Human cytomegalovirus GB polypeptide

Abstract

To provide safe and effective immunogenic compositions to protect against HCMV infection.SOLUTION: A human cytomegalovirus (HCMV) immunogenic composition comprises RNA formulated in lipid nanoparticles, where the RNA encodes an amino acid sequence of glycoprotein B (gB) protein that comprises at least one genetically engineered disulfide mutation.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 041,945, filed June 21, 2020, and U.S. Provisional Patent Application No. 63 / 208,457, filed June 8, 2021, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application was filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file was created on June 7, 2021, and contains a Sequence Listing entitled "PC72639_June2021_ST25.txt" having a size of 1,427 KB. The Sequence Listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to human cytomegalovirus (HCMV) compositions and methods thereof. [Background technology]

[0004] Human cytomegalovirus (HCMV) is a double-stranded DNA virus of the β-herpesvirus family. HCMV is a leading cause of congenital and neonatal hearing loss resulting from vertical viral transmission after infection or reactivation of latent virus in pregnant women. Furthermore, HCMV is a common opportunistic pathogen affecting immunosuppressed patients, including solid organ and stem cell transplant recipients and AIDS patients. The development of a vaccine against HCMV has been listed as a top priority by the Institute of Medicine, but no vaccine has been approved to date.

[0005] The HCMV genome encodes several envelope glycoproteins, one of which is glycoprotein B (gB). Glycoprotein B is a fusion factor required for viral entry into cells and is an important target for the neutralizing antibody (nAb) response to infection. HCMV vaccines containing gB subunit antigens are under development. Clinical trials have shown that some gB subunit-based vaccine candidates are safe and immunogenic, but improvements in protective efficacy and durability of protection are desirable. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for safe and effective immunogenic compositions to protect against HCMV infection. There is also a need for diagnostic reagents to detect immune responses to HCMV, to guide the design of gB-based HCMV vaccines, and to aid in the development of therapeutic or prophylactic antibodies against HCMV. [Means for solving the problem]

[0007] To meet these and other needs, in one aspect, the present invention relates to polypeptides that can be included in immunogenic compositions as antigens to elicit an immune response against HCMV.

[0008] The present invention provides a mutant of a wild-type cytomegalovirus (CMV) glycoprotein B (gB) protein, comprising at least two amino acid mutations compared to the amino acid sequence of the wild-type CMV gB protein, wherein the amino acid mutations are: (1) genetically engineered disulfide bond mutations; (2) additional mutations; and (3) a combination of at least one engineered disulfide mutation and at least one additional mutation; The present invention provides a mutant selected from the group consisting of:

[0009] In one embodiment, the amino acid mutations comprise a combination of at least two engineered disulfide mutations and at least one additional mutation. In another embodiment, the mutant wild-type CMV gB protein is in the form of a trimer.

[0010] In another embodiment of the invention, the engineered disulfide mutations are selected from the group consisting of D217C and Y589C; M371C and W506C; and N524C and M684C.

[0011] In a further aspect of the invention, the additional mutation is (1) Substitution of YIH at positions 155–157 by GHR; (2) substitution of W at position 240 by A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; and (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648–653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) Substitution of Y at position 696 and V at position 697 with C is selected from the group consisting of:

[0012] In another embodiment of the invention, the mutant is secreted. In another embodiment of the invention, the mutant is soluble.

[0013] The present invention also provides pharmaceutical compositions comprising (i) a CMV gB protein mutant according to the embodiments and aspects described herein and (ii) a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition is a vaccine.

[0014] The present invention also provides a method of reducing a CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0015] The present invention also provides a method of eliciting an immune response against CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0016] The present invention also provides a method for preventing CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0017] In another aspect, the invention relates to a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB). In some embodiments, the polypeptide comprises a conformation that is not the HCMV gB post-fusion conformation.

[0018] In another aspect, the invention relates to polypeptides that bind to HCMV gB prefusion-specific antibodies.

[0019] In another aspect, the invention relates to polypeptides that bind to a bis(aryl)thiourea compound. In some embodiments, the compound is N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide.

[0020] In some embodiments, the polypeptide is characterized by structural coordinates that include the root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed onto the backbone atoms described by the structural coordinates in Table 1A, i.e., the pdb file "Table1A-prefusion_gB_121918pdb," which is incorporated herein by reference in its entirety.

[0021] In some embodiments, the polypeptide is characterized by structural coordinates that include the root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed onto the backbone atoms described by the structural coordinates in Table 1B, i.e., the pdb file "Table1B-refine_apply_ncs_14pdb," which is incorporated herein by reference in its entirety.

[0022] In one aspect, the invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a cysteine ​​substitution.

[0023] In another aspect, the present invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a mutation that allows for disulfide bond formation.

[0024] In another aspect, the invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises an electrostatic mutation.

[0025] In another aspect, the invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a phenylalanine substitution.

[0026] In another aspect, the invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a leucine substitution.

[0027] In another aspect, the invention relates to a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the mutation stabilizes the prefusion conformation of the polypeptide, and wherein the polypeptide specifically binds to an HCMV gB prefusion-specific antibody.

[0028] In another aspect, the invention relates to a polypeptide comprising a cysteine ​​at any one of the amino acid positions listed in column (ii) of Table 2, relative to SEQ ID NO:1.

[0029] In another aspect, the invention relates to a polypeptide comprising an amino acid substitution at any one of the amino acid positions listed in column (ii) of Table 3, relative to SEQ ID NO:1.

[0030] In another aspect, the present invention relates to a polypeptide comprising the mutations Q98C and I653C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations T100C and S269C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations D217C and F584C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations Y242C and K710C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations Y242C and D714C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations S367C and L499C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations T372C and W506C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations S550C and D652C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations T608C and D679C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising the mutations K695C and K724C according to the numbering of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43, wherein the polypeptide comprises mutations compared to SEQ ID NO: 1.

[0031] In some embodiments, the polypeptide does not comprise a mutation at any one of the following positions: R562, P577, S587, Y588, G592, G595, L601 / H605, C610, L612, P613, Y625, Y627, F632, and K633.

[0032] In some embodiments, the polypeptide does not include the cytoplasmic tail of HCMV gB. In some embodiments, the polypeptide does not include the transmembrane region. In some embodiments, the polypeptide includes the cytoplasmic tail of HCMV gB, but does not include the transmembrane region.

[0033] In some embodiments, the polypeptide does not contain an insect cell glycosylation pattern.

[0034] In some embodiments, the polypeptide exhibits improved solubility or stability compared to native gB in the post-fusion conformation.

[0035] In some embodiