide; (d) mRNA polynucleotide containing an open reading frame encoding hCMV UL130 polypeptide; (e) mRNA polynucleotide containing an open reading frame encoding hCMV UL131A polypeptide; and (f) mRNA polynucleotide containing an open reading frame encoding hCMV gB polypeptide, thereby inducing an antigen-specific immune response to hCMV or hCMV antigen in the human subject, wherein the amount of the hCMV immunogenic composition is 25 μg to 300 μg. The present invention relates to a method in which, when administered in mRNA doses, the proportion of human subjects whose anti-pentamer-conjugated antibody (bAb) increases by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0031] Further aspects of the present disclosure relate to a method for inducing an antigen-specific immune response to human cytomegalovirus (hCMV) in a subject, comprising administering an effective amount of an hCMV immunogenic composition to a human subject, the composition comprising: (a) messenger ribonucleic acid (mRNA) polynucleotide including an open reading frame encoding hCMV gH polypeptide; (b) mRNA polynucleotide including an open reading frame encoding hCMV gL polypeptide; (c) mRNA polynucleotide including an open reading frame encoding hCMV UL128 polypeptide; (d) mRNA polynucleotide including an open reading frame encoding hCMV UL130 polypeptide; (e) mRNA polynucleotide including an open reading frame encoding hCMV UL131A polypeptide; and (f) mRNA polynucleotide including an open reading frame encoding hCMV gB polypeptide, thereby inducing an antigen-specific immune response to hCMV or hCMV antigen in the human subject, wherein the amount of the hCMV immunogenic composition is 25 μg to 300 μg The present invention relates to a method in which, when administered at mRNA doses, the proportion of human subjects whose anti-gB binding antibody (Ab) increases more than twofold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0032] In some embodiments, the geometric mean ratio (GMR) of neutralizing antibodies against epithelial cell infection measured in human subjects is approximately 8 to 14 at a time point after administration of the hCMV immunogenic composition. In some embodiments, the geometric mean ratio (GMR) of anti-pentamer-conjugated antibodies (bAb) in human subjects is approximately 6 to 10 at a time point after administration of the hCMV immunogenic composition.

[0033] In some embodiments, the geometric mean ratio (GMR) of neutralizing antibodies against fibroblast infection in human subjects is approximately 2 at a time point after administration of the hCMV immunogenic composition. In some embodiments, the geometric mean ratio (GMR) of anti-gB binding antibodies (Ab) in human subjects is approximately 2 at a time point after administration of the hCMV immunogenic composition.

[0034] Each limitation of the present invention may encompass various embodiments of the present invention. Therefore, each limitation of the present invention involving any one element or combination of elements is expected to be included in each aspect of the present invention. In its application, the present invention is not limited to the configuration details or arrangement of components shown in the following description or illustrated in the drawings. Other embodiments of the present invention are possible and can be implemented or performed in various ways.

[0035] The attached drawings are not drawn to a consistent scale. In the drawings, identical or nearly identical components shown in various diagrams are represented by similar numbers. For clarity, not all components are labeled in every drawing. [Brief explanation of the drawing]

[0036] [Figure 1] This schematic diagram illustrates that the hCMV immunogenic composition described herein contains mRNA encoding the viral antigen gB and the pentamer (gH / gL / UL128 / UL130 / UL131A). The tropism of CMV is associated with various glycoproteins. gB and the pentamer are important targets of neutralizing antibodies. The majority of neutralizing antibodies in serologically positive individuals are against the pentamer. [Figure 2A]This graph shows the components within an hCMV immunogenic composition (e.g., hCMV mRNA vaccine) containing mRNAs encoding gB, gH, gL, UL128, UL130, and UL131A. The graph also shows that the degradation rate (measured k) of each mRNA component in the vaccine correlates with the length of the mRNA construct. As shown, gH and gB, which are large mRNA molecules, were associated with high degradation rates. [Figure 2B] This graph shows the components within an hCMV immunogenic composition (e.g., an hCMV mRNA vaccine) containing mRNA encoding gB, gH, gL, UL128, UL130, and UL131A. It shows the amount of each mRNA component in a 100 μg RNA dose of an immunogenic composition containing hCMV mRNA, either on an equal mass basis (left column) or on a pre-specified molar ratio basis (right column). The amounts are shown in both mass (μg) and moles (nanomoles). [Figure 3A] This graph shows that molar ratios of hCMV mRNA components containing 2X gH and 2X gB for each of gL, UL128, UL130, and UL131A increase the expression levels of hCMV pentamers and hCMV gB in vitro (indicated by Emax) and increase the relative potency of hCMV pentamers and hCMV gB compared to using hCMV mRNA components in equal mass ratios. [Figure 3B] This graph shows that molar ratios of hCMV mRNA components containing 2X gH and 2X gB for each of gL, UL128, UL130, and UL131A increase the expression levels of hCMV pentamers and hCMV gB in vitro (indicated by Emax) and increase the relative potency of hCMV pentamers and hCMV gB compared to using hCMV mRNA components in equal mass ratios. [Figure 4]This graph shows that formulations designed based on molar content can maintain a strong antibody response in mice even at extremely low levels of gL, UL130, UL128, and UL131. In the upper panel, the content of the two formulations (Group 8 and Group 1) is shown in mass (mg) and moles (picomoles), and the height of the bars reflects the relative amount of each mRNA. In Group 1, the gH content is a limiting factor for pentamer formation (0.6 picomoles / dose). In Group 8, the content of gL, UL130, UL128, and UL131 is also designed to be 0.6 picomoles / dose, so the pentamer dose is consistent with Group 1 on a molar basis. In the lower panel, the pentamer-specific antibody response is graphed as a function of the total mass dose of Group 8 and Group 1. While the response levels were similar, Group 8 used a lower total dose (2.3 mg vs 3 mg), resulting in significantly lower levels of gL, UL130, UL128, and UL131. Furthermore, gB was 1.2 times higher in Group 8 compared to Group 1. [Figure 5] This graph shows the effect of individually increasing the molar ratio of each hCMV pentamer component on the anti-pentamer IgG response. The results indicate that excessive γH increases anti-pentamer IgG in mice. [Figure 6A] This graph shows the dose-response of anti-gB and anti-pentamer antibodies in mice for three formulations of mRNA components based on specified molar ratios (lots #1, 2, and 3), compared to an equal-mass ratio (lot #4). The amounts of each mRNA molecule are shown in μg and picomoles when the hCMV mRNA immunogenic composition is based on an equal-mass ratio or the proposed molar ratio. [Figure 6B] This graph shows the dose-response of anti-gB antibodies and anti-pentamer antibodies in mice for three formulations of mRNA components based on specified molar ratios (lots #1, 2, and 3), compared to an equal-mass ratio (lot #4). It also shows the dose-response of anti-gB antibodies when the relative molar content is adjusted in a series of hCMV mRNA immunogenic compositions. An increase in antibody response is observed with increasing gB content. [Figure 6C]This graph shows the dose-response of anti-gB and anti-pentamer antibodies in mice for three formulations of mRNA components based on specified molar ratios (lots #1, 2, and 3), compared to an equal-mass ratio (lot #4). It also shows the dose-response of anti-pentamer antibodies when the relative molar content is adjusted in a series of hCMV mRNA immunogenic compositions. An increase in antibody response is observed with increasing gH content. [Figure 6D] This graph shows the dose-response of anti-gB and anti-pentameric antibodies in mice for three formulations of mRNA components based on specified molar ratios (lots #1, #2, and #3), compared to an equal-molecular-weight ratio (lot #4). It illustrates the dose-response of anti-pentameric antibodies when relative molar content is adjusted in a series of hCMV mRNA immunogenic compositions. In lots #1, #2, and #3, UL131A is the limiting component of the pentamer, and in these groups, an increase in antibody response is observed with increasing UL131A content. In lot #4, gH is the limiting component of the pentamer, and UL131A was administered in significant excess. Although the molar content of pentamer-limiting gH in lot #4 is equal to the molar content of pentamer-limiting UL131A in lot #1, the antibody response in lot #4 is smaller than that in lot #1, indicating that excess gH is effective in maximizing the antibody response. The hCMV pentamer is formed using gH as the base component, and other pentameric polypeptides are assembled on top of it. Therefore, the absence of gH inhibits pentamer formation. [Figure 7] This graph shows the theoretical inactivation of components in an hCMV mRNA vaccine over time, when mRNA components are formulated based on specified molar ratios of mRNA components (upper panel) or based on equal mass components (lower panel). The rate of inactivation is based on the rate-to-length relationship shown in Figure 2A. The upper panel shows the prediction that in an hCMV mRNA vaccine with 2X gB and 2X gH relative to other mRNA components, gH and gB will be maintained in excess for at least 36 months. On the other hand, the lower panel shows that in formulations with equal mass components, inactivation occurs over time, and gH and gB are not maintained in equal mass ratios. [Figure 8]This graph plots the neutralizing antibody response to the hCMV mRNA vaccine in humans in a Phase I clinical trial on a molar-based X-axis. The values ​​are plotted using an hCMV vaccine formulated based on equal mass of mRNA components, but are shown here in calculated picomoles. Arrows along the X-axis indicate where microgram-based alternative formulations overlap on the X-axis. This X-axis allows for plotting two formulations with different designs on the same continuous axis, enabling dose selection for subsequent trials. On the other hand, plotting against total mass dose (mg) results in discontinuous dose-response curves between the two formulations. Dose escalation phases A and B, and dose selection phase B (30, 90, 180 μg) are graphed for individual subjects. Data for dose selection phase C (300 μg) are graphed as GMT ± 95% confidence intervals until the trial is unblinded. A represents neutralizing antibodies (nAbs) against epithelial cell infection (primarily pentamer-specific nAbs). B exhibits neutralizing antibodies (nAbs) against fibroblast infection (primarily gB-specific nAbs). [Figure 9A] This graph shows the nominal dose selection to ensure that hCMV vaccine vials remain effective until their expiration date. It displays neutralizing antibodies (nAbs) against epithelial cell infection. The normal Gaussian distribution shown is the theoretical distribution of the batch based on purity. [Figure 9B] This graph shows the nominal dose selection to ensure that hCMV vaccine vials remain effective until their expiration date. It displays neutralizing antibodies (nAbs) against fibroblast infection. The normal Gaussian distribution shown is the theoretical distribution of the batch based on purity. [Figure 10] This graph shows the degradation model of 22 batches of hCMV vaccine at 5°C over time, based on the purity percentage of gB. The data represents a first-order rate equation. [Figure 11]This graph shows the anti-pentamer response (upper panel) and anti-gB response (lower panel) at various gB purity levels in mice. The graph indicates that there is no effect on the immune response to either pentamers or gB until the gB purity falls below 49% around 26 months. This result suggests that gB mRNA can function as a single indicator of batch potency over time. [Figure 12] This is a schematic diagram illustrating the design of a Phase II clinical trial. Abbreviations: IST: Internal Safety Team; mRNA: Messenger Ribonucleic Acid. [Figure 13] This graph shows the pH shift due to high dilution of a lyophilized preparation prepared with physiological saline (N. saline). The pH range of commercially available USP physiological saline is 4.5 to 7.0, and the measured pH of the physiological saline was 6.3. [Figure 14A] This graph shows that two formulations based on molar ratio increase the expression levels of hCMV pentamer and hCMV gB in vitro. [Figure 14B] This graph shows that two formulations based on molar ratio increase the expression levels of hCMV pentamer and hCMV gB in vitro. [Figure 15] This graph shows the dose-response of two formulations of mRNA components (0.4g and 1g scales) in mice, based on molar ratios compared to an equal mass ratio (0.03g scale), for anti-gB and anti-pentamer antibodies. A shows the dose-response of the anti-gB antibody. B shows the dose-response of the anti-pentamer antibody. The scales refer to the batch sizes used to produce mRNA using in vitro transcription (IVT). [Figure 16]This graph compares CMV neutralizing antibody titers drawn by different formulations from different manufacturing batches, using either 0.03g or 1.0g scale mRNA in vitro transcription (IVT) manufacturing. A shows that in epithelial cells, the molar-ratio based large-scale lyophilized formulation (1g scale) drew higher CMV neutralizing antibody titers than the equal-mass ratio based small-scale liquid formulation (0.03g scale). B shows that the molar-ratio based large-scale lyophilized formulation (1g scale) drew higher CMV neutralizing antibody titers at a dose of 2 μg mRNA. [Figure 17] The neutralizing antibody titers against epithelial cell infection up to 3 months (1 month after the second vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatus and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 18] The neutralizing antibody titers against fibroblast infection up to 3 months (1 month after the second vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatus and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 19]The neutralizing antibody titers against epithelial cell infection up to 7 months (1 month after the third vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatus and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 20] The neutralizing antibody titers against fibroblast infection up to 7 months (1 month after the second vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatus and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 21] Anti-pentamer conjugate antibody titers up to 7 months (1 month after the second vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatus and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 22]Anti-gB-binding antibody titers up to 7 months (1 month after the second vaccination) in the Phase II trial are shown per protocol set for antibody-mediated immunogenicity, separated by CMV serostatism and vaccination group. Neg: CMV seronegative, Pos: CMV seropositive. 50 μg, 100 μg, and 150 μg refer to mRNA vaccine doses. Confidence intervals (CI) were calculated using the t-distribution of log-transformed values. The black reference line shows the baseline GMT at baseline for all CMV seropositive subjects. [Figure 23] This is a schematic diagram showing the trial flow chart for the Phase III trial described in Example 7. Abbreviations: B = Blood collection for seroconversion due to primary CMV infection (CMV sero-negative cohort); CMV = Cytomegalovirus; D = Number of days; EOS = End of trial; I = Serum collection for antibody-mediated immunogenicity; M = Number of months; n = Total number of participants; U = Urine collection for CMV excretion in the CMV sero-positive cohort. [Figure 24] This is a schematic diagram showing the prediction of congenital CMV infection in the infant outcome preliminary study described in Example 8. [Figure 25] This is a schematic diagram showing the neonatal sample collection scheme in the infant outcome preliminary study described in Example 8. [Modes for carrying out the invention]

[0037] HCMV immunogenic compositions (e.g., vaccines such as mRNA vaccines) containing mRNA encoding the hCMV pentamer (gH, gL, UL128, UL130, and UL131A) and gB in equal mass ratios (e.g., an mRNA mass ratio of approximately 1:1:1:1:1:1 for gH:gL:UL128:UL130:UL131A:gB) have been shown to be effective in inducing neutralizing antibodies against hCMV in Phase I clinical trials. However, when all mRNA components are used in equal mass ratios, differences in molecular weight result in some mRNA components being overexpressed on a molar basis and others being underexpressed. Surprisingly, this specification shows that specifying the molar ratio of specific mRNA components in an hCMV immunogenic composition containing gH, gL, UL128, UL130, UL131A, and gB improves the properties. In detail, using gL, UL128, UL130, and UL131A in nearly equal molar ratios, and increasing the molar ratio of gB and / or gH compared to other mRNA components in the hCMV immunogenic composition, resulted in improved pentamer expression, improved gB expression, improved anti-pentamer antibody response, and improved anti-gB antibody response. Furthermore, such molar ratios allow for an increased shelf life of the hCMV immunogenic composition by maintaining the potency of the product.

[0038] The rationale for altering the mRNA ratio within a polyvalent hCMV mRNA vaccine is, at least in part, based on the molar stoichiometry of hCMV pentamer formation when mRNA within the vaccine is translated into protein. Providing individual mRNA constructs in ratios consistent with molar stoichiometry maximizes protein expression per unit mass of mRNA administered to the patient. gB and gH are the largest glycoprotein components in the immunogenic composition, with gH being the basal structure where smaller pentameric proteins complex to form the mature pentamer. Furthermore, adjusting the ratio based on the relative mRNA degradation rate during storage (which depends at least in part on the length of the mRNA construct) ensures optimal functional performance throughout the entire shelf life of the drug. As shown in the examples, the maximum mRNA can be used as a surrogate indicator of the overall efficacy of the vaccine batch. As long as the gB component remained above 49% unaffected by degradation, the overall immunogenicity of the vaccine was not lost. Modified ratios containing nearly equal molar amounts of UL128, gL, UL130, and UL131A, along with excess (e.g., at least 1.5X, or at least 2X) molar amounts of gB and gH, have been demonstrated herein to be effective in vitro and in mice. For a given total dose, a molar-ratio based hCMV immunogenic composition (e.g., mRNA vaccine) can result in increased potency and / or an improved antibody response, as indicated by protein expression, compared to an hCMV mRNA vaccine based on equal mass ratios.

[0039] In some embodiments, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can result in an increase in potency of about 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%, 50%, or more than 50%. In certain embodiments, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can result in an increase in potency of about 40%. In some embodiments, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can result in cost reductions of about 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%, 50%, or more than 50%. In a particular embodiment, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can result in cost reductions of about 40%. In some embodiments, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can yield increased tolerability with a reduction in lipid dosage of approximately 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%, 50%, or more than 50%. In certain embodiments, molar-ratio based hCMV immunogenic compositions (e.g., mRNA vaccines) can yield increased tolerability with a reduction in lipid dosage of approximately 40%.

[0040] antigen An antigen is a protein or polysaccharide capable of inducing an immune response (for example, causing the immune system to produce antibodies against an antigen). In this specification, unless otherwise specified, the term antigen encompasses immunogenic proteins and immunogenic fragments that induce (or are capable of inducing) an immune response to hCMV. It should be understood that the term "protein" encompasses peptides, while the term "antigen" encompasses antigenic fragments.

[0041] HCMV contains several surface glycoproteins involved in the attachment and entry of the virus into different cell types. The pentamer complex (PC) is composed of gH / gL / UL128 / UL130 / UL131A (Hahn et al., 2004; Ryckman et al., 2008; Wang and Shenk, 2005b (each of which is incorporated herein by reference)) and mediates entry into endothelial cells, epithelial cells, and myeloid cells.

[0042] The HCMV proteins UL128, UL130, and UL131A assemble with the gH and gL proteins to form a heterologous pentamer complex called gH / gL / UL128-131A, which is found on the surface of HCMV. Analysis of native variants, deletions, and mutations suggests that the proteins of the gH / gL / UL128-131A complex have the ability to infect certain cell types (e.g., endothelial cells, epithelial cells, and leukocytes).

[0043] HCMVs invade cells by fusing their envelope to the cell membrane (fibroblasts) or the endosomal membrane (epithelial and endothelial cells). HCMVs initiate cell invasion by attaching to heparan sulfate proteoglycans on the cell surface using envelope glycoprotein M (gM) or gB. This step is followed by interaction with cell surface receptors, inducing invasion or initiating intracellular signaling. Invasion receptor function is provided by gH / gL glycoprotein complexes. Different gH / gL complexes are known to promote invasion into epithelial cells, endothelial cells, or fibroblasts. For example, invasion into fibroblasts requires a gH / gL heterodimer, while invasion into epithelial and endothelial cells requires a pentameric complex of gH / gL / UL128 / UL130 / UL131 in addition to gH / gL. Thus, different gH / gL complexes bind to different invasion receptors on epithelial / endothelial cells and fibroblasts. Following receptor binding, membrane fusion occurs, a process mediated by gB and gH / gL. Early antibody studies have supported the idea that both gB and gH / gL play crucial roles in hCMV entry. gB is essential for entry and cell transmission. Both gB and gH / gL are necessary and sufficient for cell fusion and therefore constitute the "core fusion mechanism" of HCMV. This is conserved among other herpesviruses as well. Thus, the four glycoprotein complexes play a critical role in viral attachment, binding, fusion, and entry into host cells.

[0044] Studies on the gH / gL / UL128-131A complex have shown that the hCMV glycoproteins gB, gH, gL, gM, and gN, as well as the UL128, UL130, and UL131A proteins, are immunogenic and involved in immune stimulatory responses across various cell types. Furthermore, the UL128, UL130, and UL131A genes are relatively conserved among hCMV isolates, making them attractive targets for vaccination. In addition, recent studies have shown that antibodies against epitopes within the pentameric gH / gL / UL128-131 complex neutralize entry into endothelial, epithelial, and other cell types, inhibiting hCMV's ability to infect multiple cell types.

[0045] While we do not wish to be bound by any theory, it is thought that the majority of neutralizing antibodies target envelope glycoproteins (Britt et al., 1990; Fouts et al., 2012; Macagno et al., 2010; Marshall et al., 1992 (incorporated herein by reference)), while robust T cell responses are thought to target tegument protein pp65 and non-structural proteins such as IE1 and IE2 (Blanco-Lobo et al., 2016; Borysiewicz et al., 1988; Kern et al., 2002 (incorporated herein by reference)).

[0046] The HCMV envelope glycoprotein complex (e.g., gH / gL / UL128 / UL130 / UL131A) is a major antigenic target of the antiviral immune response. Embodiments of this disclosure provide RNA (e.g., mRNA) vaccines comprising a polynucleotide encoding an HCMV antigen, more specifically, an HCMV antigen derived from one of the HCMV glycoprotein complexes. Embodiments of this disclosure provide RNA (e.g., mRNA) vaccines comprising at least one polynucleotide encoding at least one hCMV antigen polypeptide. Using the HCMV RNA vaccines provided herein, a balanced immune response, encompassing both cellular and humoral immunity, can be induced without many of the risks associated with DNA vaccines and live attenuated vaccines.

[0047] International patent applications PCT / US2015 / 027400 (WO2015 / 164674) (title "Nucleic Acid Vaccines"), international patent applications PCT / US2016 / 058310 (WO2017 / 070613) (title "HUMAN CYTOMEGALOVIRUS VACCINE"), international patent applications PCT / US2017 / 057748 (WO2018 / 075980) (title "HUMAN CYTOMEGALOVIRUS VACCINE"), U.S. Patent No. 10,064,935 (title "HUMAN CYTOMEGALOVIRUS VACCINE"), and U.S. Patent No. 10,383,937 (title "HUMAN CYTOMEGALOVIRUS VACCINE"), U.S. Patent No. 10,064,935 (title "HUMAN The entire contents of U.S. Patent No. 10,716,846 (titled "HUMAN CYTOMEGALOVIRUS VACCINE") and U.S. Patent No. 10,716,846 (titled "HUMAN CYTOMEGALOVIRUS VACCINE") are incorporated herein by reference.

[0048] The hCMV antigens of the immunogenic compositions (e.g., vaccines (e.g., mRNA vaccines)) of the present disclosure are shown in Table 13 of this specification. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) comprises (a) a messenger ribonucleic acid (mRNA) polynucleotide including an open reading frame encoding the hCMV gH polypeptide, (b) an mRNA polynucleotide including an open reading frame encoding the hCMV gL polypeptide, (c) an mRNA polynucleotide including an open reading frame encoding the hCMV UL128 polypeptide, (d) an mRNA polynucleotide including an open reading frame encoding the hCMV UL130 polypeptide, (e) an mRNA polynucleotide including an open reading frame encoding the hCMV UL131A polypeptide, and (f) an mRNA polynucleotide including an open reading frame encoding the hCMV gB. In some embodiments, the components of the hCMV vaccine include the sequences shown in Table 13.

[0049] In some embodiments, the mRNA encoding the hCMV gH protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 5, or having more than 99% identity.

[0050] In some embodiments, the mRNA encoding the hCMV gL protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 6, or greater than 99% identity.

[0051] In some embodiments, the mRNA encoding the hCMV UL128 protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 2, or having more than 99% identity.

[0052] In some embodiments, the mRNA encoding the hCMV UL130 protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 3, or greater than 99% identity.

[0053] In some embodiments, the mRNA encoding the hCMV UL131A protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 4, or having more than 99% identity.

[0054] In some embodiments, the mRNA encoding the hCMV gB protein contains a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the nucleotide sequence of Sequence ID No. 1, or having more than 99% identity.

[0055] In some embodiments, the mRNA encoding the hCMV gH polypeptide contains the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the mRNA encoding the hCMV gL polypeptide contains the open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the mRNA encoding the hCMV UL128 polypeptide contains the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the mRNA encoding the hCMV UL130 polypeptide contains the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the mRNA encoding the hCMV UL131A polypeptide contains the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the mRNA encoding the hCMV gB polypeptide contains the nucleotide sequence of SEQ ID NO: 1.

[0056] In some embodiments, the open reading frame encoding the hCMV gH polypeptide includes a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of SEQ ID NO: 11, or more than 99% identity.

[0057] In some embodiments, the open reading frame encoding the hCMV gL polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of SEQ ID NO: 12, or more than 99% identity.

[0058] In some embodiments, the open reading frame encoding the hCMV UL128 polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of Sequence ID No. 8, or having more than 99% identity.

[0059] In some embodiments, the open reading frame encoding the hCMV UL130 polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of Sequence ID No. 9, or having more than 99% identity.

[0060] In some embodiments, the open reading frame encoding the hCMV UL131A polypeptide includes a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of Sequence ID No. 10, or having more than 99% identity.

[0061] In some embodiments, the mRNA encoding the hCMV gH polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the mRNA encoding the hCMV gL polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the mRNA encoding the hCMV UL128 polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the mRNA encoding the hCMV UL130 polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the mRNA encoding the hCMV UL131A polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the mRNA encoding the hCMV gB polypeptide includes an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 7.

[0062] In some embodiments, the hCMV gB polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the sequence of SEQ ID NO: 7, or greater than 99% identity.

[0063] In some embodiments, the hCMV gH polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 19, or greater than 99% identity.

[0064] In some embodiments, the hCMV gL polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 20, or greater than 99% identity.

[0065] In some embodiments, the hCMV UL128 polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 16, or greater than 99% identity.

[0066] In some embodiments, the hCMV UL130 polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 17, or greater than 99% identity.

[0067] In some embodiments, the hCMV UL131A polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 18, or greater than 99% identity.

[0068] In some embodiments, the hCMV gB polypeptide contains an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 15, or greater than 99% identity.

[0069] In some embodiments, the hCMV gH polypeptide contains the amino acid sequence of SEQ ID NO: 19. In some embodiments, the hCMV gL polypeptide contains the amino acid sequence of SEQ ID NO: 20. In some embodiments, the hCMV UL128 polypeptide contains the amino acid sequence of SEQ ID NO: 16. In some embodiments, the hCMV UL130 polypeptide contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, the hCMV UL131A polypeptide contains the amino acid sequence of SEQ ID NO: 18. In some embodiments, the hCMV gB polypeptide contains the amino acid sequence of SEQ ID NO: 15.

[0070] In some embodiments, the mRNA components of an hCMV immunogenic composition (e.g., mRNA vaccine) are present in equal masses. In other embodiments, the mRNA components of an hCMV immunogenic composition (e.g., mRNA vaccine) are not present in equal masses. It has been found herein that when mRNA components are included in equal masses, the expression of some of the longer mRNA constructs is insufficient, at least partially due to degradation. Therefore, as described in the examples, alternative approaches based on the molar ratio of mRNA components are developed herein for the formulation of mRNA components of an hCMV immunogenic composition (e.g., mRNA vaccine).

[0071] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) comprises: (a) messenger ribonucleic acid (mRNA) polynucleotides including an open reading frame encoding the hCMV gH polypeptide; (b) mRNA polynucleotides including an open reading frame encoding the hCMV gL polypeptide; (c) mRNA polynucleotides including an open reading frame encoding the hCMV UL128 polypeptide; (d) mRNA polynucleotides including an open reading frame encoding the hCMV UL130 polypeptide; (e) mRNA polynucleotides including an open reading frame encoding the hCMV UL131A polypeptide; and (f) hCMV The immunogenic composition comprises mRNA polynucleotides containing an open reading frame encoding a gB polypeptide, the molar ratio of (a):(f) in the immunogenic composition is approximately 1:1, the molar ratio of (b):(c):(d):(e) in the immunogenic composition is approximately 1:1:1:1, and the molar ratio of each of (a) and (f) to any one of (b), (c), (d), or (e) in the immunogenic composition is approximately 1.5:1~2: 1 (for example, 1.5:1~2:1, 1.5:1~1.9:1, 1.5:1~1.8:1, 1.5:1~1.7:1, 1.5:1~1.6:1, 1.6:1~2:1, 1.6:1~1.9:1, 1.6:1~1.8:1, 1.6:1~1.7:1, 1.7:1~2:1, 1.7:1~1.9:1, 1.7:1~1.8:1, 1.8:1~2:1, 1.8:1~1.9:1, or 1.9:1~2:1).

[0072] In some embodiments, the molar ratio of each of (a) and (f) in the immunogenic composition to any one of (b), (c), (d), or (e) is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1). In some embodiments, the molar ratio of (f) to any one of (b), (c), (d), or (e) in the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1). In some embodiments, the molar ratio of (a) to any one of (b), (c), (d), or (e) in the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1), and the molar ratio of (f) to any one of (b), (c), (d), or (e) in the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1). In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 1.5:1:1:1:1:1.5. In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:2.

[0073] In some embodiments, the mRNA associated with the hCMV immunogenic composition described herein may further comprise a 5' cap (e.g., 7mG(5')ppp(5')NlmpNp), a poly(A) tail (e.g., about 100 nucleotides), or a 5' cap and a poly(A) tail.

[0074] It should be understood that the hCMV immunogenic compositions (e.g., mRNA vaccines) of this disclosure may include signal sequences. It should also be understood that the hCMV mRNA vaccines of this disclosure may include any 5' untranslated region (UTR) and / or any 3' UTR. Exemplary UTR sequences are shown in Table 13, but other UTR sequences may be used or replaced with any UTR sequences described herein. UTRs may also be omitted from the vaccine constructs provided herein.

[0075] While we do not wish to be bound by any theory, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which the amount of each mRNA component is determined using molar ratios may have increased stability compared to hCMV immunogenic compositions (e.g., mRNA vaccines) in which the mRNA components are present in equal mass. This increased stability may help ensure that the hCMV immunogenic compositions remain stable throughout the shelf life of the drug containing these components and remain sufficiently effective for administration to the target until the designated expiration date of the drug.

[0076] The stability of mRNA constructs can be measured by any means known to those skilled in the art. In some embodiments, the stability of mRNA constructs is calculated based on the degradation and / or purity of the mRNA constructs. Long mRNA constructs in the hCMV immunogenic compositions described herein, e.g., gH and gB, are expected to degrade more rapidly than short mRNA constructs in the same hCMV immunogenic compositions. Therefore, in some embodiments, the stability of the hCMV immunogenic compositions described herein is measured by the degradation and / or purity of gH and / or gB. As used herein, “purity” refers to the amount of full-length intact mRNA (e.g., gB-coding mRNA) relative to the total input of mRNA (e.g., gB-coding mRNA).

[0077] In some embodiments, all mRNA components of the hCMV immunogenic compositions described herein maintain a purity of at least 45% (e.g., at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 60%, at least 70%, at least 80%, or more) throughout the expected shelf life (e.g., up to 2 years) under appropriate storage conditions (e.g., temperatures above 0°C and below 10°C). In some embodiments, gH and / or gB maintain a purity of at least 45% (e.g., at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 60%, at least 70%, at least 80%, or more) throughout the expected shelf life (e.g., up to 2 years) under appropriate storage conditions (e.g., temperatures above -80°C and below 10°C, e.g., -80°C, -70°C, -40°C, -20°C, 0°C, 5°C, or 10°C).

[0078] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least three months (for example, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteenteen months, at least twenty months, at least twenty months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, at least twenty-five months, at least twenty-six months, at least twenty-seven months, at least twenty-eight months, at least twenty-nine months, at least thirty months, at least thirty months, at least thirty-one months, at least thirty-two months, at least thirty-three months, at least thirty-four months, at least thirty-five months, at least twenty-seven months, at least twenty-eight months, at least twenty-nine months, at least thirty-five months, at least thirty-one months, at least thirty-two months, at least thirty-three months, at least thirty-four months, at least thirty-five months, at least thirty-five months, at least thirty-six months, at least twenty-seven months, at least twenty-eight months, at least twenty-nine months, at least thirty-five months, at least thirty-one months, at least thirty-two months, at least thirty-three months, at least thirty-four months, at least thirty-five months, at least thirty-six months, at least thirty-six months, at least thirty-five months, at least thirty-six months, at least thirty-five months, at In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stored at a temperature of approximately -80°C, -70°C, -40°C, -20°C, 0°C, 5°C, or 10°C. In some embodiments, the immunogenic composition (e.g., vaccine) is stored at a temperature of approximately -80°C. In some embodiments, the immunogenic composition (e.g., vaccine) is stored at a temperature of approximately -20°C. In some embodiments, the immunogenic composition (e.g., vaccine) is stored at a temperature of approximately 5°C. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least 3 months (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 30 months, or at least 36 months) when stored at a temperature of approximately -80°C, -20°C, or 5°C.

[0079] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least 12 to 18 months when stored at temperatures above -80°C and below 10°C (e.g., -80°C, -70°C, -40°C, -20°C, 0°C, 5°C, or 10°C). In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least 12 to 18 months when stored at a temperature of about -80°C. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least 12 to 18 months when stored at a temperature of about -20°C. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is stable for at least 12 to 18 months when stored at a temperature of about 5°C.

[0080] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which the mRNA components are based on a specified molar ratio have increased stability (e.g., at least 20%, at least 50%, at least 80%, at least 100%, at least 2x, at least 5x, at least 10x or more) than hCMV immunogenic compositions in which the mRNA components are present in approximately equivalent masses. In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on a specified molar ratio have increased stability (e.g., at least 20%, at least 50%, at least 80%, at least 100%, at least 2x, at least 5x, at least 10x or more) than hCMV immunogenic compositions in which mRNA components are present in substantially equivalent masses when stored at temperatures above -80°C and below 10°C (e.g., -80°C, -70°C, -40°C, -20°C, 0°C, 5°C, or 10°C) for at least 3 months, for example, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, or at least 36 months) than hCMV immunogenic compositions in which mRNA components are present in substantially equivalent masses.

[0081] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on a specified molar ratio have increased stability (e.g., at least 20%, at least 50%, at least 80%, at least 100%, at least 2x, at least 5x, at least 10x or more) than hCMV immunogenic compositions in which mRNA components are present in approximately equivalent masses when stored for at least 24 months at temperatures above -80°C and below 10°C (e.g., -80°C, -70°C, -40°C, -20°C, 0°C, 5°C, or 10°C). In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on a specified molar ratio have increased stability (e.g., at least 20%, at least 50%, at least 80%, at least 100%, at least 2x, at least 5x, at least 10x or more) than hCMV immunogenic compositions in which mRNA components are present in approximately equivalent masses when stored for at least 24 months at a temperature of about -80°C, -20°C, or 5°C.

[0082] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on specified molar ratios result in increased pentameric expression (e.g., in vitro or in vivo) compared to hCMV immunogenic compositions in which mRNA is present in approximately equivalent mass (e.g., increased by at least 10%, at least 20%, at least 50%, at least 90%, at least 2-fold, or at least 10-fold).

[0083] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on specified molar ratios result in increased gB expression (e.g., at least 10%, at least 20%, at least 50%, at least 90%, at least 2-fold, or at least 10-fold) compared to hCMV immunogenic compositions in which mRNA is present in approximately equivalent mass.

[0084] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on a specified molar ratio induce increased anti-pentamer antibody levels (e.g., increased by at least 10%, at least 20%, at least 50%, at least 90%, at least 2-fold, or at least 10-fold) compared to hCMV immunogenic compositions in which mRNA is present in approximately equivalent mass.

[0085] In some embodiments, the hCMV immunogenic compositions described herein (e.g., mRNA vaccines) in which mRNA components are based on a specified molar ratio induce increased anti-gB antibody levels (e.g., increased by at least 10%, at least 20%, at least 50%, at least 90%, at least 2-fold, or at least 10-fold) compared to hCMV immunogenic compositions in which mRNA is present in approximately equivalent mass.

[0086] nucleic acid The hCMV immunogenic compositions of this disclosure (e.g., mRNA vaccines) comprise at least one (one or more) ribonucleic acid (RNA) having an open reading frame encoding at least one hCMV antigen. In some embodiments, the RNA is messenger RNA (mRNA) having an open reading frame encoding at least one hCMV antigen. In some embodiments, the RNA (e.g., mRNA) further comprises at least one 5'UTR, 3'UTR, poly-A tail, and / or 5' cap.

[0087] Nucleic acids include polymers (also called polynucleotides) of nucleotides (nucleotide monomers). Nucleic acids may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA; including LNA having a β-D-ribo structure, α-LNA having an α-L-ribo structure (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras and / or combinations thereof, and may include these.

[0088] "Messenger RNA" (mRNA) is any ribonucleic acid that codes for (at least one) protein (natural, unnatural, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the coded protein. Those skilled in the art will understand that, unless otherwise stated, nucleic acid sequences described in this application may enumerate "T"s within representative DNA sequences, but when the sequence corresponds to RNA (e.g., mRNA), "T"s are replaced with "U". Accordingly, any DNA disclosed herein and identified by a specific sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to that DNA, in which case each "T" in the DNA sequence is replaced with a "U".

[0089] An open reading frame (ORF) is a continuous sequence of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. The sequences disclosed herein may further include additional elements (e.g., 5' and 3' UTR), but it should be understood that these elements, unlike ORFs, are not necessarily required to be present in the vaccines disclosed herein.

[0090] variant In some embodiments, the hCMV immunogenic composition of this disclosure (e.g., mRNA vaccine) comprises mRNA encoding an hCMV antigen variant. An antigen variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from that of the wild-type, native, or reference sequence. An antigen / polypeptide variant may have substitutions, deletions, and / or insertions at specific positions within its amino acid sequence compared to the native or reference sequence. Typically, a variant has at least 50% identity with the wild-type, native, or reference sequence. In some embodiments, a variant shares at least 80% or at least 90% identity with the wild-type, native, or reference sequence.

[0091] The variant antigens / polypeptides encoded by the nucleic acids of this disclosure may include amino acid changes that confer any of several desirable properties, such as enhancing their immunogenicity, enhancing their expression, and / or improving their stability or PK / PD properties within a subject. The variant antigens / polypeptides can be prepared using conventional mutagenesis techniques and, as appropriate, assayed to evaluate whether they possess the desired properties. Assays for evaluating expression levels and immunogenicity are well known in the art. Similarly, the PK / PD properties of protein variants can also be measured using techniques recognized in the art (e.g., by evaluating antigen expression over time within a vaccinated subject and / or by confirming the persistence of the induced immune response). The stability of the protein(s) encoded by the variant nucleic acid can be measured by analyzing thermal stability or stability under urea denaturation, or by using in silico prediction. Methods for such experiments and in silico evaluations are well known in the art.

[0092] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) comprises an mRNA ORF having a nucleotide sequence identified by any one of the sequences provided herein (see, for example, Table 13), or an mRNA ORF having a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (including all values ​​in between) identical to a nucleotide sequence identified by any one of the sequences provided herein.

[0093] The term "identity" refers to the relationship between two or more polypeptide (e.g., antigens) or polynucleotide (nucleic acid) sequences, determined by comparing these sequences. Identity also refers to the degree of sequence relevance between sequences, determined by the number of matching amino acid residues or nucleic acid residues between chains of two or more sequences. Identity measures the percentage of identical matches between the smaller of two or more sequences that have gap alignments (if any) processed by a specific mathematical model or computer program (e.g., "algorithm"). The identity of the relevant antigen or nucleic acid can be readily calculated by known methods. The "percent identity (%)" applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid or nucleic acid sequence of the second sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage identity. Methods and computer programs for such alignment are well known in the art. Identity depends on the calculation of the percentage identity, but it is understood that the value may differ depending on the gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., an antigen) exhibit at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art, but with less than 100% sequence identity. Tools for such alignment include the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402).Another well-known local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) “Identification of common molecular subsequences.” J.Mol.Biol.147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J.Mol.Biol.48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to align nucleotide and protein sequences more comprehensively and faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0094] Therefore, polynucleotides encoding peptides or polypeptides, including substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to a reference sequence, particularly a polypeptide (e.g., antigen) sequence disclosed herein, are included within the scope of this disclosure. For example, a sequence tag or amino acids (e.g., one or more lysines) can be added to a peptide sequence (e.g., to its N-terminus or C-terminus). Sequence tags can be used for the detection, purification, or localization of peptides. Lysines can be used to increase the solubility of peptides or to enable biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to result in a cleaved sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) can be optionally deleted depending on the intended use of the sequence (e.g., as part of a larger sequence that is soluble or for expression of a sequence bound to a solid support). In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., fold-on regions), etc., can be replaced with alternative sequences that achieve the same or similar functions. In some embodiments, stability can be improved by filling cavities within the protein core, for example, by introducing larger amino acids. In other embodiments, stability can be improved by replacing buried hydrogen bond networks with hydrophobic residues. In yet another embodiment, glycosylation sites can be removed and replaced with appropriate residues. Such sequences are readily identifiable to those skilled in the art. It should also be understood that some of the sequences provided herein include sequence tags or terminal peptide sequences (e.g., at the N-terminus or C-terminus) that can be deleted before use, for example, in the preparation of RNA (e.g., mRNA) vaccines.

[0095] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the range of the target hCMV antigen. For example, this specification provides protein fragments of any reference protein (meaning polypeptide sequences that are at least one amino acid residue shorter than the reference antigen sequence but otherwise identical), which are immunogenic and conditioned to confer a protective immune response against the hCMV pathogen. In addition to variants that are identical to the reference protein but cleaved, in some embodiments the antigen may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations in any of the sequences provided or referred to herein. The length of the antigen / antigen polypeptide may range from about 4, 6, or 8 amino acids to full-length proteins.

[0096] Stabilizing element Natural eukaryotic mRNA molecules may contain stabilizing elements, including, but are not limited to, the untranslated regions (UTRs) of their 5' end (5'UTR) and / or 3' end (3'UTR), in addition to other structural features such as a 5'-cap structure or a 3'-poly(A) tail, both of which are typically transcribed from genomic DNA and are elements of immature mRNA. Structural features specific to mature mRNA, such as the 5'-cap and 3'-poly(A) tail, are usually added to transcribed (immature) mRNA during mRNA processing.

[0097] In some embodiments, an hCMV immunogenic composition (e.g., an mRNA vaccine) comprises at least one RNA polynucleotide having an open reading frame encoding at least one antigen polypeptide having at least one modification (at least one 5' terminal cap), and is formulated within lipid nanoparticles. The 5'-cap of the polynucleotide can be generated by simultaneously completing the following chemical RNA cap analogs during an in vitro transcription reaction according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G[ARCA cap];G(5')ppp(5')A;G(5')ppp(5')G;m7G(5')ppp(5')A;m7G(5')ppp(5')G to produce a 5'-guanosine cap structure (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA can be completed post-transcriptionally using vaccinia virus capping enzyme to produce the "cap 0 structure": m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The cap 1 structure can be produced using both vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be produced from the cap 1 structure by 2'-O-methylating the third 5'-nucleotide using 2'-O methyltransferase. The cap 3 structure can be produced from the cap 2 structure by 2'-O-methylating the fourth 5'-nucleotide using 2'-O methyltransferase. The enzymes may be derived from recombinant sources.

[0098] The 3'-poly(A) tail is typically a sequence of adenine nucleotides attached to the 3' end of transcribed mRNA. In some cases, it can contain approximately 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail can be an essential element with respect to the stability of individual mRNAs.

[0099] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) includes one or more stabilizing elements. These stabilizing elements may include, for example, histone stem-loops. A 32 kDa stem-loop binding protein (SLBP) has been reported. It is associated with the histone stem-loop at the 3' end of histone messages in both the nucleus and cytoplasm. Its expression level is regulated by the cell cycle, peaking in the S phase, at which time histone mRNA levels also increase. This protein has been shown to be essential for the efficient 3' end processing of histone premRNA by U7 snRNP. SLBP continues to associate with the stem-loop after processing, and then facilitates the translation of mature histone mRNA into histone proteins in the cytoplasm. The RNA-binding domain of SLBP is conserved throughout metazoans and protists, and its binding to the histone stem-loop depends on the structure of the loop. The minimal binding site includes at least three nucleotides at the 5' and two nucleotides at the 3' to the stem-loop.

[0100] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) comprises a coding region, at least one histone stem-loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally enhances the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selection protein (e.g., alpha-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0101] In some embodiments, combinations of poly(A) sequences or polyadenylation signals with at least one histone stem-loop, each representing an alternative mechanism, act synergistically to increase protein expression beyond the levels observed with any of the individual elements. The synergistic effect of the poly(A) and at least one histone stem-loop combination is independent of the order of the elements or the length of the poly(A) sequence.

[0102] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) does not contain a histone downstream element (HDE). A "histone downstream element (HDE)" is a chain of approximately 15-20 nucleotides of purine-rich polynucleotides located at 3' of the natural stem-loop, which corresponds to the binding site of U7 snRNA involved in the processing of histone premRNA to mature histone mRNA. In some embodiments, the nucleic acid does not contain introns.

[0103] hCMV immunogenic compositions (e.g., mRNA vaccines) may or may not contain enhancer and / or promoter sequences, which may or may not be modified, activated or not activated. In some embodiments, a histone stem-loop generally originates from a histone gene and contains intramolecular base pairs of two adjacent, partially or entirely reverse-complementary sequences separated by a spacer consisting of a short sequence, which form the loop of this structure. An unpaired loop region typically cannot base-pair with any of the stem-loop elements. This is often present in RNA, as it is an important component of many RNA secondary structures, but it can also be present in single-stranded DNA. The stability of the stem-loop structure generally depends on its length, the number of mismatches or bulges, and the base composition of the paired regions. In some embodiments, fluctuating base pairs (non-Watson-Crick base pairs) may occur. In some embodiments, at least one histone stem-loop sequence contains a length of 15 to 45 nucleotides.

[0104] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) has one or more AU-rich sequences removed. These sequences (sometimes referred to as AURES) are destabilizing sequences found in the 3'UTR. AURES may be removed from the RNA vaccine, or they may remain in the RNA vaccine.

[0105] Signal peptide In some embodiments, an hCMV immunogenic composition (e.g., an mRNA vaccine) contains mRNA having an ORF encoding a signal peptide fused with the hCMV antigen. The signal peptide comprises 15–60 amino acids from the N-terminus of the protein and is typically required for membrane translocation on the secretory pathway, thus universally regulating the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates transmembrane transport of the growth peptide chain for processing. ER processing produces a mature protein, and the signal peptide is typically cleaved from the precursor protein by the host cell's ER-resident signal peptidase, or retained uncleaved, and functions as a membrane anchor. The signal peptide can also facilitate the targeting of proteins to the cell membrane.

[0106] The length of a signal peptide can range from 15 to 60 amino acids. For example, the length of a signal peptide can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In the initial step, the length of the signal peptide is 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35- The amino acids are 50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20.

[0107] Signal peptides derived from heterologous genes (essentially regulating the expression of genes other than the hCMV antigen) are known in the art and can be tested for desired properties before being incorporated into the nucleic acids of this disclosure. In some embodiments, the signal peptide may comprise one of the following sequences: MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 25), MDWTWILFLVAAATRVHS (SEQ ID NO: 26); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 13); MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 27); MKCLLYLAFLFIGVNCA (SEQ ID NO: 28); MWLVSLAIVTACAGA (SEQ ID NO: 29).

[0108] Array optimization In some embodiments, ORFs encoding the antigens of this disclosure are codon-optimized. Methods for codon optimization are known in the art. For example, one or more ORFs of any of the sequences provided herein may be codon-optimized. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding, to bias the GC content to increase mRNA stability or reduce secondary structures, to minimize tandem repeat codons or base sequences that may impair gene organization or expression, to customize transcription and translation regulatory regions, to insert or remove protein transport sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, to add, remove or replace protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to regulate translation rates so that various domains of the protein fold properly, or to reduce or remove problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using optimization algorithms.

[0109] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with a natural or wild-type sequence ORF (e.g., a natural or wild-type mRNA sequence encoding the hCMV antigen). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with a natural or wild-type sequence (e.g., a natural or wild-type mRNA sequence encoding the hCMV antigen). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with a natural or wild-type sequence (e.g., a natural or wild-type mRNA sequence encoding the hCMV antigen). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with a natural or wild-type sequence (e.g., a natural or wild-type mRNA sequence encoding the hCMV antigen). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with a natural or wild-type sequence (e.g., a natural or wild-type mRNA sequence encoding the hCMV antigen).

[0110] In some embodiments, the codon-optimized mRNA sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with the natural or wild-type sequence (e.g., the natural or wild-type mRNA sequence encoding the hCMV antigen). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity with the natural or wild-type sequence (e.g., the natural or wild-type mRNA sequence encoding the hCMV antigen).

[0111] In some embodiments, the codon-optimized mRNA sequence encodes an antigen that is immunogenic to or more immunogenic than the hCMV antigen encoded by the non-codon-optimized sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% higher).

[0112] Modified mRNA, when transfused into mammalian host cells, exhibits stability for 12–18 hours, or even longer than 18 hours, for example, 24, 36, 48, 60, 72 hours, or longer than 72 hours, and can be expressed by mammalian host cells.

[0113] In some embodiments, codon-optimized RNA may have enhanced G / C levels. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect RNA stability. RNA with increased amounts of guanine (G) and / or cytosine (C) residues may be more functionally stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modification within the coding region. Due to the degeneracy of the genetic code, the modification is made by substituting existing codons with codons that further enhance RNA stability without altering the resulting amino acids. This approach is limited to the coding region of RNA.

[0114] Unmodified nucleotides In some embodiments, at least one RNA (e.g., mRNA) of the hCMV immunogenic composition of the Disclosure (e.g., mRNA vaccine) is unchemically modified and comprises a standard ribonucleotide consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the Disclosure comprise standard nucleoside residues (e.g., residues present in transcription RNA (e.g., A, G, C, or U)). In some embodiments, the nucleotides and nucleosides of the Disclosure comprise standard deoxyribonucleosides (e.g., deoxyribonucleosides present in DNA (e.g., dA, dG, dC, or dT)).

[0115] chemical modification The hCMV immunogenic compositions (e.g., mRNA vaccines) of the Disclosure, in some embodiments, comprise at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one hCMV antigen, wherein the nucleic acid comprises nucleotides and / or nucleosides that may be standard (unmodified) or modified as known in the Art. In some embodiments, the nucleotides and nucleosides of the Disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally modified nucleotides and nucleosides or unnaturally modified nucleotides and nucleosides. Such modifications may include modifications to the sugar, backbone, or nucleic acid base portion of nucleotides and / or nucleosides that are recognized in the Art.

[0116] In some embodiments, the naturally modified nucleotides or nucleosides of this disclosure are those commonly known or recognized in the art. Non-limiting examples of such naturally modified nucleotides and nucleosides can be found, among other things, in the widely recognized MODOMICS database.

[0117] In some embodiments, the unnatural modified nucleotides or nucleosides of this disclosure are commonly known or recognized in the art. Non-limiting examples of such unnatural modified nucleotides and nucleosides can be found, among others, in published U.S. Patent Applications PCT / US2012 / 058519, PCT / US2013 / 075177, PCT / US2014 / 058897, PCT / US2014 / 058891, PCT / US2014 / 070413, PCT / US2015 / 36773, PCT / US2015 / 36759, PCT / US2015 / 36771, or PCT / IB2017 / 051367 (all of which are incorporated herein by reference).

[0118] Therefore, the nucleic acids of this disclosure (e.g., DNA nucleic acids and RNA nucleic acids (e.g., mRNA nucleic acids)) may include standard nucleotides and nucleosides, natural nucleotides and nucleosides, unnatural nucleotides and nucleosides, or any combination thereof.

[0119] The nucleic acids of this disclosure (e.g., DNA nucleic acids and RNA nucleic acids (e.g., mRNA nucleic acids)) include, in some embodiments, various (multiple) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of the nucleic acid includes one, two, or more (arbitrarily different) types of standard and / or modified nucleotides and nucleosides.

[0120] In several embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms are shown to exhibit reduced degradation within the cells or organisms compared to unmodified nucleic acids, including standard nucleotides and nucleosides.

[0121] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms may exhibit reduced immunogenicity in cells or organisms compared to unmodified nucleic acids containing standard nucleotides and nucleosides (e.g., reduced spontaneous response).

[0122] Nucleic acids (e.g., RNA nucleic acids (e.g., mRNA nucleic acids)) include, in some embodiments, unnaturally modified nucleotides introduced during or after the synthesis of the nucleic acid to achieve a desired function or property. The modifications may be present in internucleotide bonds, purine or pyrimidine bases, or sugars. The modifications may be introduced by chemical synthesis or by polymerase enzymes at the ends of the chain or at any location within the chain. Any region of the nucleic acid can be chemically modified.

[0123] This disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids (e.g., mRNA nucleic acids)). “Nucleoside” means a compound comprising a combination of a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred herein as “nucleic acid base”). “Nucleotide” means a nucleoside containing a phosphate group. Modified nucleotides, containing one or more modified or unnatural nucleosides, can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly. Nucleic acids may contain regions of linked nucleosides. Such regions may have a variety of skeletal bonds. These bonds may be standard phosphodiester bonds, in which case the nucleic acid would contain nucleotide regions.

[0124] Base pairing of modified nucleotides includes not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors enables hydrogen bonding between non-standard and standard bases, or between two complementary non-standard base structures, as in the case of nucleic acids having at least one chemical modification. An example of such a non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of this disclosure.

[0125] In some embodiments, the modified nucleic acid bases in nucleic acids (e.g., RNA nucleic acids (e.g., mRNA nucleic acids)) include 1-methyl-psoidouridine (m1ψ), 1-ethyl-psoidouridine (e1ψ), 5-methoxyuridine (mo5U), 5-methylcytidine (m5C), and / or pseudouridine (ψ). In some embodiments, the modified nucleic acid bases in nucleic acids (e.g., RNA nucleic acids (e.g., mRNA nucleic acids)) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpsoidouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleic acid bases (including, but not limited to, chemical modifications).

[0126] In some embodiments, the mRNA of the Disclosure comprises a 1-methylpsoiduridine (m1ψ) substitution at one or more or all uridine positions of the nucleic acid.

[0127] In some embodiments, the mRNA of the Disclosure comprises 1-methylpsoiduridine (m1ψ) substitutions at one or more uridine positions in the nucleic acid, and 5-methylcytidine substitutions at one or more cytidine positions in the nucleic acid.

[0128] In some embodiments, the mRNA of the Disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0129] In some embodiments, the mRNA of the Disclosure comprises pseudouridine (ψ) substitutions at one or more uridine positions in the nucleic acid, and 5-methylcytidine substitutions at one or more cytidine positions in the nucleic acid.

[0130] In some embodiments, the mRNA of this disclosure contains uridine at one or more or all of the uridine positions of the nucleic acid.

[0131] In some embodiments, mRNA is uniformly modified with a particular modification (e.g., completely modified, modified throughout the entire sequence). For example, a nucleic acid may be uniformly modified with 1-methylpsoidouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methylpsoidouridine. Similarly, a nucleic acid can be uniformly modified by replacing any type of nucleoside residue present in the sequence with a modifying residue such as those described above.

[0132] The nucleic acids of this disclosure can be modified partially or completely along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purines or pyrimidines, or one or more or all of A, G, U, and C) can be uniformly modified within the nucleic acids of this disclosure or within a given sequence region thereof (e.g., within mRNA with or without a poly-A tail). In some embodiments, all nucleotides X within the nucleic acids of this disclosure (or their sequence region) are modified nucleotides, where X may be one of the nucleotides A, G, U, or C, or one of the following combinations: A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0133] Nucleic acids are characterized by approximately 1% to 100% modified nucleotides (in terms of overall nucleotide content, or in terms of one or more types of nucleotides (i.e., one or more of A, G, U, or C)), or any range of percentages (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%). This may include 0%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%. Any remaining percentage will be determined by the presence of unqualified A, G, U, or C.

[0134] mRNA may contain modified nucleotides ranging from 1% to 100%, or any percentage within that range (e.g., at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides). For example, nucleic acids may contain modified uracil or modified pyrimidines such as cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the nucleic acid is replaced with modified uracil (e.g., 5-substituted uracil). Modified uracil can be replaced with a single compound having a unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the nucleic acid is replaced with modified cytosine (e.g., 5-substituted cytosine). The modified cytosine can be replaced with a compound having a single unique structure or with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0135] Untranslated area (UTR) The mRNAs of this disclosure may contain one or more regions or portions that act or function as untranslated regions. If the mRNA is designed to encode at least one target antigen, the nucleic acid may contain one or more of these untranslated regions (UTRs). The wild-type untranslated regions of nucleic acids are transcribed but not translated. In mRNA, the 5'UTR begins at the transcription start site and extends to the start codon, but does not contain the start codon. The 3'UTR, on the other hand, begins immediately after the stop codon and extends to the transcription termination signal. There is growing evidence for the regulatory role that UTRs play with respect to the stability and translation of nucleic acid molecules. The regulatory function of UTRs can be incorporated into the polynucleotides of this disclosure, particularly to enhance molecular stability. Specific functions can also be incorporated to ensure control of downregulation of transcripts in case the transcript is misdirected to an undesirable organ site. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

[0136] The 5'UTR refers to the region of mRNA located just upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not code for proteins (it is non-coding). Natural 5'UTRs have a function that plays a role in translation initiation. These have signatures such as the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has a common CCR(A / G)CCAUGG (SEQ ID NO: 30), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G" after the start codon. The 5'UTR is also known to form secondary structures involved in the binding of elongation factors.

[0137] In some embodiments of this disclosure, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature in relation to a different ORF. In another embodiment, the 5'UTR is a synthetic UTR, i.e., one that does not exist in nature. Synthetic UTRs include mutated UTRs to improve their properties (e.g., to increase gene expression) and fully synthetic ones. Exemplary 5'UTRs include α-globin or β-globin of Xenopus or human origin (US8278063;US9012219), human cytochrome b-245α polypeptide, and hydroxysteroid (17b) dehydrogenase, and tobacco etch virus (US8278063, 9012219). The CMV pre-early 1 (IE1) gene (US20140206753, WO2013 / 185069), sequence GGGAUCCUACC (SEQ ID NO: 18) (WO2014144196) may also be used. In another embodiment, the 5'UTR of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif (oligopyrimidine tract) (e.g., WO / 2015101414, WO2015101415, WO / 2015 / 062738, WO2015024667, WO2015024667; the 5'UTR element derived from the ribosomal protein Large32 (L32) gene (WO / 2015101414, WO201 5'UTR elements derived from the 5'UTR of the hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (5101415, WO / 2015 / 062738), or 5'UTR elements derived from the 5'UTR of ATP5A1 (WO2015024667) can be used. In some embodiments, the internal ribosome entry site (IRES) is used instead of the 5'UTR.

[0138] In some embodiments, the 5'UTR of the present disclosure comprises the nucleotide sequence of SEQ ID NO: 13.

[0139] The 3'UTR is a region of mRNA located downstream (3') of the stop codon (the codon in the mRNA transcript that signals the end of translation). The 3'UTR does not code for proteins (it is non-coding). Natural or wild-type 3'UTRs are known to have a series of embedded adenosine and uridine molecules. These AU-rich signatures are particularly widespread in genes with high turnover. Based on sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the AU-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A)(SEQ ID NO: 18) notamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are not as well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and Myogenin are two well-studied examples within this class. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, particularly HuR, have been reported to increase mRNA stability. HuR binds to all three classes of AREs. Manipulating the HuR-specific binding site within the 3'UTR of nucleic acid molecules leads to HuR binding and, consequently, in vivo message stabilization.

[0140] 3'UTRs may be heterologous or synthetic. Regarding 3'UTRs, globin UTRs (including Xenopus β-globin UTRs and human β-globin UTRs) are known in the art (US8278063, US9012219, US20110086907). Modified β-globin constructs with enhanced stability in several cell types have been developed by cloning two consecutive human β-globin 3'UTRs head-to-tail, and are well known in the art (US2012 / 0195936, WO2014 / 071963). In addition, α2-globin, α1-globin, UTRs, and their variants are also known in the art (WO2015101415, WO2015024667). Other 3'UTRs described in non-patent literature using mRNA constructs include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other exemplary 3'UTRs include those of bovine or human growth hormone (wild-type or modified) (WO2013 / 185069, US20140206753, WO2014152774), and rabbit β-globin and hepatitis B virus (HBV), α-globin 3'UTRs and viral VEEV 3'UTR sequences are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014144196) is used. In some embodiments, 3'UTRs of human and mouse ribosomal proteins are used. Other examples include rps9 3'UTR (WO2015101414), FIG4 (WO2015101415), and human albumin 7 (WO2015101415).

[0141] In some embodiments, the 3'UTR of the present disclosure comprises the nucleotide sequence of SEQ ID NO: 14.

[0142] Those skilled in the art will understand that heterogeneous or synthetic 5'UTRs can be used with any desired 3'UTR sequence. For example, a heterogeneous 5'UTR can be used with a synthetic 3'UTR containing a heterogeneous 3'UTR.

[0143] The combination of features can be included in the flanking region or within other features. For example, the ORF may be flanked by a 3'UTR which may contain a 5'UTR that may contain a potent Kozak translation initiation signal and / or an oligo(dT) sequence for template addition of a poly(A) tail. The 5'UTR may contain a first polynucleotide fragment and a second polynucleotide fragment derived from the same and / or different genes, such as the 5'UTR described in U.S. Patent Application Publication No. 20100293625 and PCT / US2014 / 069155 (which is incorporated herein by reference in its entirety).

[0144] RNA in vitro transcription The polynucleotide-encoding cDNAs described herein can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO / 2014 / 152027, which is incorporated herein by reference in its entirety.

[0145] In some embodiments, the RNA transcript is produced using an unamplified linearized DNA template in an in vitro transcription reaction for generating the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of RNA polynucleotides (e.g., hCMV mRNA, but not limited to these). In some embodiments, cells (e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells) are transfused using the plasmid DNA template. In some embodiments, the transfused cells are cultured to replicate the plasmid DNA, and then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5' to the target gene and responsively bound to the target gene.

[0146] In some embodiments, the in vitro transcription template encodes the 5' untranslated (UTR) region, includes an open reading frame, and encodes the 3' UTR and poly-A tail. The specific nucleic acid sequence composition and length of the in vitro transcription template depend on the mRNA encoded by the template.

[0147] If an RNA transcript is generated, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art. Please understand that the vaccines of this disclosure do not contain such promoter sequences.

[0148] Poly(A) tails may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, poly(A) tails contain 50 to 250 adenosine monophosphates. In relevant biological environments (e.g., intracellular, in vivo), poly(A) tails function to protect mRNA from enzymatic degradation (e.g., in the cytoplasm), assist in transcription termination, and / or transport and translation of mRNA from the nucleus.

[0149] In some embodiments, nucleic acids contain 200 to 3,000 nucleotides. For example, nucleic acids may contain 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0150] In some embodiments, RNA transcripts are capped via enzymatic capping. In some embodiments, the RNA contains a 5' end cap, e.g., 7mG(5')ppp(5')NlmpNp.

[0151] chemical synthesis Solid-phase chemical synthesis. The nucleic acids of this disclosure can be produced in whole or in part using solid-phase technology. Solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reaction solution. Solid-phase synthesis is useful for site-specific introduction of chemical modifications to nucleic acid sequences.

[0152] Liquid-phase chemical synthesis. The synthesis of nucleic acids according to this disclosure by sequential addition of monomer building blocks may be carried out in the liquid phase.

[0153] Combinations of synthesis methods. Each of the synthesis methods discussed above has its own advantages and limitations. Attempts have been made to combine these methods in order to overcome these limitations. Such combinations of methods are within the scope of this disclosure. Combining solid-phase or liquid-phase chemical synthesis with enzymatic ligation provides an efficient method for purifying long-chain nucleic acids that cannot be obtained by chemical synthesis alone.

[0154] Nucleic acid region or subregion ligation Nucleic acid assembly using ligases may also be used. DNA or RNA ligases facilitate intermolecular ligation of the 5' and 3' ends of polynucleotide chains via the formation of phosphodiester bonds. Nucleic acids, such as chimeric polynucleotides and / or cyclic nucleic acids, can be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by ligase-catalyzed reactions to produce recombinant DNA with different functions. Two oligodeoxynucleotides (one containing a 5' phosphoryl group and the other containing a free 3' hydroxyl group) serve as substrates for DNA ligases.

[0155] purification The nucleic acid purification described herein may include, but is not limited to, nucleic acid cleanup, quality assurance, and quality control. Cleanup may be carried out by, but is not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), Poly-T beads, LNA® oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, but is not limited to, methods known in the art, such as strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” as used in reference to nucleic acids, such as “purified nucleic acid,” means that the nucleic acid has been separated from at least one impurity. “Impurity” is any substance that transforms the nucleic acid into another unsuitable, impure, or inferior substance. Therefore, purified nucleic acids (e.g., DNA and RNA) exist in a form or configuration different from that found in nature, or in a form or configuration different from that which existed before being subjected to processing or purification.

[0156] Quality assurance and / or quality control checks may be performed using methods such as gel electrophoresis, UV absorbance, or analytical HPLC, but are not limited to these.

[0157] In some embodiments, nucleic acids may be sequenced by methods including, but not limited to, reverse transcriptase PCR.

[0158] Quantification In some embodiments, the nucleic acids of the present disclosure can be quantified in exosomes or when derived from one or more bodily fluids. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, sputumous fluid, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or bulbourethral gland fluid, sweat, feces, hair, tears, cystic fluid, pleural and ascites fluid, pericardial fluid, lymph, atherosclerotic fluid, chyle, bile, interstitial fluid, menstrual fluid, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, exosomes may be obtained from organs selected from the group consisting of the lungs, heart, pancreas, stomach, intestines, bladder, kidneys, ovaries, testes, skin, colon, breasts, prostate, brain, esophagus, liver, and placenta.

[0159] Assays can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, and exosomes can be isolated using immunohistochemistry such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, fractionation centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity purification, microfluidic separation, or a combination thereof.

[0160] These methods allow researchers to monitor the residual or delivered levels of nucleic acids in real time. This is possible because, in some embodiments, the nucleic acids of this disclosure differ from their endogenous forms due to structural or chemical modifications.

[0161] In some embodiments, nucleic acids may be quantified using methods such as ultraviolet-visible spectroscopy (UV / Vis), though not limited to these. An example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acids may be analyzed to determine whether they are of appropriate size and to confirm whether nucleic acid degradation has occurred. Nucleic acid degradation may be confirmed by methods such as agarose gel electrophoresis, HPLC-based purification methods, including, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0162] Lipid nanoparticles (LNPs) In some embodiments, the hCMV immunogenic composition of this disclosure (e.g., mRNA vaccine) is formulated within one or more lipid nanoparticles (LNPs). The lipid nanoparticles typically comprise ionizable amino(cationic) lipids, non-cationic lipids, sterols, and PEG lipid components together with the nucleic acid cargo of interest. The lipid nanoparticles of this disclosure are based on components, compositions, and methods commonly known in the art (e.g., PCT / US2016 / 052352;PCT / US2016 / 068300;PCT / US2017 / 037551;PCT / US2015 / 027400;PCT / US2016 / 047406;PCT / US2016000129;PCT / US2016 / 014280;PCT / US2016 / 014280; It can be generated using references to PCT / US2017 / 038426;PCT / US2014 / 027077;PCT / US2014 / 055394;PCT / US2016 / 52117;PCT / US2012 / 069610;PCT / US2017 / 027492;PCT / US2016 / 059575 and PCT / US2016 / 069491 (all of which are incorporated herein by reference in their entirety).

[0163] The vaccines of this disclosure are typically formulated within lipid nanoparticles. The vaccines can be manufactured using mixing processes such as microfluidics and T-type mixing of two fluid flows (one containing mRNA and the other containing lipid components). In some embodiments, the vaccines are prepared by combining ionizable aminolipids, phospholipids (such as DOPE or DSPC), PEG lipids (such as 1,2-dimiristoyl-OT-glycerol methoxypolyethylene glycol, also known as PEG-DMG), and structural lipids (such as cholesterol) in alcohol (e.g., ethanol). The lipids can be combined to obtain a desired molar ratio and diluted with water and alcohol (e.g., ethanol) to a final lipid concentration of, for example, about 5.5 mM to about 25 mM.

[0164] Vaccines containing mRNA and lipid components can be prepared, for example, by combining a lipid solution and an mRNA solution in a wt:wt ratio of lipid components to mRNA of approximately 5:1 to approximately 50:1. Using a microfluidic-based system (e.g., NanoAssemblr), a suspension (e.g., water-to-alcohol ratios of approximately 1:1 and approximately 4:1) can be obtained by rapidly injecting the lipid solution into the mRNA solution at a flow rate of, for example, approximately 10 ml / min to approximately 18 ml / min.

[0165] The vaccine can be treated by dialysis to remove alcohol (e.g., ethanol) and perform buffer exchange. The formulation can be dialyzed against phosphate-buffered saline (PBS) at pH 7.4, for example, in a larger volume than the primary product (e.g., using a Slide-A-Lyzer cassette (Thermo Fisher Scientific Inc., Rockford, IL)) and for example, using a molecular weight cutoff of 10 kD. The exemplary methods described above induce nanoprecipitation and particle formation. The same nanoprecipitation can also be achieved using alternative processes, including, but not limited to, T-type and direct injection.

[0166] The vaccines of this disclosure are typically formulated within lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise at least one ionizable aminolipid, at least one noncationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0167] The lipid nanoparticles of this disclosure consist of a mixture of lipids, the amount of which is measured according to the mole fraction or mole percentage of each lipid component in the lipid nanoparticles. The mole percentage is obtained by multiplying the mole fraction by 100%. When the lipid mixture is described numerically, mRNA and any water are not indicated.

[0168] In some embodiments, the lipid nanoparticles comprise a mixture of lipids containing 20–60 mol% of ionizable aminolipids. For example, the lipid nanoparticles may contain 20–50 mol%, 20–40 mol%, 20–30 mol%, 30–60 mol%, 30–50 mol%, 30–40 mol%, 40–60 mol%, 40–50 mol%, or 50–60 mol% of ionizable aminolipids in mole percent. In some embodiments, the lipid nanoparticles comprise 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% of ionizable aminolipids.

[0169] While ionizable aminolipids are sometimes referred to as cationic lipids in the literature, this document uses the term "ionizable aminolipids" to reflect that these lipids are cationic only under specific pH conditions.

[0170] In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 5-25 mol% of noncationic lipids. For example, the lipid nanoparticles may comprise noncationic lipids containing 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% of noncationic lipids. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% of noncationic lipids.

[0171] In some embodiments, the lipid nanoparticles comprise a mixture of lipids containing 25-55 mol% sterols. For example, the lipid nanoparticles may comprise sterols containing 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterols. In some embodiments, the lipid nanoparticles contain 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% of sterols in mole percent.

[0172] In some embodiments, the lipid nanoparticles comprise a mixture of lipids containing 0.5 to 15 mol% of PEG-modified lipids. For example, the lipid nanoparticles contain 0.5 to 10 mol%, 0.5 to 5 mol%, 1 to 15 mol%, 1 to 10 mol%, 1 to 5 mol%, 2 to 15 mol%, 2 to 10 mol%, 2 to 5 mol%, 5 to 15 mol%, 5 to 10 mol%, or 10 to 15 mol% of PEG-modified lipids in mole percent. In some embodiments, the lipid nanoparticles contain 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% of PEG-modified lipids in mole percent.

[0173] In some embodiments, the lipid nanoparticles contain 20-60% ionizable aminolipids, 5-25% noncationic lipids, 25-55% sterols, and 0.5-15% PEG-modified lipids in molar ratio.

[0174] In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 49 mol% ionizable aminolipid, 38.5 mol% cholesterol, 10 mol% DSPC, and 2.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 48 mol% ionizable aminolipid, 38.5 mol% cholesterol, 11 mol% DSPC, and 2.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticles comprise a lipid mixture containing 47 mol% ionizable aminolipid, 38.5 mol% cholesterol, 11.5 mol% DSPC, and 3 mol% DMG-PEG.

[0175] In some embodiments, the ionizable aminolipids of this disclosure include compounds having the following structures: [ka]

[0176] In some embodiments, the ionizable aminolipids of this disclosure include compounds having the following structures: [ka]

[0177] In some embodiments, the noncationic lipids of the present disclosure include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn- Lysero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3 -Phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0178] In some embodiments, the PEG-modified lipids of the present disclosure include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipids are DMG-PEG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0179] In some embodiments, the sterols of the present disclosure include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0180] In some embodiments, the LNP of the present disclosure comprises an ionizable aminolipid of compound 1, where the noncationic lipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG.

[0181] In some embodiments, the lipid nanoparticles contain 45 to 55 mole percent of ionizable aminolipids. For example, the lipid nanoparticles may contain 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mole percent of ionizable aminolipids.

[0182] In some embodiments, the lipid nanoparticles contain 5 to 15 mole percent of DSPC. For example, the lipid nanoparticles may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mole percent of DSPC.

[0183] In some embodiments, the lipid nanoparticles contain 35 to 40 mole percent of cholesterol. For example, the lipid nanoparticles may contain 35, 36, 37, 38, 39, or 40 mole percent of cholesterol.

[0184] In some embodiments, the lipid nanoparticles contain 1 to 2 mole percent of DMG-PEG. For example, the lipid nanoparticles may contain 1, 1.5, or 2 mole percent of DMG-PEG.

[0185] In some embodiments, the lipid nanoparticles contain 50 mole percent of ionizable aminolipids, 10 mole percent of DSPC, 38.5 mole percent of cholesterol, and 1.5 mole percent of DMG-PEG.

[0186] In some embodiments, the LNPs of this disclosure include N:P ratios ranging from about 2:1 to about 30:1.

[0187] In some embodiments, the LNPs of this disclosure include an N:P ratio of approximately 6:1.

[0188] In some embodiments, the LNPs of this disclosure include an N:P ratio of approximately 3:1.

[0189] In some embodiments, the LNPs of this disclosure comprise ionizable aminolipid components:RNA in a wt / wt ratio of about 10:1 to about 100:1.

[0190] In some embodiments, the LNPs of this disclosure comprise ionizable aminolipid components:RNA in a wt / wt ratio of approximately 20:1.

[0191] In some embodiments, the LNPs of this disclosure comprise ionizable aminolipid components:RNA in a wt / wt ratio of approximately 10:1.

[0192] In some embodiments, the average diameter of the LNPs in this disclosure is approximately 50 nm to approximately 150 nm.

[0193] In some embodiments, the average diameter of the LNPs in this disclosure is approximately 70 nm to approximately 120 nm.

[0194] Multivalent vaccine The hCMV immunogenic compositions (e.g., mRNA vaccines) provided herein may comprise mRNA or a plurality of mRNAs encoding two or more antigens of the same or different hCMV species. In some embodiments, the hCMV immunogenic compositions (e.g., mRNA vaccines) comprise RNA or a plurality of RNAs encoding two or more antigens. In some embodiments, the mRNA of the hCMV immunogenic composition (e.g., mRNA vaccine) may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more antigens.

[0195] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) comprises at least one RNA encoding hCMV gH, hCMV gL, hCMV UL128, hCMV UL130, hCMV UL131A, and hCMV gB.

[0196] In some embodiments, two or more different RNAs encoding antigens (e.g., mRNAs) may be formulated within the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens may be formulated within separate lipid nanoparticles (e.g., each RNA is formulated within a single lipid nanoparticle). The lipid nanoparticles can then be administered either combined as a single vaccine composition (e.g., a vaccine composition containing multiple RNAs encoding multiple antigens) or separately.

[0197] Pharmaceutical preparations This specification provides compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention or treatment of hCMV in humans and other mammals. hCMV immunogenic compositions (e.g., mRNA vaccines) can be used as therapeutic or prophylactic agents. These can be used in the medical field for the prevention and / or treatment of infectious diseases.

[0198] In some embodiments, an hCMV immunogenic composition (e.g., an mRNA vaccine) containing the mRNA described herein can be administered to a subject (e.g., a mammalian subject such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigen polypeptide (antigen).

[0199] The “effective dose” of an hCMV immunogenic composition (e.g., mRNA vaccine) is based at least in part on the target tissue, target cell type, administration method, physical properties of the RNA (e.g., length, nucleotide composition, and / or degree of modified nucleosides), other components of the vaccine, and other determinants, such as the age, weight, height, sex, and overall health status of the subject. Typically, the effective dose of an hCMV immunogenic composition (e.g., mRNA vaccine) provides an induced or boosted immune response as a function of antigen production within the target cells. In some embodiments, the effective dose of an hCMV immunogenic composition (e.g., mRNA vaccine) containing an RNA polynucleotide having at least one chemical modification is more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may be demonstrated by increased cellular translocation (percentage of cells translocated with the RNA vaccine), increased protein translation and / or expression from the polynucleotide, decreased nucleolysis (e.g., indicated by increased duration of protein translation from modified polynucleotides), or altered antigen-specific immune responses in host cells.

[0200] The term “pharmaceutical composition” refers to a combination of an active agent and an inactive or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. A “pharmaceutically acceptable carrier” is one that does not cause undesirable physiological effects after or during administration to a subject. The carrier in a pharmaceutical composition must also be “acceptable” in the sense that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizers may be used as a pharmaceutical carrier for the delivery of the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, excipients, and diluents for achieving compositions usable as dosage forms. Other examples of carriers include colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0201] In some embodiments, immunological compositions according to this disclosure (e.g., RNA vaccines comprising polynucleotides and their encoded polypeptides) can be used for the treatment or prevention of hCMV infection. hCMV immunogenic compositions (e.g., mRNA vaccines) can be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or during the early stages of infection in the incubation period or during active infection after symptom onset. In some embodiments, the amount of the hCMV immunogenic composition (e.g., mRNA vaccine) of this disclosure provided to cells, tissues, or subjects may be an effective amount for immunoprevention.

[0202] hCMV immunogenic compositions (e.g., mRNA vaccines) can be administered together with other prophylactic or therapeutic compounds. In non-limiting examples, the prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, the term “booster” refers to an additional dose of the prophylactic (vaccine) composition, in the case of a prophylactic composition such as a vaccine. A booster (or booster vaccine) may be administered after an earlier dose of the prophylactic composition. The time between the initial dose of the prophylactic composition and the booster is not limited to 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 It can be a day, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the time between the initial dose of the prophylactic composition and the booster may be, but is not limited, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, or one year. In some embodiments, multiple boosters (e.g., one, two, three, or more) are administered. In some embodiments, two boosters are administered after the initial dose (e.g., one around the beginning of the second month and one around the beginning of the sixth month).

[0203] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) can be administered intramuscularly (e.g., deltoid muscle), nasally, or intradermally, similar to the administration of inactivated vaccines known in the art.

[0204] hCMV immunogenic compositions (e.g., mRNA vaccines) can be used in a variety of settings depending on the prevalence of infection or the degree or level of unmet medical needs. As a non-limiting example, RNA vaccines can be used to treat and / or prevent a variety of infectious diseases. RNA vaccines have superior properties in that they produce much higher antibody titers, better neutralizing immunity, a more sustained immune response, and / or an earlier response than commercially available vaccines.

[0205] This specification provides pharmaceutical compositions comprising an hCMV immunogenic composition (e.g., an mRNA vaccine) and / or a complex, in combination with one or more pharmaceutically acceptable excipients of any choice.

[0206] hCMV immunogenic compositions (e.g., mRNA vaccines) can be formulated or administered alone or in combination with one or more other components. For example, an hCMV immunogenic composition (e.g., an mRNA vaccine) may contain other components (including, but not limited to, adjuvants).

[0207] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is adjuvant-free. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) contains an adjuvant. Any known adjuvant suitable for use in a vaccine can be used. In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) contains the MF59 adjuvant system (e.g., as described in O'Hagan et al., Expert Rev Vaccines. 2007 Oct;6(5):699-710 (incorporated herein by reference)).

[0208] hCMV immunogenic compositions (e.g., mRNA vaccines) can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the vaccine composition includes at least one additional active substance (e.g., a therapeutic active substance, a prophylactic active substance, or a combination of both). The vaccine composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations for the formulation and / or manufacture of pharmaceutical agents (e.g., vaccine compositions) can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety).

[0209] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is administered to a human (e.g., a human patient or subject). For the purposes of this disclosure, the expression “active ingredient” generally refers to the RNA vaccine or the polynucleotides contained therein, for example, the RNA polynucleotide encoding the antigen (e.g., mRNA polynucleotide).

[0210] The formulations of the vaccine compositions described herein can be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods include the steps of associating an active ingredient (e.g., mRNA polynucleotide) with excipients and / or one or more auxiliary components, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into desired single or multi-dose units.

[0211] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any further ingredients in a pharmaceutical composition according to this disclosure will vary depending on the specificity, size, and / or condition of the target being treated, and the route used to administer the composition. For example, a composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) of the active ingredient.

[0212] In some embodiments, an hCMV immunogenic composition (e.g., an mRNA vaccine) is formulated with one or more excipients to (1) enhance stability, (2) increase cellular translocation, (3) enable sustained or delayed release (e.g., from a depot), (4) alter in vivo distribution (e.g., target specific tissues or cell types), (5) increase in vivo translation of the encoded protein, and / or (6) alter the in vivo release profile of the encoded protein (antigen). In addition to conventional excipients (e.g., any solvent, dispersion medium, diluent, or other liquid vehicle, dispersant or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative), excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with hCMV immunogenic compositions (e.g., mRNA vaccines) (e.g., for transplantation into a subject), hyaluronidases, nanoparticle mimics, and combinations thereof.

[0213] In some embodiments, the hCMV immunogenic composition described herein (e.g., mRNA vaccine) is formulated into an LNP formulation and lyophilized. The lyophilized composition can be reconstituted with a solution suitable for administration. In some embodiments, the lyophilized composition is reconstituted with a solution containing 0.9% sodium chloride. In some embodiments, the reconstituted composition is then diluted with trissucrose diluent SD-0724 to a concentration that delivers an appropriate dose level in an appropriate volume (e.g., 0.5 mL).

[0214] Dosage / Administration This specification provides compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of hCMV infection in humans and other mammals. hCMV immunogenic compositions (e.g., mRNA vaccines) can be used as therapeutic or prophylactic agents. In some embodiments, the hCMV immunogenic compositions (e.g., mRNA vaccines) of this disclosure are used to provide prophylactic protection from hCMV. In some embodiments, the hCMV immunogenic compositions (e.g., mRNA vaccines) of this disclosure are used to treat hCMV infection. In some embodiments, the hCMV immunogenic compositions (e.g., mRNA vaccines) of this disclosure are used in priming immune effector cells, for example, to ex vivo activate peripheral blood mononuclear cells (PBMCs) and then inject (reinject) them into a target.

[0215] The subjects can be any mammal (including non-human primates and humans). Typically, the subjects are human subjects.

[0216] In some embodiments, an hCMV immunogenic composition (e.g., an mRNA vaccine) is administered to a subject (e.g., a mammalian subject such as a human subject) in an amount effective to induce an antigen-specific immune response. The RNA encoding the hCMV antigen is expressed and translated in vivo to produce the antigen, which stimulates an immune response in the subject. The subject may be hCMV serologically positive (e.g., previously had a spontaneous hCMV infection) or hCMV serologically negative (e.g., never had a spontaneous hCMV infection) prior to administration of the hCMV mRNA vaccine.

[0217] Prophylactic protection from hCMV can be achieved after administration of the hCMV immunogenic composition of this disclosure (e.g., mRNA vaccine). The vaccine may be administered one, two, three, four, or more times, but one dose of the vaccine (optionally followed by one or more boosters) may be sufficient. Although not desirable, it is also possible to administer the vaccine to an infected individual to achieve a therapeutic response. The dosage setting may need to be adjusted accordingly.

[0218] Methods for eliciting an immune response in a subject to hCMV are provided in various embodiments of this disclosure. The method involves administering an hCMV immunogenic composition (e.g., mRNA vaccine) described herein to a subject, thereby inducing an immune response in the subject that is specific to the hCMV antigen (e.g., hCMV gH, gL, UL128, UL130, UL131A, and / or gB). In some embodiments, the immune response is the induction of neutralizing antibodies against the hCMV antigen (e.g., hCMV gH, gL, UL128, UL130, UL131A, and / or gB). In some embodiments, the anti-antigen antibody titer in the subject increases after vaccination compared to the anti-antigen antibody titer in the subject vaccinated with a prophylactic effective dose of a conventional vaccine against hCMV. "Anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0219] In some embodiments, the effective prophylactic dose is the effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, the effective dose is the dose listed in the vaccine's package insert. In some embodiments, the effective dose is sufficient to produce detectable levels of hCMV antigen (e.g., gH, gL, UL128, UL130, UL131A, and / or gB polypeptides) in the serum of the subject 1 to 72 hours after administration of the hCMV immunogenic composition (e.g., mRNA vaccine) (e.g., 1 to 72 hours, 1 to 60 hours, 1 to 45 hours, 1 to 30 hours, 1 to 15 hours, 15 to 72 hours, 15 to 60 hours, 15 to 45 hours, 15 to 30 hours, 30 to 72 hours, 30 to 60 hours, 30 to 45 hours, 45 to 72 hours, 45 to 60 hours, or 60 to 72 hours). In some embodiments, the effective amount is sufficient to produce a neutralizing titer produced by a neutralizing antibody against the hCMV antigen (e.g., gH, gL, UL128, UL130, UL131A, and / or gB polypeptide) in a measurement of the serum of a target 1 to 72 hours after administration of an hCMV immunogenic composition (e.g., mRNA vaccine) (e.g., 1 to 72 hours, 1 to 60 hours, 1 to 45 hours, 1 to 30 hours, 1 to 15 hours, 15 to 72 hours, 15 to 60 hours, 15 to 45 hours, 15 to 30 hours, 30 to 72 hours, 30 to 60 hours, 30 to 45 hours, 45 to 72 hours, 45 to 60 hours, or 60 to 72 hours).

[0220] As used herein, "conventional vaccine" refers to vaccines other than the mRNA vaccines of this disclosure. Examples of conventional vaccines include, but are not limited to, live microbial vaccines, inactivated microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, and virus-like particle (VLP) vaccines. In exemplary embodiments, a conventional vaccine is a vaccine that has achieved regulatory approval and / or is registered with a national drug regulatory agency (e.g., the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA)).

[0221] In some embodiments, the anti-antigen antibody titer within a subject increases by 1 log to 10 log after vaccination compared to the anti-antigen antibody titer within a subject vaccinated with a prophylactic effective dose of a conventional vaccine against hCMV or within an unvaccinated subject. In some embodiments, the anti-antigen antibody titer within a subject increases by 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log after vaccination compared to the anti-antigen antibody titer within a subject vaccinated with a prophylactic effective dose of a conventional vaccine against hCMV or within an unvaccinated subject.

[0222] A method for eliciting an immune response in a subject to hCMV is provided in another aspect of this disclosure. The method involves administering an hCMV immunogenic composition (e.g., mRNA vaccine) described herein to a subject, thereby inducing an immune response specific to the hCMV antigen in the subject, the immune response being equivalent to that of a subject administered with a conventional vaccine against hCMV at a drug dose level 2 to 100 times higher than that of the RNA vaccine.

[0223] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject administered with a conventional vaccine at twice the drug dose level of the hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in a subject is equivalent to the immune response in a subject administered with a conventional vaccine at three times the drug dose level of the hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in a subject is equivalent to the immune response in a subject administered with a conventional vaccine at four, five, ten, fifty, or 100 times the drug dose level of the hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in a subject is equivalent to the immune response in a subject administered with a conventional vaccine at 10 to 1000 times the drug dose level of the hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in subjects is equivalent to the immune response in subjects administered with a conventional vaccine at a drug dose level 100 to 1000 times higher than that of the hCMV immunogenic composition (e.g., mRNA vaccine).

[0224] In other embodiments, the immune response is assessed by determining the titer of [protein] antibodies within the subject. In other embodiments, the ability of serum or antibodies from an immunized subject is tested for its ability to neutralize viral uptake or reduce hCMV transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response is measured using techniques recognized in the art.

[0225] Other aspects of the present disclosure provide a method for eliciting an immune response to hCMV in a subject by administering an hCMV mRNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one hCMV antigen to the subject, thereby inducing an immune response specific to the hCMV antigen in the subject, the immune response in the subject being induced 2 days to 10 weeks earlier than the immune response induced in subjects vaccinated with a prophylactic effective dose of a conventional vaccine against hCMV. In some embodiments, the immune response in the subject is induced in subjects vaccinated with a prophylactic effective dose of a conventional vaccine at a drug dose level 2 to 100 times higher than that of the RNA vaccine.

[0226] In some embodiments, the immune response in a subject is induced 2, 3, 1, 2, 3, 5, or 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactic effective dose of a conventional vaccine.

[0227] hCMV immunogenic compositions (e.g., mRNA vaccines) can be administered via any route that yields a therapeutically effective outcome. Such routes include, but are not limited to, intradermal, intramuscular, intranasal, and / or subcutaneous administration. This disclosure provides a method for administering an RNA vaccine to a subject in need. The exact amount required will vary from subject to subject depending on the subject's race, age, and general condition, the severity of the disease, the specific composition, its mode of administration, its mode of activity, etc. hCMV immunogenic compositions (e.g., mRNA vaccines) are typically formulated in unit dosage forms for ease of administration and uniformity of drug dose. However, it will be understood that the total daily dose of hCMV immunogenic compositions (e.g., mRNA vaccines) may be determined by the attending physician within reasonable medical judgment. The specific therapeutic effective dose level, prophylactic effective dose level, or appropriate imaging dose level for any particular patient depends on a variety of factors, including the disorder being treated and its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the timing, route of administration, and excretion rate of the specific compound used, the duration of treatment, any drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field.

[0228] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) contains approximately 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, and 24 μg. 25μg, 26μg, 27μg, 28μg, 29μg, 30μg, 31μg, 32μg, 33μg, 34μg, 35μg, 36μg g, 37μg, 38μg, 39μg, 40μg, 41μg, 42μg, 43μg, 44μg, 45μg, 46μg, 47μg, 4 8μg, 49μg, 50μg, 51μg, 52μg, 53μg, 54μg, 55μg, 56μg, 57μg, 58μg, 59μg , 60μg, 61μg, 62μg, 63μg, 64μg, 65μg, 66μg, 67μg, 68μg, 69μg, 70μg, 71 It is administered in doses of μg, 72μg, 73μg, 74μg, 75μg, 76μg, 77μg, 78μg, 79μg, 80μg, 81μg, 82μg, 83μg, 84μg, 85μg, 86μg, 87μg, 88μg, 89μg, 90μg, 95μg, 100μg, 110μg, 120μg, 130μg, 140μg, 150μg, 160μg, 170μg, 180μg, 190μg, 200μg, 250μg, 300μg, 350μg, 400μg, 450μg, or 500μg (including all values ​​in between).

[0229] As used herein, the dose of the hCMV immunogenic composition (e.g., mRNA vaccine) of this disclosure refers to the total μg mRNA in the lipid nanoparticle formulation. As used herein, "total μg mRNA" refers to the total or nominal dose for a single administration, provided that RNA impurities, mRNA degradation products, and other inactive mRNA are included in the total. The weight of lipid components is not included in the doses of this disclosure.

[0230] In some embodiments, the hCMV immunogenic composition (e.g., mRNA vaccine) is administered in a dose of about 50–150 μg. In some embodiments, only a single dose is administered, while in other embodiments, multiple doses (e.g., one, two, or three doses) are administered. In embodiments where multiple doses are administered, the doses may be the same or different between the first and subsequent doses. In some embodiments, the effective dose of the hCMV immunogenic composition provided herein (e.g., mRNA vaccine containing mRNA encoding gH / gL / UL128 / UL130 / UL131A / gB) may be as low as 150 μg and administered, for example, as a single dose.

[0231] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 50 to 150 μg. For example, the effective doses of the hCMV immunogenic composition (e.g., mRNA vaccine) are 50 μg, 51 μg, 52 μg, 53 μg, 54 μg, 55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 7 The single dose can be 4 μg, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg (including all values ​​in between). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 50 μg. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 100 μg. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 150 μg.

[0232] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is one of 50 μg, 100 μg, or 150 μg. In some embodiments, the effective dose is administered as a primary immunization, followed by a single booster of the same effective dose. In some embodiments, the effective dose is administered as a primary immunization, followed by two consecutive booster immunizations of the same effective dose. In some embodiments, the effective dose is 50 μg of the hCMV immunogenic composition (e.g., mRNA vaccine), administered as a primary immunization of 50 μg, followed by two consecutive booster immunizations of 50 μg each. In some embodiments, the effective dose is 100 μg of the hCMV immunogenic composition (e.g., mRNA vaccine), administered as a primary immunization of 100 μg, followed by two consecutive booster immunizations of 100 μg each. In some embodiments, the effective dose is 150 μg of an hCMV immunogenic composition (e.g., mRNA vaccine), administered as a 150 μg primary immunization, followed by two consecutive booster immunizations of 150 μg each. In some embodiments, the booster immunizations are spaced at least two weeks apart.

[0233] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 50-150 μg. For example, the effective doses of the hCMV immunogenic composition (e.g., mRNA vaccine) are 50 μg, 51 μg, 52 μg, 53 μg, 54 μg, 55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 7 The dose can be two doses of 4 μg, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg (including all values ​​in between). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 50 μg. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 100 μg. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 150 μg.

[0234] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is three doses of 50-150 μg. For example, the effective doses of the hCMV immunogenic composition (e.g., mRNA vaccine) are 50 μg, 51 μg, 52 μg, 53 μg, 54 μg, 55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 7 The dose can be 4 μg, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg (including all values ​​in between). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is 50 μg in 3 doses. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is 100 μg in 3 doses. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is 150 μg in 3 doses.

[0235] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a dose of 50 μg to 150 μg in more than three doses (e.g., four, five, or more doses). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a dose of 50 μg in more than three doses (e.g., four, five, or more doses). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a dose of 100 μg in more than three doses (e.g., four, five, or more doses). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a dose of 150 μg in more than three doses (e.g., four, five, or more doses).

[0236] In some embodiments, the effective dose of an hCMV immunogenic composition (e.g., mRNA vaccine) is related to the amount of integral mRNA in the composition. As used herein, “integral mRNA” refers to an intact mRNA transcript that can produce the hCMV antigen and / or induce an immune response to the antigen within a subject. The amount of integral mRNA in an hCMV immunogenic composition (e.g., mRNA vaccine) is related to its length, degradation rate, and the length of time since the immunogenic composition was produced. When determining an effective dose from clinical results, the dose may be referred to in terms of the total mRNA present in the hCMV immunogenic composition (e.g., mRNA vaccine) (i.e., total dose) or in terms of the integral mRNA present.

[0237] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) of pentameric components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) of gB mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) of pentameric components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) of gB mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is 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 pmol (including all values ​​in between). This is a single dose of the pentameric components and gB mRNA in amounts of 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, and 50 pmol (including all values ​​in between).

[0238] In certain embodiments, the effective amount or dose of an hCMV immunogenic composition (e.g., mRNA vaccine) does not require equal picomoles of each component. For example, mRNA encoding an endogenous transmembrane domain containing components such as gH and gB may require a larger picomolecal dose to ensure that these components are not limited. In addition, in some embodiments, the picomolecal dose of each of the six mRNAs may be determined individually for stability or other biochemical or biophysical requirements.

[0239] In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) of pentameric components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) of gB mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) of pentameric components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) of integral gB mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is 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 pmol (including all values ​​in between). This is a two-dose formulation of the pentameric components and gB mRNA in amounts of 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, and 50 pmol (including all values ​​in between).

[0240] In some embodiments, the effective amount of the hCMV immunogenic composition (e.g., mRNA vaccine) is 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) of pentameric components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) of integral GB This is a three-dose of mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a three-dose of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) of pentameric components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) of integral gB mRNA. In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is a three-dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 pmol (including all values ​​in between) of pentameric components. The components and three doses of integral gB mRNA in amounts of 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, and 50 pmol (including all values ​​in between).

[0241] In some embodiments, the effective amount of the hCMV immunogenic composition (e.g., mRNA vaccine) is 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) of pentameric components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) of integral GB The effective dose of mRNA is more than three doses (e.g., four, five, or more doses). In some embodiments, the effective dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is more than three doses (e.g., four, five, or more doses) of pentameric components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) of integral gB mRNA. In some embodiments, the effective amount of the hCMV immunogenic composition (e.g., mRNA vaccine) is a pentamer of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 pmol (including all values ​​in between). The constituents and doses of integral gB mRNA in quantities of 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, and 50 pmol (including all values ​​in between) that are greater than three doses (e.g., four, five, or more doses).

[0242] In some embodiments, at the specific integral mRNA doses described herein, the hCMV immunogenic composition (e.g., mRNA vaccine) contains mRNA in a molar ratio of 2:2:1:1:1:1 gB:gH:gL:UL128:UL130:UL131A.

[0243] In some embodiments, one, two, three, or more than three doses of an hCMV immunogenic composition (e.g., mRNA vaccine) (in any dose described herein) are administered to the subject. In some embodiments, the doses are administered on day 1, around the beginning of the second month (e.g., day 29), and around the beginning of the sixth month (e.g., day 169).

[0244] In some embodiments, the dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is as follows: Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, Day 15, Day 16, Day 17, Day 18, Day 19, Day 20, Day 21, Day 22, Day 23, Day 24, Day 25, Day 26, Day 27, Day 28, Day 29, Day 30, Day 31, Day 32, Day 33, Day 34, Day 35, Day 36, Day 37, Day 38, Day 39, Day 40, Day 41, Day 42, Day 43 Day 44, Day 45, Day 46, Day 47, Day 48, Day 49, Day 50, Day 51, Day 52, Day 53, Day 54, Day 55, Day 56, Day 57, Day 58, Day 59, Day 60, Day 61, Day 62, Day 63, Day 64, Day 65, Day 66, Day 67, Day 68, Day 69, Day 70, Day 71, Day 72, Day 73, Day 74, Day 75, Day 76, Day 77, Day 78, Day 79, Day 80, Day 81, Day 82, Day 83, Day 84, Day 85, Day 86, Day 87, Day 88, Day 89, Day 90, Day 91, Day 92, Day 93 Day 94, Day 95, Day 96, Day 97, Day 98, Day 99, Day 100, Day 101, Day 102, Day 103, Day 104, Day 105, Day 106, Day 107, Day 108, Day 109, Day 110, Day 111, Day 112, Day 113, Day 114, Day 115, Day 116, Day 117, Day 118, Day 119, Day 120, Day 121, Day 122, Day 123, Day 124, Day 125, Day 126, Day 127, Day 128, Day 129, Day 130, Day 131, Day 132, Day 133, Day 134, Day 135, 13 Day 6, Day 137, Day 138, Day 139, Day 140, Day 141, Day 142, Day 143, Day 144, Day 145, Day 146, Day 147, Day 148, Day 149, Day 150, Day 151, Day 152, Day 153, Day 154, Day 155, Day 156, Day 157, Day 158, Day 159, Day 160, Day 161, Day 162, Day 163, Day 164, Day 165, Day 166, Day 167, Day 168, Day 169, Day 170, Day 171, Day 172, Day 173, Day 174, Day 175, Day 176, Day 177,The drug is administered to the subjects on days 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, and 199. In some embodiments, the dose of the hCMV immunogenic composition (e.g., mRNA vaccine) is administered to the subjects after day 199.

[0245] The hCMV mRNA vaccines described herein can be formulated in dosage forms described herein or known in the art, such as intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreous, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0246] Vaccine effectiveness Some aspects of this disclosure provide formulations of hCMV mRNA vaccines in which the hCMV mRNA vaccine is formulated in an amount effective to induce an antigen-specific immune response (e.g., production of antibodies specific to the anti-hCMV antigen) within a subject. “Effective amount” means the dose of hCMV mRNA vaccine effective to induce an antigen-specific immune response. The Specified also provides methods for inducing an antigen-specific immune response within a subject.

[0247] As used herein, an immune response to a vaccine or LNP of this disclosure means the occurrence of a humoral and / or cellular immune response within a subject to one or more hCMV proteins present in the vaccine. For the purposes of this disclosure, “humoral” immune response means an immune response mediated by antibody molecules (e.g., including secretory (IgA) or IgG molecules), while “cellular” immune response means an immune response mediated by T lymphocytes (e.g., CD4+ helper and / or CD8+ T cells (e.g., CTLs) and / or other leukocytes). One important aspect of cellular immunity involves antigen-specific responses by cytolytic T cells (CTLs). CTLs are specific to peptide antigens expressed on the cell surface, presented with proteins encoded by major histocompatibility complexes (MHC). CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T cells. Helper T cells stimulate the function of nonspecific effector cells against cells that present peptide antigens on their surface along with MHC molecules, helping to focus their activity. The cellular immune response also triggers the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes (including those derived from CD4+ and CD8+ T cells).

[0248] In some embodiments, the antigen-specific immune response is characterized by measuring the anti-hCMV antigen antibody titer produced in a subject administered with the hCMV mRNA vaccine provided herein. Antibody titer is a measure of the amount of antibody (e.g., antibody specific to a particular antigen (e.g., anti-hCMV antigen) or an epitope of the antigen) present in the subject. Antibody titer is typically expressed as the reciprocal of the maximum dilution that yields a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay for quantifying antibody titer.

[0249] In some embodiments, antibody titers are used to assess whether a subject has contracted an infection or whether immunization is necessary. In some embodiments, antibody titers are used to quantify the strength of an autoimmune response, to determine whether booster immunization is necessary, to determine whether a previous vaccine was effective, and to confirm a recent or past infection. According to this disclosure, antibody titers can be used to quantify the strength of the immune response induced in a subject by an hCMV mRNA vaccine.

[0250] In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is at least 1 log higher than that of the control. For example, the anti-hCMV antigen antibody titer produced in the subject may be at least 1.5, at least 2, at least 2.5, at least 3 log, at least 4 log, or at least 5 log or more higher than that of the control. In some embodiments, the anti-hCMV antigen antibody produced in the subject is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 log higher than that of the control. In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is 1 to 5 log higher than that of the control. For example, the anti-hCMV antigen antibody titer produced within a subject may increase by 1-1.5, 1-2, 1-2.5, 1-3, 1-4, 1-5, 1.5-2, 1.5-2.5, 1.5-3, 1.5-4, 1.5-5, 2-2.5, 2-3, 2-4, 2-5, 2.5-3, 2.5-4, 2.5-5, 3-4.3-5, or 4-5 log compared to the control.

[0251] In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is at least twofold higher than that of the control. For example, the anti-hCMV antigen antibody titer produced in the subject may be at least threefold, at least fourfold, at least fivefold, at least sixfold, at least sevenfold, at least eightfold, at least ninefold, or at least tenfold higher than that of the control. In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is 2, 3, 4, 5, 6, 7, 8, 9, or tenfold higher than that of the control. In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is 2 to 10fold higher than that of the control. For example, the anti-hCMV antigen antibody titer produced within a subject may increase by 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times compared to the control.

[0252] In some embodiments, the antigen-specific immune response is measured as the ratio of geometric mean titers (GMTs) of serum neutralizing antibody titers against hCMV (referred to as the geometric mean ratio (GMR)). The geometric mean titer (GMT) is the average antibody titer in a group of subjects, calculated by multiplying all values ​​and taking the nth root of that number (where n is the number of subjects for which data is available).

[0253] In some embodiments, administration of an effective dose of an hCMV immunogenic composition (e.g., mRNA vaccine) elicits a serum neutralizing antibody titer against hCMV. In some embodiments, administration of a single dose (e.g., any dose described herein) or multiple doses of an hCMV immunogenic composition (e.g., mRNA vaccine) elicits a serum neutralizing antibody titer against hCMV.

[0254] In some embodiments, an effective amount of the hCMV immunogenic composition described herein (e.g., mRNA vaccine) is sufficient to produce the geometric mean titer (GMT) and associated GMR of serum neutralizing anti-CMV antibody against epithelial cell hCMV infection at 1, 29, 56, 84, 168, or 196 postimmunization days. In some embodiments, an effective amount of the hCMV immunogenic composition described herein (e.g., mRNA vaccine) is sufficient to produce the GMR of serum neutralizing antibody titer and associated GMR of serum neutralizing antibody against fibroblast hCMV infection at 1, 29, 56, 84, 168, or 196 postimmunization days.

[0255] In some embodiments, the GMT of serum neutralizing antibodies against hCMV increases by at least twofold (e.g., at least twofold, at least threefold, at least fourfold) from baseline in subjects administered with an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against hCMV increases by two to tenfold from baseline in subjects after administration of a single dose (e.g., ≥50 μg, e.g., a single dose of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against hCMV increases by two to tenfold from baseline in subjects after administration of two doses (e.g., ≥50 μg, e.g., two doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against hCMV increases 2- to 10-fold from baseline in subjects after administration of three doses (e.g., ≥50 μg, e.g., three doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine).

[0256] In some embodiments, the neutralizing antibody (nAb) GMT against epithelial cell infection increases by at least 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, 50, or 51 times compared to the baseline GMT at some point after administration of the hCMV immunogenic composition. In some embodiments, this point in time is after the administration of two doses of the immunogenic composition. In some embodiments, this point in time is after the administration of three doses of the immunogenic composition.

[0257] In some embodiments, the proportion of human subjects in which the nAb for epithelial cell infection increases by more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, or 13-fold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at some point after administration of the hCMV immunogenic composition.

[0258] In some embodiments, administration of an effective dose of an hCMV immunogenic composition (e.g., mRNA vaccine) elicits a serum neutralizing antibody titer against the hCMV gB protein. In some embodiments, administration of a single dose (e.g., any dose described herein) or multiple doses of an hCMV immunogenic composition (e.g., mRNA vaccine) elicits a serum neutralizing antibody titer against the hCMV gB protein.

[0259] In some embodiments, the GMT of serum neutralizing antibodies against the hCMV gB protein increases by at least twofold (e.g., at least twofold, at least threefold, at least fourfold) from baseline in subjects administered with an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against the hCMV gB protein increases by two to tenfold from baseline in subjects after administration of a single dose (e.g., ≥50 μg, e.g., a single dose of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against the hCMV gB protein increases by two to tenfold from baseline in subjects after administration of two doses (e.g., ≥50 μg, e.g., two doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of serum neutralizing antibodies against the hCMV gB protein increases 2- to 10-fold from baseline in subjects after administration of three doses (e.g., ≥50 μg, e.g., three doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine).

[0260] In some embodiments, the proportion of human subjects in which nAbs for fibroblast infection increase more than twofold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at some point after administration of the hCMV immunogenic composition.

[0261] In some embodiments, administration of an effective amount of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against hCMV. In some embodiments, administration of a single dose (e.g., any dose described herein) or multiple doses of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against hCMV. In some embodiments, administration of an effective amount of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against the hCMV gB protein. In some embodiments, administration of a single dose (e.g., any dose described herein) or multiple doses of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against the hCMV gB protein. In some embodiments, administration of an effective amount of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against the hCMV pentamer. In some embodiments, administration of a single dose (e.g., any dose described herein) or multiple doses of a hCMV immunogenic composition (e.g., an mRNA vaccine) elicits an antigen-specific T cell response against the hCMV pentamer. In some embodiments, the T cell response (e.g., against hCMV, the hCMV gB protein, or the hCMV pentamer) includes secretion of interferon-γ (IFN-γ).

[0262] The control / baseline is, in some embodiments, the anti-hCMV antigen antibody titer produced in a subject not administered the hCMV mRNA vaccine. In some embodiments, the control / baseline is the anti-hCMV antigen antibody titer produced in a subject with natural hCMV infection (i.e., a subject seropositive for hCMV prior to administration of the hCMV mRNA vaccine). In some embodiments, the control / baseline is the anti-hCMV antigen antibody titer produced in a subject that was seronegative for hCMV prior to administration of the hCMV mRNA vaccine. In some embodiments, the geometric mean titer (GMT) of serum neutralizing antibodies against hCMV increases in a dose-dependent manner.

[0263] In some embodiments, the geometric mean titer (GMT) of the binding antibody response against the hCMV pentamer (anti-pentamer antibody titer) increases by at least 2-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold) over baseline in a subject administered an hCMV immunogenic composition (e.g., an mRNA vaccine). In some embodiments, the GMT of the binding antibody response against the hCMV pentamer increases 2- to 10-fold over baseline in a subject after administration of a single dose (e.g., a single dose of ≧50 μg, e.g., a single dose of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., an mRNA vaccine). In some embodiments, the GMT of the binding antibody response against the hCMV pentamer increases 2- to 10-fold over baseline in a subject after administration of two doses (e.g., two doses of ≧50 μg, e.g., two doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., an mRNA vaccine). In some embodiments, the GMT of the binding antibody response against hCMV increases 2- to 10-fold over baseline in a subject after administration of three doses (e.g., three doses of ≧50 μg, e.g., three doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., an mRNA vaccine). In some embodiments, the GMT of the anti-pentamer binding antibody (bAb) increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold over the baseline GMT at a certain time point after administration of the hCMV immunogenic composition. In some embodiments, that time point is after administration of two doses of the immunogenic composition. In some embodiments, that time point is after administration of three doses of the immunogenic composition.

[0264] In some embodiments, the percentage of human subjects in which the anti-pentamer binding antibody (bAb) increases by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more over baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0265] In some embodiments, the GMT of the binding antibody response to gB increases by at least twofold (e.g., at least twofold, at least threefold, at least fourfold) from baseline in subjects administered with an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of the binding antibody response to anti-gB increases by two to tenfold from baseline in subjects after administration of a single dose (e.g., ≥50 μg, e.g., a single dose of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of the binding antibody response to anti-gB increases by two to tenfold from baseline in subjects after administration of two doses (e.g., ≥50 μg, e.g., two doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of the anti-gB binding antibody response increases 2 to 10 times from baseline within a subject after administration of three doses (e.g., ≥50 μg, e.g., three doses of 50 μg, 100 μg, or 150 μg) of an hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the GMT of the anti-gB binding antibody (Ab) increases by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times from baseline GMT at some point after administration of the hCMV immunogenic composition. In some embodiments, this point in time is after administration of a single dose of the immunogenic composition. In some embodiments, this point in time is after administration of two doses of the immunogenic composition. In some embodiments, this point in time is after administration of three doses of the immunogenic composition. In some embodiments, the GMT response reaches its peak within about 10 days to 2 weeks after dose administration.

[0266] In some embodiments, the proportion of human subjects in which anti-gB-binding antibody (Ab) increases more than twofold from baseline is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% at one time point after administration of the hCMV immunogenic composition.

[0267] In some embodiments, the efficacy of the hCMV mRNA vaccine is measured in a mouse model. For example, the hCMV mRNA vaccine can be administered to a mouse model, and the mouse model can be evaluated for induction of neutralizing antibody titers. A viral challenge test can also be used to evaluate the efficacy of the vaccines of this disclosure. For example, the hCMV mRNA vaccine can be administered to a mouse model, the mouse model can be attacked with hCMV, and the mouse model can be evaluated for survival and / or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).

[0268] In some embodiments, the effective dose of the hCMV mRNA vaccine is a reduced dose compared to the standard therapeutic dose of the recombinant hCMV protein vaccine. As used herein, “standard treatment” refers to medical or psychological treatment guidelines, which may be general or specific. “Standard treatment” refers to appropriate treatment based on scientific evidence and collaboration among healthcare professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician / clinician should follow for a particular type of patient, disease, or clinical situation. As used herein, “standard therapeutic dose” refers to the dose of recombinant or purified hCMV protein vaccine, attenuated live or inactivated hCMV mRNA vaccine, or hCMV VLP vaccine that a physician / clinician or other healthcare professional would consider administering to a subject to treat or prevent hCMV or an hCMV-related condition, in accordance with standard treatment guidelines for treating or preventing hCMV or an hCMV-related condition.

[0269] In some embodiments, the anti-hCMV antigen antibody titer produced in subjects administered an effective dose of hCMV mRNA vaccine is equivalent to the anti-hCMV antigen antibody titer produced in control subjects administered a standard therapeutic dose of recombinant or purified hCMV protein vaccine, or attenuated live or inactivated hCMV mRNA vaccine, or hCMV VLP vaccine.

[0270] Vaccine efficacy can be evaluated using standard analyses (see, for example, Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). For example, vaccine efficacy can be measured in a double-blind, randomized, controlled clinical trial. Vaccine efficacy can be expressed as a proportional reduction in disease incidence (AR) between the incidence rate (ARU) of the unvaccinated trial cohort and the incidence rate (ARV) of the vaccinated trial cohort, and can be calculated from the relative risk (RR) of the disease in the vaccinated group using the following formula. Effect = (ARU - ARV) / ARU × 100; and Effect = (1 - RR) × 100

[0271] Similarly, vaccine efficacy can be evaluated using standard analyses (see, for example, Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). Vaccine efficacy assesses how a vaccine (which may already be known to have high efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than controlled clinical trials. Vaccine efficacy is proportional to the vaccine's effect (potency), but is also influenced by the degree of immunization of the target group within the population and by vaccine-independent factors that affect "real-world" outcomes (e.g., hospitalization, outpatient visits, or costs). For example, retrospective case-control analyses can be used to compare vaccination rates in a set of infection cases and appropriate controls. Vaccine efficacy can be expressed as a difference in proportions by using the odds ratio (OR) for developing an infection despite vaccination. Effectiveness = (1 - OR) × 100

[0272] In some embodiments, the efficacy of the hCMV mRNA vaccine is at least 60% compared to an unvaccinated control. For example, the efficacy of the hCMV mRNA vaccine may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% compared to an unvaccinated control.

[0273] Bactericidal immunity. Bactericidal immunity refers to an intrinsic immune state that prevents effective pathogen infection of a host. In some embodiments, the effective dose of the hCMV mRNA vaccine of this disclosure is sufficient to induce bactericidal immunity in a subject for at least one year. For example, the effective dose of the hCMV mRNA vaccine of this disclosure may be sufficient to induce bactericidal immunity in a subject for at least two years, at least three years, at least four years, or at least five years. In some embodiments, the effective dose of the hCMV mRNA vaccine of this disclosure is sufficient to induce bactericidal immunity in a subject at a dose at least five times lower than that of a control. For example, the effective dose may be sufficient to induce bactericidal immunity in a subject at a dose at least 10 times, 15 times, or 20 times lower than that of a control.

[0274] Detectable antigen. In some embodiments, the effective dose of the hCMV mRNA vaccine of this disclosure is sufficient to produce detectable levels of hCMV antigen in measurements in the serum of a subject 1 to 72 hours after administration.

[0275] Antibody titer. Antibody titer is a measure of the amount of antibody (for example, an antibody specific to a particular antigen, e.g., anti-hCMV antigen) in a sample. Antibody titer is typically expressed as the reciprocal of the maximum dilution that yields a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay for quantifying antibody titer.

[0276] In some embodiments, an effective dose of the hCMV mRNA vaccine of this disclosure is sufficient to produce a neutralizing antibody titer of 1,000 to 10,000 produced by neutralizing antibodies against the hCMV antigen, as measured in the serum of the subject 1 to 72 hours after administration. In some embodiments, an effective dose is sufficient to produce a neutralizing antibody titer of 1,000 to 5,000 produced by neutralizing antibodies against the hCMV antigen, as measured in the serum of the subject 1 to 72 hours after administration. In some embodiments, an effective dose is sufficient to produce a neutralizing antibody titer of 5,000 to 10,000 produced by neutralizing antibodies against the hCMV antigen, as measured in the serum of the subject 1 to 72 hours after administration.

[0277] In some embodiments, the neutralizing antibody titer is at least 100 NT. 50 For example, the neutralizing antibody titer should be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 NT. 50 This may be the case. In some embodiments, the neutralizing antibody titer is at least 10,000 NT. 50 That is the case.

[0278] In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units / milliliter (NU / mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 NU / mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU / mL.

[0279] In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is at least 1 log higher than that of the control. For example, the anti-hCMV antigen antibody titer produced in the subject may be at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 log higher than that of the control.

[0280] In some embodiments, the anti-hCMV antigen antibody titer produced in the subject increases by at least 2-fold compared to the control. For example, the anti-hCMV antigen antibody titer produced in the subject increases by at least 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to the control.

[0281] In some embodiments, the geometric mean, which is the nth root of the product of n numbers, is commonly used to explain exponential growth. The geometric mean is used in some embodiments to characterize the antibody titers produced in a subject.

Example

[0282] To better understand the invention described herein, the following examples are provided. The examples described in this application are provided to illustrate the systems and methods provided in this application and should not be construed as limiting their scope in any way.

[0283] Example 1: Selection of the optimal ratio of mRNA constructs in an hCMV mRNA immunogenic composition that can maximize efficacy throughout the duration of effectiveness An hCMV mRNA vaccine (Figures 1A and 1B) containing mRNA encoding all components of the hCMV pentamer (gH, gL, UL128, UL130, and UL131A) and gB in equal mass ratios has been shown to be effective in inducing neutralizing antibodies against hCMV in a Phase I clinical trial. However, since the molecular weights of the individual mRNA constructs are different, using equal mass ratios results in very different molar dosages of the individual mRNA components, with some being present in excess and others being under-expressed. Specifically, in formulations with equal mass ratios, the largest mRNA constructs corresponding to gB and gH are under-expressed on a molar basis.

[0284] It was hypothesized that providing individual mRNA constructs in molar stoichiometric ratios might maximize protein expression per unit mass of mRNA administered to patients. Furthermore, it was hypothesized that larger mRNA constructs corresponding to gB and gH would degrade faster than smaller mRNA constructs, and that this degradation could limit the overall stability of the mRNA-based vaccine. Therefore, we investigated whether further adjustment of the molar ratio of mRNA components, based on predictions of relative mRNA degradation rates during storage, could achieve optimal functional performance throughout the entire shelf life of the drug.

[0285] Formulations containing equimolar amounts of UL128, gL, UL130, and UL131A, along with 2X molar amounts of gB and gH, were investigated and compared in vitro and in vivo studies with conventionally used equal mass ratios. Surprisingly, formulations of mRNA components based on the proposed molar ratios of hCMV mRNA vaccine components were found to increase the protein expression of both large glycoprotein antigens (gB and pentamer) with respect to a given total dose. The increased expression of gB and pentamer was measured in in vitro cell culture experiments, and when administered to mice, improved antibody responses to both anti-gB and anti-pentamer IgG were observed compared to the use of equal mass ratios.

[0286] While we do not wish to be bound by any theory, formulations containing equimolar amounts of gL, UL128, UL130, and UL131A, along with 2X molar amounts of gB and gH, compensate for differences in the stoichiometry and mRNA degradation rates of the pentameric proteins. See Figure 2A. In addition to calculating the doses of individual components on a molar basis, further improvements were obtained by simultaneously adding 2X molar amounts of gH and gB to 1X equimolar amounts of mRNA encoding proteins such as gL, UL128, UL130, and UL131A that complex with gH. In vaccines formulated in this way, the molar doses of these high molecular weight mRNA components are higher than those of other low molecular weight mRNA components. This design was in contrast to formulations based on equal mass ratios of mRNA components (Figure 2B). In Figure 3, in vitro expression data for two separate examples show increased pentameric expression with formulations based on the molar stoichiometry of 2X gH compared to equimolar formulations. Increased expression was observed by an increase in Emax. Surprisingly, the hCMV mRNA immunogenic composition based on the above molar ratio achieved a comparable or slightly higher antibody response in mice using a lower total dose than the hCMV mRNA immunogenic composition based on equal mass (Figure 4).

[0287] When tested by increasing the molar ratio of each mRNA construct to 2X and generating an excess of additional constructs so that other mRNA components are present in equimolar ratios, it was found that excess gH had the greatest effect on increasing the anti-pentamer IgG response in mice (Figure 5). Importantly, it was found that the total dose of the hCMV mRNA immunogenic composition required to show an increase in anti-gB and anti-pentamer antibody responses in mice was lower when the mRNA components were formulated based on the molar ratios described herein than when they were formulated based on equal mass ratios (Figures 6A-6D).

[0288] Based on the calculated theoretical inactivation rates, the proposed molar ratios for mRNA components in the hCMV mRNA immunogenic compositions described herein were found to adequately compensate for the degradation of longer mRNA constructs and maintain excess levels of gH and gB for at least 36 months, corresponding to the expected shelf life of pharmaceuticals containing these components (Figure 7). The excess of longer gH and gB constructs compensated for inactivation over time while maintaining the maximum potency of the product (Figure 7).

[0289] The proposed molar ratios for mRNA in the hCMV mRNA vaccine described herein maximize efficacy throughout the entire duration of the vaccine's effectiveness. Specifically, compositions based on equimolar concentrations of small mRNAs (gL, UL130, UL128, and UL131) and excess long mRNA constructs (gH and gB) increase the gB content compared to equimolar formulations by fully utilizing the structural stoichiometry of the target protein complex, thereby compensating for the known mechanism of mRNA inactivation. This approach also allows for robust dose setting with less mRNA, which may lead to lower costs and improved tolerability. Furthermore, since gL, UL128, UL130, and UL131 all bind to membrane-immobilized gH and assemble into a large glycoprotein complex known as a pentamer, the supply of gH is not limited compared to the other smaller members of the pentamer, which is advantageous. gH forms the base structure of the pentamer, and other smaller proteins need to be assembled on top of it to form the mature pentamer complex. Therefore, a shortage of the base components will result in a shortage of the mature pentamer as well.

[0290] Example 2: Phase II dose-selection clinical trial A Phase I clinical trial was conducted using an hCMV mRNA vaccine containing pentameric components and gB in equal mass ratios. Phase I clinical trial data showed that neutralizing antibody titers against epithelial cell infection (derived from successful pentameric expression and immune response) generally exceeded those against fibroblast infection (derived from successful gB expression and immune response). Therefore, maximizing the integral gB neutralizing immune response was a key factor in evaluating Phase I data and data from naturally infected serologically positive patients to design doses for subsequent clinical trials. Although there were differences in the concentrations of mRNA components used in the Phase I and Phase II vaccine formulations, the hCMV Phase I clinical trial data was graphed on a molar-based x-axis to allow comparison between Phase I (equal mass) data and the proposed Phase II (molar-based) data on a single continuous x-axis. Graphing Phase I and Phase II data on the same continuous x-axis provides a comprehensive dose-response curve that can be used for dose selection in subsequent clinical trials. The x-axis in Figures 8A and 8B shows the picomoles of integral pentamer mRNA and integral gB mRNA, respectively. These values ​​were calculated for each Phase I dose based on the total dose (μg), the ratio of mRNA in the drug, the actual purity of the drug, and the molecular weight of each mRNA (Figures 8A-8B).

[0291] The graphs shown in Figures 8A and 8B show neutralizing antibody titers (nAb) against fibroblast (gB) and epithelial cell (pentamer) infections. In both graphs, the serologically positive benchmark level is indicated by a red horizontal line. The serologically positive benchmark levels for gB and pentamer are antibody titers specific to these antigens and are found in unvaccinated patients who have previously been infected with and recovered from hCMV. Note that the benchmark serologically positive pentamer titer is significantly higher than the benchmark gB titer. Data from Phase I dose escalation phases A and B and Phase I dose selection phase B (30, 90, 180 μg) are graphed for each individual subject. Data from post-administration data point 1 (after 1 dose) and post-administration data point 2 (after 2 doses) for Phase I 300 μg dose cohort C are graphed using the GMT ± 95% confidence interval. In the text box below the X-axis, the arrows indicate the proposed Phase II (P202) doses calculated using moles, but are represented by the total mRNA weight in the LNP (50, 100, 150 μg).

[0292] In Figure 8A, Phase II (P202) data based on the specified proposed dose predict 9–29 picomoles of integral pentamer mRNA, which corresponds to the portion of the dose-response curve previously shown in Phase I trials to elicit neutralizing antibodies generally above the serologically positive benchmark after two or three doses. At the Phase I ratio, the serologically positive benchmark was easily reached after three doses at 5 picomoles (see Figure 8A after 3 doses), and many subjects further reached the benchmark serologically positive level after two doses at 12 picomoles (see Figures 8A and B after 2 doses). In the case of epithelial cell infection, a strong neutralizing antibody response to the pentamer is induced, so it was not necessary to design a Phase II formulation with an expanded dose level to a higher picomolecular dose of pentamer mRNA components. In fact, the Phase II formulation will achieve 9–29 picomoles of integral pentamer mRNA components even with a slightly lower total mass per dose. On the other hand, molar-based dose design enabled dose selection that met these benchmark levels and provided product stability for at least 26 months. For example, the 300 μg dose in Phase I and Phase C delivered significantly more integral pentamers than the 150 μg dose in Phase II. See Figure 8A.

[0293] In contrast, Figure 8B shows that fibroblast nAb data for gB indicate that achieving serologically positive benchmarks is more difficult with gB antigen than with the pentamer due to its lower immunogenicity. Not all patients reached serologically positive benchmark nAb levels after 2 or 3 doses with 20–25 picomoles of integral gB. Therefore, the dose curve was extended to higher picomolecal doses of gB mRNA by designing the Phase II mRNA molar ratio described in Example 1 to deliver more gB mRNA per total mass dose. Indeed, formulations based on the Phase II molar ratio would deliver 16–47 picomoles of integral gB mRNA at very low total mass doses. In this case, a 300 μg dose of P101C delivers less integral gB than a 150 μg dose of P202.

[0294] Next, as shown in Figures 9A and 9B, these dose-response curves are used to visualize the planned dose for the Phase II clinical trial described herein, and together with the obtained data, the visualization techniques shown in Figures 9A and 9B are used to select the optimal dose for the Phase III clinical trial. Since the immunogenicity of the pentamer is significantly greater than that of gB, the minimum required dose is determined by maximizing the fibroblast nAb titer against gB, and according to the proposed molar ratio design, the epithelial nAb titer against the correlated pentamer is expected to be more than sufficient.

[0295] The dose-setting strategy provided herein correlates the purity of endogenous transmembrane protein-coding mRNA content with the neutralizing antibody response, without considering LNP function, other quality attributes, cellular function, immune response, or sequence-specific aspects of mRNA and protein. Based on this study, these dose-response curves can be used to define doses in future clinical trials.

[0296] Predicting the minimum required dose in each vial during its expiration date involves modeling all factors expected to affect the active ingredient content, such as concentration (e.g., in-process control (IPC) assay and dilution) and purity (e.g., degradation). The IPC assay measures total mRNA content. Subjects in Phase III clinical trials and patients receiving commercial vaccines are administered various active ingredient concentrations. The total dose of mRNA (μg) encoding endogenous transmembrane proteins was calculated as 500 μL mRNA concentration (g / L) × purity (%).

[0297] To ensure the effectiveness of vials nearing their expiration date, a nominal dose was selected (Figures 9A-9B). For example, a nominal dose of 150 μg (a distributed target dose) allows for a 2-year shelf life at 5°C in Phase III and commercial supply. At the expiration date, the vial delivers an active ingredient content within a nominal dose range of 100 μg. Figure 10 shows the degradation model at 5°C. In mouse studies, the antibody response to the equal-mass ratio hCMV mRNA vaccine decreased slightly when the gB purity dropped to 50%, and since 50% gB is predicted to last approximately 2 years at 5°C based on the degradation rate model, it was found that 50% gB purity is necessary for efficacy at the expiration date (Figure 11). Note that the immunogenicity target is maintained in mouse assays until the gB purity drops to less than 49% after 26 months. Predicting changes in immunogenicity in mice in relation to changes in human responses depends on the sensitivity of the dose-response curve. The doses tested in mice (0.5 μg and 2 μg) represent the steepest portion of the dose-response curve. Human dose selection aims for saturation of nAb levels (low sensitivity to purity is expected). Based on the current model, a dose of 150 μg is expected to be immunogenically potent and well-tolerated.

[0298] Example 3. Phase II randomized, observer-blinded, placebo-controlled dose-finding study to evaluate the safety and immunogenicity of hCMV mRNA vaccine in healthy adults. The objective of this Phase II trial was to evaluate the safety and immunogenicity of the hCMV mRNA immunogenic composition in healthy adults (18-40 years old) who were either CMV serologically negative or CMV serologically positive at enrollment. The hCMV mRNA vaccine has demonstrated preclinical safety and immunogenicity and has the potential to prevent primary human CMV infection and CMV reinfection / reactivation in CMV-positive individuals.

[0299] Explanation of the test vaccine The hCMV mRNA vaccine for CMV infection evaluated in this study consists of six distinct mRNA sequences in a lipid nanoparticle (LNP) formulation that encode six viral proteins (full-length CMV gB and the pentameric gH / gL / UL128 / UL130 / UL131A glycoprotein complex [pentamer]) that are targets of the nAb response to human CMV infection. The six mRNAs are present in the hCMV mRNA vaccine in a molar ratio of approximately 1:1:1:1:2:2 for gL:UL128:UL130:UL131A:gH:gB.

[0300] The LNP formulation contains four lipid excipients: heptadecan-9-yl=8-((2-hydroxyethyl)(6-oxo-6(undecyloxy)hexyl)amino)octanoate, an ionizable cationic aminolipid, and commercially available lipid cholesterols: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol (PEG2000 DMG) (Mui et al 2013).

[0301] The hCMV mRNA vaccine injection was supplied as a 520 μg lyophilized product in a single-use glass vial and stored at -25°C to -15°C (-13°F to 5°F) until use. After preparation to the appropriate dose, the hCMV mRNA vaccine injection was administered intramuscularly to the deltoid muscle in a volume of 0.5 mL.

[0302] The lyophilized vaccine was reconstituted with 0.6 mL of 0.9% sodium chloride injection (USP) and diluted with trisucrose diluent SD-0724 to a concentration that would deliver the specified dose level in a 0.5 mL volume. A placebo of 0.9% sodium chloride injection (USP) (normal saline) was administered in a 0.5 mL volume.

[0303] Test design The proposed design and dose levels for this Phase II trial were based on safety and immunogenicity data accumulated from the ongoing Phase I trial. Interim analyses of safety and immunogenicity data for the 30 μg, 90 μg, and 180 μg dose-level cohorts from the Phase I trial demonstrated that the hCMV mRNA vaccine was generally well-tolerated in both CMV seronegative and CMV seropositive adults. In CMV seronegative participants, neutralizing antibodies (nAbs) against both epithelial cell and fibroblast infections were observed at all dose levels after two doses of the hCMV mRNA vaccine (administered at 0 and 2 months), and the immune response was measured 1 month after the second dose. Furthermore, antibody levels against viral proteins necessary for entry into epithelial and fibroblast cells were boosted in CMV seropositive participants at all dose levels after the same vaccination schedule.

[0304] The Phase II study described herein evaluated the safety and immunogenicity of three dose levels of hCMV mRNA immunogenicity compositions in CMV seronegative and CMV seronegative adults aged 18–40 years using a dose-escalation, sequential registration design, with the aim of enabling single-dose selection for future development.

[0305] A schematic diagram of the study design is shown in Figure 12. This study included two treatment groups: a CMV seronegative group and a CMV seropositive group, and enrolled in parallel. Randomization ratios were sequentially stratified to three different dose levels based on CMV serostatism using two-way response technology (IRT). At each dose level, subjects were randomized in a 3:1 ratio to receive either hCMV mRNA vaccine or placebo, and administered in a three-dose schedule (0 months, 2 months, and 6 months).

[0306] treatment group CMV seronegative group There were 60 subjects in the CMV seronegative group at each dose level. The dose levels were 50 μg, 100 μg, or 150 μg. At each dose level, subjects were randomized in a 3:1 ratio to receive either hCMV mRNA vaccine or placebo, and administered in a three-dose schedule (0 months, 2 months, and 6 months).

[0307] CMV seropositive group The CMV serologically positive group consisted of 24 subjects at each dose level. The dose levels were 50 μg, 100 μg, or 150 μg. At each dose level, subjects were randomized in a 3:1 ratio to receive either hCMV mRNA vaccine or placebo, and administered in a three-dose schedule (0 months, 2 months, and 6 months).

[0308] Screening period Screening for each subject was performed during their first visit to the facility. Screening may be performed up to 28 days prior to the first day of the visit. Screening visits may be conducted over two separate visits.

[0309] Treatment period All subjects will receive three doses on day 1, day 56, and day 168 of their hospital visit.

[0310] Estimated examination period The trial period for each participant was approximately 18 months.

[0311] Sample size Approximately 252 subjects were enrolled, including approximately 72 CMV seropositive subjects and approximately 180 CMV seronegative subjects. The number of subjects enrolled was considered sufficient to provide a descriptive overview of the safety and immunogenicity of different dose levels of the hCMV mRNA vaccine. In total, 189 subjects received the hCMV mRNA vaccine.

[0312] Purpose and endpoint Main purpose The main objectives of this examination are as follows: 1. Evaluate the safety of different dose levels of hCMV mRNA vaccine administered in a 3-dose vaccination schedule (0 months, 2 months, 6 months). 2. Evaluate the neutralizing anti-CMV antibody response to infection of fibroblasts and epithelial cells after administration of hCMV mRNA vaccine at different dose levels in a three-dose vaccination schedule (0 months, 2 months, and 6 months).

[0313] Secondary purpose The secondary objectives of this examination are as follows: 1. Evaluate the antigen-specific antibody response after administering the hCMV mRNA vaccine at different dose levels in a three-dose vaccination schedule. 2. Evaluate the immunogenicity of the hCMV mRNA vaccine according to the CMV serostatus at the time of registration.

[0314] exploratory purpose The exploratory objectives of this study are as follows: 1. Evaluate the cell-mediated immune response after administration of hCMV mRNA vaccine at different dose levels. 2. In subjects who test positive for CMV serotype, evaluate the estimated immune response after hCMV mRNA vaccination compared to placebo.

[0315] Primary endpoints 1. Involuntary localized and systemic aortic regurgitation (AR) within 7 days following each vaccination. 2. Voluntary adverse events (AEs) within 28 days after each vaccination. 3. Adverse events requiring medical attention within 6 months following the last vaccination (MAAE) and serious adverse events (SAE) throughout the entire study period. 4. Geometric mean titers (GMT) of serum neutralizing anti-CMV antibodies against epithelial cell infection and fibroblast infection at each time point, and associated geometric mean ratios (GMR) of post-baseline / baseline titers. 5. Percentage of subjects at each time point in time when the nAb for epithelial cell infection and fibroblast infection increased by more than 2-fold, more than 3-fold, and more than 4-fold compared to baseline.

[0316] Secondary endpoints 1. GMT of anti-GBB-specific IgG and anti-pentadose-specific IgG measured by enzyme-linked immunosorbent assay (ELISA) at each time point, as well as the associated GMR of post-baseline / baseline titers. 2. GMT, GMR, and percentage of subjects in the CMV serologically positive and CMV serologically negative groups at each time point in which serum nAbs for epithelial cell infection and fibroblast infection increased by 2-fold, 3-fold, and 4-fold or more from baseline, as well as the GMT and GMR of antigen-specific IgG (ELISA) at each time point.

[0317] exploratory endpoints 1. gB and pentamer-specific interferon (IFN)γ-secreting T cells as measured by enzyme-linked immunosorbent spot (ELISpot) assay. 2. Exploratory assays to evaluate the anti-CMV immune response or primary CMV infection may be performed at the discretion of the sponsor.

[0318] analysis The following analysis will be performed. 1. A 3-month interim analysis of safety and immunogenicity data collected from day 1 to day 84 (3 months) may be performed for each dose level. Available safety or immunogenicity data up to day 196 (7 months) can also be combined as part of these interim analyses. This analysis will serve as the basis for selecting the hCMV mRNA vaccine dose level for subsequent trials. 2. A 7-month interim analysis of safety and immunogenicity data collected from day 1 to day 196 (7 months) may be performed for each dose level. Available safety or immunogenicity data up to day 336 (12 months) can also be included as part of these interim analyses. 3. Final open-label analysis of safety and immunogenicity data collected from the first day of hospital visit until the end of the trial.

[0319] Immunogenicity evaluation Serum neutralizing anti-CMV antibody titers for epithelial cell infection on days 1, 29, 56, 84, 168, 196, 336, and 504, as well as GMR after baseline / baseline titer. Serum neutralizing anti-CMV antibody titers for fibroblast infection on days 1, 29, 56, 84, 168, 196, 336, and 504, as well as GMR after baseline / baseline titer. The percentage of subjects whose nAb levels for epithelial cell infection increased by more than 2, 3, and 4 times compared to day 1 on days 29, 56, 84, 168, 196, 336, and 504. The percentage of subjects whose nAb levels for fibroblast infection increased by more than 2, 3, and 4 times compared to day 1 on days 29, 56, 84, 168, 196, 336, and 504. The percentage of subjects whose nAb titer for epithelial cell infection was higher than the nAb titer associated with spontaneous CMV infection on days 1, 29, 56, 84, 168, 196, 336, and 504. The percentage of subjects whose nAb titer for fibroblast infection was higher than the nAb titer associated with spontaneous CMV infection on days 1, 29, 56, 84, 168, 196, 336, and 504. GMT of anti-GB IgG measured by ELISA on days 1, 29, 56, 84, 168, 196, 336, and 504, as well as GMR of post-baseline / baseline titer. GMT of anti-pentamer IgG measured by ELISA on days 1, 29, 56, 84, 168, 196, 336, and 504, as well as GMR of post-baseline / baseline titer.

[0320] Example 4: Evaluation of immunogenicity, in vitro expression, and analytical characteristics of the Phase II process. Immunogenicity, in vitro expression of hCMV antigen, and analytical characteristics of the Phase II process were evaluated using lyophilized hCMV RNA vaccines at 0.4 g and 1 g scales based on molar ratios, and compared with a 0.03 g liquid formulation. Lyophilized formulations of different doses were prepared using physiological saline (pH measured at 6.3). The pH shift of the diluted formulations is shown in Figure 13.

[0321] In vitro expression levels of hCMV pentamer and hCMV gB were evaluated for two lyophilized formulations (0.4g and 1g scales) based on molar ratios of mRNA components, and a liquid formulation (0.03g scale) based on equal mass ratios. The 0.4g and 1g scale lyophilized formulations showed higher in vitro expression of the pentamer protein compared to the 0.03g scale liquid formulation (Figures 14A-14C).

[0322] Figure 15A shows the anti-pentamer IgG titers at 21 or 36 days post-immunization, and Figure 15B shows the anti-gB IgG titers at 21 or 36 days post-immunization. The lyophilized formulations at the 0.4g and 1g scales showed higher pentamer-specific and gB-specific IgG titers (15-20 times higher) at a 0.5μg dose compared to the 0.03g scale equal-mass liquid formulation. The immune response in the group receiving the 1g scale dose was similar to that of the 0.4g scale group.

[0323] The neutralizing titer for CMV was determined by comparing a large-scale (1g) lyophilized formulation with a small-scale (0.03g) liquid formulation. The neutralizing titer against CMV virus was 26 times higher when using the lyophilized formulation based on molar ratio compared to the liquid formulation based on equal mass ratio (Figures 16A and 16B).

[0324] Example 5: Evaluation of immunogenicity one month after the second vaccination in a Phase II trial. This example describes the results of a 3-month (1 month after the second vaccination) interim analysis regarding the immunogenicity of the hCMV mRNA vaccine.

[0325] Immunogenicity in CMV sero-negative and CMV sero-positive participants was measured as the neutralizing antibody (nAb) response to epithelial cell infection (an indicator of the immune response to pentameric antigens) and the nAb response to fibroblast infection (an indicator of the immune response to gB antigens). Results were summarized as mean, median, minimum, maximum, geometric mean titer (GMT), geometric mean ratio to baseline (GMR), and the corresponding 95% confidence intervals for GMT and GMR.

[0326] For microneutralization assays to measure nAbs against epithelial cell infection, VR1814 and ARPE-19 cells of CMV isolates were used. For microneutralization assays to measure nAbs against fibroblast infection, AD169 and HEL299 cells of CMV isolates were used.

[0327] Baseline neutralizing antibody In CMV seronegative participants, baseline (before the first vaccination) nAb GMT for epithelial cell infection and fibroblast infection was below the lower limit of quantification (LLOQ) in all treatment groups (reported as 8, equivalent to 0.5 × LLOQ). This indicates that there was no prior CMV infection.

[0328] In CMV seropositive participants (n=46), the baseline GMT of nAb for cell infection was 3,924 (95% CI: 2,249, 6,845), and the baseline GMT of nAb for fibroblast infection was 3,955 (95% CI: 2,197, 7,119). These values ​​serve as benchmarks for comparing the nAb GMT of naturally acquired immunity and the immune response in the CMV seronegative group within this per-protocol set.

[0329] Neutralizing antibody (nAb) response In CMV seronegative participants, neutralizing antibodies against epithelial cell infection (Figure 17) increased in a dose-dependent manner after the first vaccination. At one month (one month after the first vaccination), the GMT was 955 (95% CI 503, 1,814); 2,100 (95% CI 1,074, 4,110); and 3,109 (95% CI 2,116, 4,568) in the 50 μg, 100 μg, and 150 μg treatment groups, respectively (Table 1). In each mRNA therapy group, nAbs against epithelial cell infection further increased after the second vaccination in all therapy groups to GMTs exceeding the serologically positive benchmark GMT. The GMTs at 3 months (1 month after the second vaccination) were 57,028 (95% CI 36,725, 88,554); 49,302 (95% CI 32,141, 75,627); and 49,706 (95% CI 35,792, 69,029) for the 50 μg, 100 μg, and 150 μg therapy groups, respectively (Table 1).

[0330] In CMV seronegative participants, neutralizing antibodies against fibroblast infection (Figure 18) increased after the first vaccination, and the GMT at 1 month (1 month after the first vaccination) was 73 (95% CI 35, 151), 175 (95% CI 86, 356), and 136 (95% CI 68, 272) in the 50 μg, 100 μg, and 150 μg treatment groups, respectively (Table 1). In each mRNA treatment group, nAbs for fibroblast infection further increased after the second vaccination to GMTs close to or exceeding the serologically positive benchmark GMT in all treatment groups. The GMTs at 3 months (1 month after the second vaccination) were 3,856 (95% CI 2,726, 5,455); 3,242 (95% CI 2,009, 5,233); and 4,638 (95% CI 3,417, 6,296) for the 50 μg, 100 μg, and 150 μg treatment groups, respectively (Table 1). [Table 1]

[0331] In CMV seropositive participants, neutralizing antibodies against epithelial cell infection (Figure 17) were boosted in a dose-dependent manner after the first vaccination. The GMT at 1 month (1 month after the first vaccination) was 27,062 (95% CI 7,392, 99,073); 52,989 (95% CI 24,882, 112,847); and 116,899 (95% CI 60,899, 224,392) in the 50 μg, 100 μg, and 150 μg treatment groups, respectively, corresponding to GMRs of 12.0, 8.9, and 15.6 (Table 2). In each mRNA therapy group, nAbs against epithelial cell infection were further boosted after the second vaccination, with GMTs at 3 months (1 month after the second vaccination) being 102,850 (95% CI 64,178, 164,826); 81,111 (95% CI 40,570, 162,167); and 126,075 (95% CI 73,077, 217,509) for the 50 μg, 100 μg, and 150 μg therapy groups, respectively, corresponding to GMRs of 51.4, 13.7, and 16.9 (Table 2).

[0332] In CMV seropositive participants, neutralizing antibodies against fibroblast infection (Figure 18) were boosted in a dose-dependent manner after the first vaccination, with GMTs at 1 month (1 month after the first vaccination) being 5,686 (95% CI 1,680, 19,252); 14,251 (95% CI 5,790, 35,077); and 21,341 (95% CI 13,468, 33,817) in the 50 μg, 100 μg, and 150 μg treatment groups, respectively, corresponding to GMRs of 2.3, 2.2, and 3.0 (Table 2). In each mRNA treatment group, nAbs against fibroblast infection were further boosted after the second vaccination, with GMTs at 3 months (1 month after the second vaccination) being 9,970 (95% CI) in the 50 μg, 100 μg, and 150 μg treatment groups, respectively. The values ​​were 6,487, 15,325); 11,652 (95% CI 6,323, 21,475); and 13,208 (95% CI 8,875, 19,657), corresponding to GMRs of 4.4, 1.8, and 1.9, respectively (Table 2). [Table 2]

[0333] Conclusion on immunogenicity CMV seronegative participants (a) Neutralizing antibodies against epithelial cell infection (an indicator of the immune response to intact CMV pentameric antigen) increased in a generally dose-dependent manner after the first vaccination (1 month). After the second vaccination (3 months), nAb GMT for epithelial cell infection was boosted by at least 12-fold compared to the baseline GMT (an indicator of past naturally acquired CMV infection) in CMV seropositive participants. The 3-month nAb GMT for epithelial cell infection in the 50 μg, 100 μg, and 150 μg treatment groups was generally comparable. (b) Neutralizing antibodies against fibroblast infection (an indicator of the immune response to CMV gB antigen) did not show a significant increase after the first vaccination (1 month). After the second vaccination (3 months), nAb against fibroblast infection was boosted to a GMT (indicator of past naturally acquired CMV infection) that was roughly equivalent to the baseline GMT (indicator of past naturally acquired CMV infection) in CMV seropositive participants. The 3-month GMT was roughly equivalent for the 50 μg, 100 μg, and 150 μg treatment groups.

[0334] CMV seropositive participants (a) Baseline nAb GMT for epithelial cell infection varied among treatment groups, with GMT generally increasing between baseline and 3 months in the placebo group. In each hCMV mRNA vaccine treatment group, nAb GMT for epithelial cell infection was boosted to at least 8-fold above their respective baseline GMT levels after the first vaccination (1 month), and to at least 13-fold to over 51-fold above their respective baseline GMT levels after the second vaccination (3 months). No dose-dependent GMR response was observed after either the first or second vaccination. (b) Baseline nAb GMT for fibroblast infection varied among treatment groups, with GMT generally increasing between baseline and 3 months in the placebo group. In each hCMV mRNA vaccine treatment group, nAb GMT for fibroblast infection was boosted to at least twice the baseline level after the first and second vaccinations (1 and 3 months), but the increase in nAb GMT in the placebo group meant that there was no significant increase in GMR compared to placebo after any vaccination. Across all mRNA treatment groups, no dose-dependent GMR response was observed compared to the placebo group.

[0335] Involuntary safety Involuntary adverse reactions (ARs) after the first vaccination were recorded (Table 3). Injection site pain was the most commonly reported involuntary local adverse reaction. The most common involuntary systemic ARs in both CMV seronegative and CMV seropositive treatment groups were headache, fatigue, and myalgia. No serious adverse events (SAEs) were reported. No spontaneous events leading to discontinuation of the study occurred. [Table 3]

[0336] Involuntary adverse reactions after the second vaccination were recorded (Table 4). The distribution of the proportion and severity of involuntary ARs was generally the same in the CMV seronegative and CMV seropositive treatment groups after the second vaccination. [Table 4]

[0337] Safety conclusions (a) The vaccine was generally well-tolerated at all doses, regardless of serological status. (b) No SAEs were reported. (c) No spontaneous events occurred that would lead to the suspension of the trial. (d) The proportion of CMV seronegative and CMV seronegative participants in the treatment group who reported at least one involuntary AR after the first and second doses of vaccination was approximately the same. (e) Overall, in either the CMV seronegative or CMV seronegative treatment group, no dose-dependent pattern was observed in the proportion of participants reporting involuntary adverse reactions (ARs) after the first or second vaccination. (f) Overall, the most common involuntary localized AR was injection site pain. The most common involuntary systemic ARs in both CMV seronegative and CMV seronegative treatment groups were headache, fatigue, and muscle pain. (g) The rate of involuntary systemic AR after the first vaccination was generally lower in CMV seronegative participants. Reports of grade 3 involuntary systemic AR were few and limited to the CMV seropositive treatment group. No dose-dependent pattern was observed in the rate of involuntary AR in either the CMV seronegative or CMV seropositive treatment group. (h) The distribution of the proportion and severity of involuntary ARs in the CMV seronegative treatment group and the CMV seronegative treatment group after the second vaccination was generally the same. (i) The proportion of participants reporting spontaneous AEs overall and Grade 3 spontaneous AEs was numerically similar in the treatment groups of CMV seronegative and CMV seronegative participants. The proportion of CMV seronegative participants reporting treatment-related spontaneous AEs was numerically higher in CMV seronegative participants. None of the reported spontaneous AEs showed a clear dose-dependent pattern or a pattern related to CMV serological status.

[0338] Example 6: Evaluation of immunogenicity one month after the third vaccination in a Phase II trial. This example describes the results of a 7-month (1 month after the third vaccination) interim analysis regarding the immunogenicity of the hCMV mRNA vaccine.

[0339] Immunogenicity in CMV sero-negative and CMV sero-positive participants was measured as the neutralizing antibody (nAb) response to epithelial cell infection (an indicator of the immune response to pentameric antigen), the nAb response to fibroblast infection (an indicator of the immune response to gB antigen), anti-pentamer bound antibody titer, and anti-gB bound antibody titer. Results were summarized as mean, median, minimum, maximum, geometric mean titer (GMT), geometric mean ratio to baseline (GMR), and the corresponding 95% confidence intervals for GMT and GMR.

[0340] For microneutralization assays to measure nAbs against epithelial cell infection, VR1814 and ARPE-19 cells of CMV isolates were used. For microneutralization assays to measure nAbs against fibroblast infection, AD169 and HEL299 cells of CMV isolates were used. For binding ELISA assays to measure pentamers, recombinant human CMV pentamer protein complexes consisting of full-length UL75(gH), UL115(gL), UL128, UL130, and UL131A derived from VR1814 isolate were used. For binding ELISA assays to measure gB, recombinant human CMV gB encoding an extracellular domain linked to the cytoplasmic domain was used.

[0341] Baseline neutralizing antibody In CMV seronegative participants (n=127), baseline nAb GMT for epithelial cell infection and fibroblast infection (before the first vaccination) was less than LLOQ in all treatment groups (reported as 8, equivalent to 0.5 × LLOQ). This indicates that there was no prior CMV infection.

[0342] In CMV seropositive participants (n=62), the baseline GMT of nAb for epithelial cell infection was 4,732 (95% CI: 3,059; 7,321), and the baseline GMT of nAb for fibroblast infection was 4,045 (95% CI: 2,566; 6,374). These values ​​serve as benchmarks for comparing the nAb GMT of spontaneously acquired immunity in this PP immunogenicity set with the neutralizing antibody response in the CMV seronegative group.

[0343] Neutralizing antibody (nAb) response In CMV seronegative participants, neutralizing antibodies against epithelial cell infection increased in a dose-dependent manner after the first vaccination (Figure 19, Table 5). In each mRNA treatment group, nAbs against epithelial cell infection increased further after the second vaccination, and after the third vaccination, all treatment groups reached a GMT 20-fold above the seropositive benchmark GMT (Figure 20).

[0344] In each mRNA treatment group, nAbs for fibroblast infection increased to near or above the serologically positive benchmark GMT after the second vaccination in all treatment groups. After the third vaccination, the GMT in the 100 μg and 150 μg treatment groups was similar to the GMT after the second vaccination (Figure 20). [Table 5]

[0345] In CMV seropositive participants, neutralizing antibodies against epithelial cell infection increased in a dose-dependent manner after the first vaccination (Figure 19, Table 6). In each mRNA treatment group, nAbs against epithelial cell infection after the third vaccination were similar to GMT after the second vaccination in the 100 μg and 150 μg groups (Figure 19).

[0346] In each mRNA therapy group, the nAb for fibroblast infection after the third vaccination was similar to that after the second vaccination (Figure 20).

[0347] It should be noted that baseline GMT differed between treatment groups for both nAbs for epithelial cell infection and nAbs for fibroblast infection. Specifically, the baseline GMT in the 50 μg treatment group was numerically lower than that in the 100 μg and 150 μg treatment groups, which may have contributed to the relatively higher GMR in the 50 μg treatment group. Furthermore, the post-baseline GMT in the placebo treatment group was higher compared to their respective baseline GMTs. [Table 6]

[0348] Binding antibody (nAb) response In CMV seronegative participants, anti-pentamer bAb levels increased slightly after the first vaccination (Figure 21, Table 7), and then increased to a GMT more than 10 times higher than baseline in the CMV seropositive group after the second vaccination, but no clear dose-related relationship was observed. In all mRNA therapy groups, the GMT after the third vaccination was similar to that after the second vaccination (Figure 21).

[0349] In each mRNA treatment group, anti-gB levels did not show a significant increase after the first vaccination, but increased after the second vaccination and further increased after the third vaccination (Figure 22, Table 7). GMT levels after the third vaccination were lower than baseline GMT levels in the CMV seropositive group. No clear dose-relationships were observed across all treatment groups. [Table 7]

[0350] In serologically positive participants, anti-pentamer bAb levels were boosted in all mRNA treatment groups after the first vaccination (Figure 21, Table 8) and further increased after the second vaccination, but no clear dose-relation was observed. After the third vaccination, anti-pentamer bAb levels were comparable to those after the second vaccination in all mRNA treatment groups (Figure 22). Across all mRNA treatment groups, GMR levels were at least 6.2 after the first vaccination, at least 10.2 after the second vaccination, and at least 6.8 after the third vaccination.

[0351] Anti-gB bAb levels were boosted in all mRNA treatment groups after the first vaccination and increased after the second and third vaccinations. Across all mRNA treatment groups, GMR was at least 1.8 after each of the three vaccination doses (Figure 22, Table 8). [Table 8]

[0352] Conclusion on immunogenicity CMV seronegative participants (a) Neutralizing antibody response. Neutralizing Ab GMT for epithelial cell infection increased in all CMV seronegative mRNA treatment groups after the third vaccination compared to after the second vaccination, and was 20-fold higher than the baseline GMT in the CMV seropositive group. nAb GMT for fibroblast infection after the second vaccination approached or exceeded the baseline GMT of the CMV seropositive group in all mRNA treatment groups. After the third vaccination, nAb GMT in the 100 μg and 150 μg treatment groups was similar to the GMT after the second vaccination. (b) Binding antibody response. Anti-pentamer-bound Ab GMT increased to a level 10-fold above baseline GMT in the CMV seropositive group after the second vaccination, and GMT after the third vaccination was similar to that after the second vaccination, with no clear dose relationship. Anti-gB-bound Ab GMT was numerically higher after the third vaccination compared to the second vaccination in the mRNA treatment group, but there was no clear overall dose relationship, and it was numerically lower than baseline GMT in the CMV seropositive group.

[0353] CMV seropositive participants (a) Neutralizing antibody response. In the mRNA therapy group, nAb GMR for epithelial cell infection was boosted to at least 8.6 after the first vaccination, at least 14.0 after the second vaccination, and at least 12.4 after the third vaccination, but no clear overall dose relationship was observed. Neutralizing Ab GMR for fibroblast infection was boosted to at least 2.2 after the first vaccination, at least 2.1 after the second vaccination, and at least 1.9 after the third vaccination, but no clear overall dose relationship was observed. (b) Binding antibody response. In the mRNA treatment group, anti-pentamer bAb GMT was boosted to at least 6.2 after the first vaccination, at least 10.2 after the second vaccination, and at least 6.8 after the third vaccination, but there was no clear overall dose relationship. In each mRNA treatment group, anti-gB-bound Ab GMT was boosted to at least 1.8 GMR after the first vaccination across all mRNA treatment groups, and the GMR was roughly equivalent after the second and third vaccinations, but there was no clear dose relationship.

[0354] Involuntary safety The most common involuntary systemic adverse reactions (ARs) in both CMV seropositive and CMV seronegative whole mRNA groups were headache, fatigue, muscle pain, arthralgia, and chills. In the CMV seronegative whole mRNA group, the percentages of participants reporting headache, fatigue, muscle pain, arthralgia, and chills after the first vaccination were 27%, 33%, 21%, 10%, and 13%, respectively. After the second vaccination, these percentages increased numerically to 47%, 42%, 44%, 33%, and 35%, respectively, and after the second vaccination, they increased to 49%, 52%, 47%, 34%, and 31%, respectively. In the CMV serologically positive whole mRNA group, the percentages of participants reporting headache, fatigue, muscle pain, arthralgia, and chills after the first vaccination (52%, 54%, 59%, 43%, and 37%, respectively) were roughly the same as those reporting after the second vaccination (51%, 61%, 70%, 44%, and 47%, respectively) and the third vaccination (56%, 62%, 46%, 41%, and 51%, respectively). In the CMV serologically negative placebo group, the percentages of headache, fatigue, muscle pain, arthralgia, and chills after the first vaccination were 37%, 33%, 16%, 7%, and 10%, respectively; after the second vaccination, they were 38%, 25%, 5%, 5%, and 2.5%, respectively; and after the third vaccination, they were 22%, 8%, 0%, 0%, and 3%, respectively. In the CMV serologically positive placebo group, the rates of headache, fatigue, muscle pain, arthralgia, and chills were 35%, 25%, 10%, 5%, and 0%, respectively, after the first vaccination; 43%, 14%, 7%, 7%, and 0%, respectively, after the second vaccination; and 20%, 7%, 0%, 0%, and 0%, respectively, after the third vaccination (Tables 9 and 10). [Table 9] [Table 10]

[0355] Safety conclusions (a) Vaccine tolerability was generally good at all doses, regardless of serological status. No trial suspension rules set out in the protocol have occurred to date. (b) One case of non-treatment-related SAE was reported in a vaccinated participant (follicular thyroid cancer). (c) No spontaneous events leading to the discontinuation of the trial occurred. TEAEs leading to the discontinuation of the trial vaccine occurred rarely. (d) After the first, second, and third doses of vaccination, the proportion of CMV serologically negative and CMV serologically positive participants in the vaccine treatment group who reported at least one involuntary AR was approximately the same. (e) In either the CMV seronegative or CMV seronegative vaccine treatment group, no dose-dependent pattern was observed in the proportion of participants who reported involuntary adverse reactions (ARs) after the first, second, or third dose of vaccination. (f) In both the CMV seronegative and CMV seronegative vaccine treatment groups, the most common involuntary localized aortic response (AR) was injection site pain. The overall frequency after the third vaccination was roughly the same as after the second vaccination in the vaccine treatment groups. The overall frequency of grade 3 severe injection site pain was similar after the second and third vaccinations in both CMV seronegative mRNA treatment groups, and numerically lower after the third vaccination compared to the second vaccination in the CMV seronegative mRNA treatment group. (g) In both CMV seronegative and CMV seronegative vaccine treatment groups, the most common involuntary systemic aortic regurgitation (AR) was headache, fatigue, muscle pain, arthralgia, and chills. The proportion of participants reporting involuntary systemic AR was generally higher after the second vaccination compared to the first vaccination, and similar after the third vaccination compared to the second vaccination. The frequency of fever after the third vaccination was numerically higher compared to the second vaccination, but the proportion of grade 3 fever was similar (defined as an oral temperature of 39.0°C to 40.0°C / 102.1 to 104.0°F). (h) There were no clear overall differences in the pattern or distribution of spontaneous AEs or MAAEs, and the proportions were roughly the same in the CMV serologically negative and CMV serologically positive groups. (i) Analysis of the test results revealed that there were no safety concerns.

[0356] Example 7. Phase III randomized, observer-blinded, placebo-controlled trial to evaluate the efficacy, safety, and immunogenicity of hCMV mRNA cytomegalovirus (CMV) vaccine in healthy adults aged 16-40 years. The objective of this Phase III trial is to evaluate the vaccine safety, efficacy, and immunogenicity of the hCMV mRNA vaccine against primary cytomegalovirus (CMV) infection in healthy women aged 16–40 years who are either CMV seronegative or CMV seropositive at enrollment.

[0357] Explanation of the test vaccine As described above, the hCMV mRNA vaccine consists of six distinct mRNA sequences (full-length CMV glycoprotein B [gB] and pentameric gH / gL / UL128 / UL130 / UL131A glycoprotein complex [pentamer]) in a lipid nanoparticle (LNP) formulation that encode key targets of the neutralizing antibody (nAb) response to human CMV infection.

[0358] The LNP formulation contains four lipid excipients: heptadecan-9-yl=8-((2-hydroxyethyl)(6-oxo-6(undecyloxy)hexyl)amino)octanoate, a proprietary ionizable aminolipid, and the commercially available lipid cholesterol 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycerol,methoxypolyethylene glycol (PEG2000-DMG).

[0359] The hCMV mRNA vaccine injection should be stored at 2°C to 8°C until use. After being prepared to the appropriate dose, the hCMV mRNA vaccine injection should be administered intramuscularly in a volume of 0.5 mL, preferably into the deltoid muscle of the non-dominant arm.

[0360] The Phase III hCMV mRNA vaccine formulation is filled and lyophilized in 2R type I glass vials with a total mRNA content of 151 μg / vial. The lyophilized drug is reconstituted with 0.7 mL of 0.9% sodium chloride injection solution before use to form a homogeneous LNP dispersion at an administration concentration of 0.2 mg / mL.

[0361] A 0.9% sodium chloride injection (physiological saline) placebo is administered intramuscularly in a volume of 0.5 mL, preferably into the deltoid muscle of the non-dominant arm.

[0362] Test design The primary efficacy objective is to demonstrate the vaccine efficacy of the hCMV mRNA vaccine against primary cytomegalovirus (CMV) infection in female subjects who are CMV sero-negative at enrollment. Primary CMV infection is defined as seroconversion of serum immunoglobulin G (IgG) against CMV, assessed from a negative result (baseline / day 1) to a positive result, measured by a platform-based automated immunoassay based on at least one of four recombinant CMV antigens (pp150, pp28, pp52, pp38) not encoded by the hCMV mRNA vaccine, starting 28 days after the third injection. Female and male subjects who are CMV sero-positive at baseline are not included in the primary efficacy analysis.

[0363] A total of approximately 8,100 subjects will be enrolled in the trial: CMV sero-negative cohort: 5,500 female subjects and 600 male subjects, and CMV sero-positive cohort: 1,400 female subjects and 600 male subjects.

[0364] Participants will be randomly assigned in a 1:1 ratio to receive either 100 μg of hCMV mRNA vaccine or placebo. Randomization will be blinded using a concentrated two-way response technique on day 1 of hospital visit, according to a pre-developed randomization schedule. Randomization will be stratified for each serostatus cohort (CMV seronegative, CMV seropositive) by sex (female, male) and age (16 to under 18 years, 18 to under 20 years, and 20 to under 40 years) to ensure equal treatment allocation. At least 400 enrolled CMV seronegative female participants will be assigned to the 16 to under 18 year group. Each participant will participate in two phases: the vaccination phase (day 1 to 7 months [day 197; 28 days after the third injection]) and the follow-up phase (from 7 months onward [day 198] to 30 months [day 887; approximately 24 months after the third injection]). Once the vaccination phase is complete, participants will make scheduled trial visits approximately every three months during the follow-up phase for safety and trial evaluation.

[0365] All subjects receive their first injection on day 1, and the same treatment for their second injection at 2 months (day 57) and third injection at 6 months (day 169). Subjects receive approximately 14 trial visits and approximately 18 safety telephone calls / electronic diary (eDiary) safety and monitoring prompts during the study period. Local and systemic involuntary adverse reactions (ARs) are collected using the eDiary for 7 days after each injection during the vaccination phase. Any involuntary ARs that persist beyond day 7 are recorded until they cease to be reported, not exceeding 28 days. Spontaneous adverse events (AEs) are collected from the day of each injection for 28 days after each injection. MAAEs requiring medical attention are collected from day 1 to 6 months after the last injection. Serious adverse events (SAEs) are collected from consent until end of study (EOS). Death, AESI, and AEs resulting in additional injections or withdrawal from the study are collected until EOS. For all subjects who become pregnant during the trial from day 1 to end-of-surgery (EOS), pregnancy safety and overall outcome data, including spontaneous miscarriage, involuntary abortion, and birth defects, will be collected. Pregnancy safety and overall outcome data may also be collected after EOS (i.e., pregnancies continuing after EOS visit).

[0366] During the study period, participants will undergo scheduled blood draws to assess eligibility, seroconversion due to primary CMV infection (CMV seronegative cohort), vaccine-induced antibody response, and antibody persistence. A more detailed outline of the procedures and visits can be found in the Evaluation Schedule (SOA) (Tables 11 and 12). [Table 11-1] [Table 11-2] [Table 12-1] [Table 12-2]

[0367] The trial is an observer-blinded study in which only the delegated open-label investigator responsible for the preparation, administration, and / or management of the study vaccine has access to the assignment of study treatments. Participants, their parent(s) / legally authorized representatives(s) (LARs), the principal investigator, and the site staff responsible for study evaluation / safety will not have access to treatment assignments during the trial. The principal investigator may deblind in emergency situations.

[0368] Participants may experience adverse events (AEs) that require unscheduled visits, and these AEs will be recorded as MAAEs. Following the reporting of an AE, the principal investigator may request participants to make unscheduled follow-up visits. During these visits, additional tests may be conducted as needed to ensure safety and the health of the participants. An electronic case report form must be completed each time an unscheduled visit occurs.

[0369] All subjects were followed for safety for a minimum of 30 months (24 months after the third injection) for seroconversion due to primary CMV infection (CMV seronegative cohort), CMV excretion (CMV seropositive cohort), pregnancy outcomes, and SAEs up to EOS and MAAEs in the 6 months after the last injection (Table 12).

[0370] Subjects who withdraw from further vaccinations after either the first or second vaccination without withdrawing their consent will be tracked for safety as described above and will be provided with blood samples for immunogenicity and seroconversion due to primary CMV infection (CMV seronegative cohort) as well as CMV excretion (CMV seropositive cohort) (Table 12).

[0371] Cytomegalovirus seronegative cohort: Cytomegalovirus seronegative cohort subjects will provide blood samples for seroconversion assessment due to primary CMV infection and urine samples for pregnancy testing at the following trial visits: day 1, month 2 (day 57), month 3 (day 85), month 6 (day 169), month 7 (day 197), month 10 (day 287), month 12 (day 347), month 15 (day 437), month 18 (day 527), month 21 (day 617), month 24 (day 707), month 27 (day 797), and month 30 (day 887, EOS). (Tables 11 and 12.)

[0372] Participants who meet the primary endpoint of seroconversion due to primary CMV infection between month 3 and endoscopy (EOS) will be contacted to undergo an unscheduled trial visit (seroconversion visit) for clinical evaluation, including blood, urine, and saliva collection for CMV polymerase chain reaction (PCR), after which they will return to their scheduled visit schedule. After the seroconversion visit, urine will be collected for CMV PCR from all subsequent scheduled trial visits until EOS. Once a participant meets the criteria for seroconversion due to primary CMV infection, serum collection for testing for primary CMV infection will not be performed at subsequent visits.

[0373] In the CMV seronegative cohort, the majority of seroconversions due to primary CMV infection are asymptomatic and are expected to be detected by scheduled serological surveillance during planned study visits. Subjects will be assessed for symptoms meeting the criteria for possible symptomatic primary CMV infection according to protocol-defined criteria via planned study visits, safety phone calls, and eDiary safety surveillance prompts (starting at month 8), beginning at month 3. Subjects meeting the protocol-defined criteria for possible symptomatic primary CMV infection during planned study visits, safety phone calls, or via eDiary during the follow-up phase will be instructed to return to the study site (unplanned visit) for a CMV disease evaluation visit. The CMV disease evaluation visit will include physical examination; collection of blood samples for hematology, chemistry, seroconversion due to primary CMV infection, and Epstein-Barr virus (EBV) panels; and a urine sample for CMV PCR. At the discretion of the principal investigator, testing for HIV antibodies, HIV viral load, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be performed. In addition, subjects who report symptoms suggestive of symptomatic primary CMV infection according to the criteria defined in the protocol may also receive an electronic patient-reported outcome (ePRO) questionnaire, and participant-reported health status and health-related productivity information may be collected. The questionnaire may include the EuroQol 5-item, 5-level (EQ 5D 5L) and the Health-Related Productivity Questionnaire (HRPQ), and may be administered based on symptoms reported via CMV disease assessment visits or safety eDiaries, and based on the availability and timeframe of the ePRO questionnaire.

[0374] Cytomegalovirus seropositive cohort: Cytomegalovirus seropositive cohort subjects underwent urine collection for CMV excretion and urinary pregnancy testing at the following trial visits: day 1, month 2 (day 57), month 3 (day 85), month 6 (day 169), month 7 (day 197), month 10 (day 287), month 12 (day 347), month 15 (day 437), month 18 (day 527), month 21 (day 617), month 24 (day 707), month 27 (day 797), and month 30 (day 887, EOS). (Tables 11 and 12).

[0375] Exam period: The trial period for each participant is approximately 30 months.

[0376] Sample size: The sample size for CMV seropositive subjects will be determined by safety considerations. Approximately 2,000 CMV seropositive female and male subjects will be randomly assigned in a 1:1 ratio to receive either the hCMV vaccine or a placebo. If 1,000 CMV seropositive subjects are exposed to the hCMV vaccine, there is at least a 90% probability that at least one CMV seropositive participant will experience an adverse event (AE) with a true AE rate of 0.25% in this trial.

[0377] The sample size for CMV seronegative male subjects will be determined by safety considerations. Approximately 600 CMV seronegative male subjects will be randomly assigned in a 1:1 ratio to receive either the hCMV vaccine or a placebo. If 300 CMV seronegative male subjects are exposed to the hCMV vaccine, there is at least a 95% probability that at least one CMV seronegative male participant will be observed to have an adverse event (AE) with a true 1% AE rate in this trial.

[0378] Purpose and endpoint Main purpose: The main objectives of this examination are as follows: 1) To demonstrate the effectiveness of hCMV mRNA vaccine in preventing primary CMV infection in CMV seronegative female subjects. 2) Evaluate the safety and reactiongenicity of the hCMV mRNA vaccine when administered in a three-dose injection schedule to all subjects.

[0379] Secondary purpose: The secondary objectives of this examination are as follows: 1) Evaluate the immunogenicity of the hCMV mRNA vaccine when administered in a three-dose injection schedule to all subjects. 2) Evaluate the persistence of immunogenicity to the hCMV mRNA vaccine in all subjects up to 24 months after the third injection.

[0380] Exploratory purpose: The exploratory objectives of this study are as follows: 1) To evaluate the effect of hCMV mRNA vaccine on CMV infection kinetics in CMV seronegative subjects who meet the endpoint of seroconversion due to primary CMV infection, as measured by CMV PCR in blood, urine, and saliva. 2) Evaluate the effect of the hCMV mRNA vaccine on the frequency and level of urinary CMV excretion in CMV seropositive subjects. 3) In CMV seronegative female subjects who received the hCMV mRNA vaccine, evaluate the levels of antigen-specific nAbs and binding antibodies in subjects who meet the endpoint for seroconversion due to primary CMV infection and in subjects who do not meet the endpoint for seroconversion due to primary CMV infection. 4) Evaluate the effect of hCMV mRNA vaccine on the incidence of symptomatic primary CMV infection in CMV seronegative subjects. 5) Evaluate immune markers after receiving the hCMV mRNA vaccine as a factor associated with the risk of primary CMV infection in CMV seronegative subjects. 6) Describe the effect of the hCMV mRNA vaccine in preventing primary CMV infection in CMV seronegative subjects who received two of the three doses of the regimen. 7) In CMV seronegative subjects, evaluate the impact of symptomatic primary CMV infection on participant-reported health status and health-related productivity. 8) Evaluate the effect of the hCMV mRNA vaccine on the incidence of primary CMV infection in CMV serologically negative male subjects.

[0381] Primary endpoints The primary endpoint of this study is as follows: 1) Primary CMV infection defined as seroconversion, where serum IgG, measured by a platform-based automated immunoassay based on at least one of four recombinant CMV antigens (pp150, pp28, pp52, pp38) not encoded by the hCMV mRNA vaccine, changes from a negative result to a positive result 28 days after the third injection. 2) Involuntary adverse events (ARs) up to 7 days after each injection, spontaneous adverse events (AEs) up to 28 days after each injection, mandatory adverse events (MAAEs) from day 1 to 6 months after the last injection, adverse adverse events (AESIs) from day 1 to end-of-surgery (EOS), and serious adverse events (SAEs) from the time of consent to end-of-surgery (EOS).

[0382] Secondary endpoints The secondary endpoints of this study are as follows: 1) Antigen-specific nAb and conjugated antibody GMT at day 1, month 3, month 7, and month 12. 2) Antigen-specific nAb and conjugated antibody GMT at 18 months, 24 months, and 30 months.

[0383] analysis Immunogenicity evaluation The immunogenicity of this study is evaluated as follows: 1) Serum functional antibody levels against vaccine antigens, measured by nAb titers for epithelial cell infection and nAb titers for fibroblast infection. 2) Serum-bound antibody titer against vaccine antigens, measured by an enzyme-linked immunosorbent assay specific to gB and pentameric proteins.

[0384] Safety evaluation Safety assessment includes the following monitoring and recording for each subject: 1) Involuntary local and systemic aortic regurgitation (AR) occurring within 7 days after each injection (i.e., the day of injection and the following 6 days). Any involuntary AR continuing beyond day 7 will be recorded until no further reports are made, not exceeding 28 days. Involuntary AR will be recorded daily using eDiary. a) Involuntary local axillary regurgitation (AR) includes injection site pain, erythema [redness] at the injection site, swelling / induration [hardness] at the injection site, and local axillary swelling or tenderness on the same side as the vaccinated arm. b) Involuntary systemic aortic regurgitation (AR) includes headache, fatigue, muscle pain (generalized muscle pain), arthralgia (pain in some joints), nausea, fever, and chills. 2) Starting at 8 months, subjects will receive safety eDiary prompts at designated points in the evaluation schedule. The safety eDiary will prompt participants to report the occurrence of relevant safety events. If a relevant safety event is identified in the eDiary prompt, trained site staff will contact the participant within 1 business day, or within 2 business days after the eDiary completion window closes if the participant has not completed the eDiary, to collect information on MAAEs (up to 6 months after the last injection), AEs leading to dropout, SAEs, and concomitant medications associated with these events. In addition, only in the CMV seronegative cohort, the safety eDiary will prompt participants to report symptoms that may be consistent with symptomatic CMV. For CMV seronegative subjects who did not experience seroconversion due to primary CMV infection, site staff will review the symptoms reported in the safety eDiary and determine whether a CMV disease evaluation visit is necessary. 3) Spontaneous adverse events observed or reported during the first 28 days after each injection (i.e., the first day of each injection and the following 27 days). 4) AEs resulting from administration from day 1 to 30 months (day 887) or until withdrawal from the study and / or discontinuation of participation in the study. 5) MAAE from day 1 to 6 months after the final injection or until withdrawal from the study. 6) AESI from day 1 to 30 months (day 887) or until dropping out of the test. 7) SAEs from the time of consent until 30 months (887 days) or until withdrawal from the study. 8) Measure vital signs. 9) Physical examination findings. 10) Pregnancy testing at each trial visit. For subjects who become pregnant during the trial from day 1 to end-of-surgery (EOS), pregnancy safety and outcome data will be collected. For pregnancies that continue beyond the EOS visit, data may be collected even after the EOS.

[0385] Example 8: Preliminary study of infant outcomes in subjects who became pregnant during Phase III participation. The objective of this preliminary study is to evaluate cytomegalovirus (CMV)-related outcomes in live births of female subjects who became pregnant during the major Phase III trial of the hCMV mRNA vaccine described in Example 7. All objectives and endpoints in this preliminary study are exploratory.

[0386] The primary trial of the hCMV mRNA vaccine enrolls a population of women at risk of CMV infection and reinfection, as close exposure to infants is a registration requirement. The ultimate goal of hCMV mRNA vaccine development is to prevent congenital CMV infection by establishing or enhancing prenatal immunity against CMV in mothers planning pregnancy; therefore, this preliminary trial provides a unique opportunity to collect information on infant outcomes in a maternal population at high risk of CMV infection or reinfection. The objectives of this hCMV mRNA vaccine preliminary trial are to evaluate CMV excretion in all enrolled neonates, assess the diagnosis of congenital CMV (cCMV) based on clinical records, and evaluate placental transfer of vaccine-induced antibodies in maternal / infant blood sample pairs.

[0387] Considering that the major hCMV mRNA vaccine Phase III trial is expected to enroll 6,900 women (5,500 CMV seronegative and 1,400 CMV seropositive), and based on publicly available estimates of the annual pregnancy rate, live birth rate, and primary CMV infection rate in CMV seronegative pregnant women, as well as the cCMV infection rate in both CMV seronegative and CMV seropositive pregnant women, it is estimated that there will be a maximum of approximately 1,200 live births during the participation period of the major hCMV mRNA vaccine Phase III trial, and that the number of newborns infected with cCMV is expected to be extremely small (Figure 24).

[0388] Test design This preliminary trial can enroll all subjects who have received at least one trial dose of the hCMV mRNA vaccine in the main Phase III trial, who become pregnant at any point during their participation in the trial, and who intend to continue the pregnancy until delivery. Pregnancy is defined as a positive urine pregnancy test obtained during participation in the main Phase III trial of the hCMV mRNA vaccine. All subjects who become pregnant during participation in the main Phase III trial of the hCMV mRNA vaccine will not receive further trial injections, but will continue to be followed up according to the main protocol for safety, pregnancy outcomes, immunogenicity, seroconversion due to primary CMV infection (CMV seronegative subjects), and CMV excretion (CMV seropositive subjects), and will be provided for the hCMV mRNA vaccine preliminary trial. In addition, as part of the main Phase III trial of the hCMV mRNA vaccine, pregnant CMV seronegative subjects may return to the trial site more frequently (monthly) for blood tests to check for seroconversion due to primary CMV infection.

[0389] This preliminary trial does not involve randomization procedures. All staff at the trial sites, the CRO blinding teams, and the trial sponsors remain blinded to individual treatment assignments.

[0390] The participation period may be from the time when an hCMV mRNA vaccine subject meets the definition of pregnancy, consents to participate in the hCMV mRNA vaccine preliminary trial, and their eligibility is determined, until 6 weeks postpartum. Participation in this preliminary trial may be extended beyond the EOS visit for the hCMV mRNA vaccine (parental trial) if the date of delivery is less than 6 weeks after the EOS visit, or occurs after the EOS visit.

[0391] Up to approximately 1,200 subjects and their infants are expected to be eligible to participate in this preliminary study. Subjects will provide individual consent for each procedure of the preliminary study for themselves and their infants.

[0392] The preliminary examination procedure may include the following: 1. A single urine and saliva sample from the newborn within three weeks of birth. 2. Request and review of medical records of subjects and / or infants. 3. Within the operational capacity of the subject's testing facility, one pair of samples consisting of maternal venous blood obtained within 72 hours of birth and neonatal umbilical cord blood or neonatal venous blood (if permitted) obtained within 72 hours of birth. * Collection. * Maternal and neonatal samples do not need to be collected simultaneously, as long as both are taken within 72 hours of birth. This preliminary test will be conducted in accordance with the protocol, GCP, and all applicable regulatory requirements.

[0393] Purpose and endpoint exploratory purpose 1. Evaluate the incidence of CMV excretion in newborns of all subjects who became pregnant during the Phase III primary trial of the hCMV mRNA vaccine, separated by treatment group. 2. In infants who meet any of the following criteria, assess a) whether or not there is a clinical diagnosis of cCMV infection, and b) whether or not there is a clinical diagnosis of cCMV disease and its severity: i) Abnormalities have been reported in pregnancy reports from the primary trial of the hCMV mRNA vaccine; ii) The exploratory endpoint of Objective 1 is met due to a positive result from CMV PCR; iii) Born to a CMV sero-negative subject who meets the primary endpoint of seroconversion due to primary CMV infection before or during pregnancy. 3. Measure the placental transfer efficiency of vaccine-induced pentamer-specific and gB-specific IgG in live births of subjects who tested negative for CMV serotype at the time of registration.

[0394] exploratory endpoints 1. Positive CMV PCR of saliva and urine from a newborn, or positive CMV PCR of urine from a newborn collected within 3 weeks of birth. 2. Clinical diagnosis of cCMV infection based on review of the medical records of infants and / or subjects, recording test results that confirm the meeting of the public criteria for cCMV infection. In infants meeting the clinically diagnosed criteria for cCMV infection, clinical diagnosis of cCMV disease and assessment of the severity of cCMV disease are performed based on the infant's medical records. 3. Neonatal:maternal ratio of pentamer-specific binding antibody titers, gB-specific binding antibody titers, and nAb titers in paired samples of maternal blood obtained within 72 hours of birth and neonatal umbilical cord blood or neonatal venous blood obtained within 72 hours of birth.

[0395] analysis Test evaluation and procedures For subjects in the preliminary trial, the following test evaluations and procedures may be performed: 1. Urinary pregnancy test (if a positive urinary pregnancy test is not recorded in the primary hCMV mRNA vaccine primary trial or other medical records). 2. A single sample of neonatal urine or saliva (+ / -) for CMV PCR within three weeks postpartum. These samples may be collected at either the birthing facility or the testing facility, as appropriate (Figure 24). If the CMV PCR test is positive on the saliva sample, a urine sample is subsequently taken for CMV PCR testing. If the neonatal urine collection is performed at the testing facility, a urine bag is used. Saliva samples are collected using an oral swab at least 90 minutes after the last feeding. If a CMV PCR result is obtained on the infant's urine or saliva sample, the principal investigator shall immediately refer the subject and the infant to the subject's pediatrician and inform the subject's obstetrician. The clinical diagnosis of cCMV infection and disease is based on clinical assessments and diagnoses recorded in the medical records of infants ± subjects. 3. If any of the following occurs or is reported, request and review the medical records of the subject and / or infant: a. Infants whose CMV PCR result for a urine or saliva sample collected within three weeks of birth is positive (as described above); b. Congenital or fetal abnormalities reported in pregnancy reports completed as part of the hCMV mRNA vaccine primary trial; c. Infants born to CMV seronegative subjects who meet the primary endpoint of seroconversion due to primary CMV infection before or during pregnancy. Medical records are reviewed based on criteria that support the diagnosis of cCMV infection and disease. 4. Collection of one pair of samples* of maternal blood obtained within 72 hours of birth and neonatal umbilical cord blood or neonatal venous blood obtained within 72 hours of birth, within the operational capacity of the subject's laboratory. Immunogenicity assessment of these samples may include: a. Serum-bound antibody levels to pentamer and gB vaccine antigens as measured by enzyme-linked immunosorbent assay. b. Serum functional antibody levels against pentamer and gB vaccine antigens, as measured by nAb titers for epithelial cell infection and nAb titers for fibroblast infection.

[0396] Immunogenicity evaluation This study will not evaluate clinical efficacy. The purpose of this preliminary study is entirely exploratory.

[0397] Safety evaluation Safety will be evaluated as described in the hCMV mRNA vaccine major Phase III trial (Example 7).

[0398] array It should be understood that any mRNA sequence described herein may include a 5'UTR and / or 3'UTR. The UTR sequence may be selected from the following sequences, but other known UTR sequences may also be used. It should also be understood that any mRNA construct described herein may further include a poly(A) tail and / or cap (e.g., 7mG(5')ppp(5')NlmpNp). Furthermore, it should be understood that many mRNAs and encoded antigen sequences described herein include a signal peptide and / or peptide tag (e.g., a C-terminal His tag), but the indicated signal peptide and / or peptide tag may be substituted with a different signal peptide and / or peptide tag, or the signal peptide and / or peptide tag may be omitted. 5'UTR:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC(Sequence ID 13) 3'UTR:UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 14) [Table 13-1] [Table 13-2] [Table 13-3] Table 13-4 Table 13-5 Table 13-6 Table 13-7 Table 13-8 Table 13-9

[0399] Synonyms All references, patents, and patent applications disclosed herein are incorporated herein by reference with respect to the subject matter they refer to, and in some cases may encompass the entire document. This application is a compilation of the following: PCT International Publication No. WO2017 / 070613 (title "HUMAN CYTOMEGALOVIRUS VACCINE") filed on October 21, 2016; PCT International Publication No. WO2018 / 075980 (title "HUMAN CYTOMEGALOVIRUS VACCINE") filed on October 20, 2017; PCT International Publication No. WO2021 / 050864 (title "HUMAN CYTOMEGALOVIRUS VACCINE") filed on September 11, 2020; U.S. Patent No. 10,064,935 (title "HUMAN CYTOMEGALOVIRUS RNA VACCINES") granted on September 4, 2018; and U.S. Patent No. 10,383,937 (title "HUMAN") granted on August 20, 2019. All content, including all drawings and all parts of the specifications (including sequence listings or amino acid / polynucleotide sequences) of U.S. Patent No. 10,716,846 (titled "HUMAN CYTOMEGALOVIRUS RNA VACCINES"), granted on 21 July 2020, and U.S. Patent No. 10,695,419 (titled "HUMAN CYTOMEGALOVIRUS VACCINE"), is incorporated by reference.

[0400] In this specification, the indefinite articles "a" and "an" used in the specification and claims should be understood to mean "at least one" unless the opposite is explicitly stated.

[0401] Furthermore, unless explicitly stated otherwise, it should be understood that in any method comprising two or more steps or acts claimed herein, the order of the steps or acts of such method is not necessarily limited to the order in which the steps or acts of such method are described.

[0402] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and "composed of," should be understood to be open-ended, meaning they include but are not limited to what follows. Only the transitional phrases "consist of" and "essentially consist of" are closed or semi-closed transitional phrases, as described in Section 2111.03 of the U.S. Patent and Trademark Office Examination Guidelines.

[0403] The terms "approximately" and "effectively" preceding a number mean ±10% of the stated number.

[0404] Where a range of values ​​is given, each value between the upper and lower limits of the range is specifically intended and described herein.

[0405] Those skilled in the art will be able to identify or confirm numerous equivalents to the specific embodiments of the present disclosure described herein by means of ordinary experiments. Such equivalents are intended to be covered by the following claims.

Claims

1. An hCMV vaccine comprising: (a) messenger ribonucleic acid (mRNA) polynucleotide containing an open reading frame encoding human cytomegalovirus (hCMV) gH polypeptide; (b) mRNA polynucleotide containing an open reading frame encoding hCMV gL polypeptide; (c) mRNA polynucleotide containing an open reading frame encoding hCMV UL128 polypeptide; (d) mRNA polynucleotide containing an open reading frame encoding hCMV UL130 polypeptide; (e) mRNA polynucleotide containing an open reading frame encoding hCMV UL131A polypeptide; and (f) mRNA polynucleotide containing an open reading frame encoding hCMV gB polypeptide. The molar ratio of (a):(f) in the vaccine is approximately 1:

1. The molar ratio of (b):(c):(d):(e) in the vaccine is approximately 1:1:1:

1. The molar ratio of each of (a) and (f) in the vaccine to any one of (b), (c), (d), or (e) is approximately 1.5:1 to 2:

1. The aforementioned vaccine.

2. The vaccine according to claim 1, wherein the molar ratio of (a):(b):(c):(d):(e):(f) is approximately 2:1:1:1:1:

2.

3. The hCMV vaccine according to claim 1 or 2, wherein the hCMV gH polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19, the hCMV gL polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20, the hCMV UL128 polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16, the hCMV UL130 polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 17, the hCMV UL131A polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18, and the hCMV gB polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

15.

4. The hCMV vaccine according to claim 1 or 2, wherein the hCMV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 19, the hCMV gL polypeptide comprises the amino acid sequence of SEQ ID NO: 20, the hCMV UL128 polypeptide comprises the amino acid sequence of SEQ ID NO: 16, the hCMV UL130 polypeptide comprises the amino acid sequence of SEQ ID NO: 17, the hCMV UL131A polypeptide comprises the amino acid sequence of SEQ ID NO: 18, and the hCMV gB polypeptide comprises the amino acid sequence of SEQ ID NO:

15.

5. The hCMV vaccine according to any one of claims 1 to 4, wherein the open reading frame encoding the hCMV gH polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO: 11, the open reading frame encoding the hCMV gL polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO: 12, the open reading frame encoding the hCMV UL128 polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO: 8, the open reading frame encoding the hCMV UL130 polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO: 9, the open reading frame encoding the hCMV UL131A polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO: 10, and the open reading frame encoding the hCMV gB polypeptide includes a sequence having at least 90% identity with the sequence of SEQ ID NO:

7.

6. The hCMV vaccine according to any one of claims 1 to 4, wherein the open reading frame encoding the hCMV gH polypeptide includes SEQ ID NO: 11, the open reading frame encoding the hCMV gL polypeptide includes SEQ ID NO: 12, the open reading frame encoding the hCMV UL128 polypeptide includes SEQ ID NO: 8, the open reading frame encoding the hCMV UL130 polypeptide includes SEQ ID NO: 9, the open reading frame encoding the hCMV UL131A polypeptide includes SEQ ID NO: 10, and the open reading frame encoding the hCMV gB polypeptide includes SEQ ID NO:

7.

7. The hCMV vaccine according to any one of claims 1 to 6, wherein the hCMV vaccine is stable for at least three months when stored at temperatures above 0°C and below 10°C.

8. The hCMV vaccine according to any one of claims 1 to 6, wherein the hCMV vaccine is stable for at least 12 to 18 months when stored at temperatures above 0°C and below 10°C.

9. The hCMV vaccine according to any one of claims 1 to 6, wherein the hCMV vaccine is stable for at least 24 months when stored at temperatures above 0°C and below 10°C.

10. The hCMV vaccine according to any one of claims 1 to 9, wherein at least one of the mRNA polynucleotides of (a) to (f) contains a chemical modification.

11. The hCMV vaccine according to claim 10, wherein at least 80% of the uracil in the open reading frames of the mRNA polynucleotides (a) to (f) has a chemical modification selected from N1-methyl-psoidouridine or N1-ethyl-psoidouridine.

12. The hCMV vaccine according to any one of claims 1 to 11, further comprising lipid nanoparticles.

13. The hCMV vaccine according to claim 12, wherein the lipid nanoparticles comprise a mixture of lipids including an ionizable aminolipid, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-sn-glycerol,methoxypolyethylene glycol (DMG-PEG).

14. The hCMV vaccine according to claim 13, wherein the ionizable aminolipid comprises compound I. 【Chemistry 1】

15. The hCMV vaccine according to claim 13 or 14, wherein the lipid nanoparticles comprise a lipid mixture containing 20 to 60 mol% ionizable aminolipids, 25 to 55 mol% cholesterol, 5 to 25 mol% DSPC, and 0.5 to 15 mol% DMG-PEG.

16. The hCMV vaccine according to claim 13 or 14, wherein the lipid nanoparticles comprise a lipid mixture containing 45 to 55 mol% ionizable amino lipids, 35 to 40 mol% cholesterol, 5 to 15 mol% DSPC, and 1 to 2 mol% DMG-PEG.

17. An hCMV vaccine according to any one of claims 1 to 16, for inducing an antigen-specific immune response to hCMV in human subjects.

18. The hCMV vaccine according to claim 17, wherein the human subject is CMV serologically positive before being administered the hCMV vaccine.

19. The hCMV vaccine according to claim 17, wherein the human subject is CMV serologically negative before being administered the hCMV vaccine.

20. The hCMV vaccine according to any one of claims 1 to 16 for preventing or treating hCMV in human subjects.

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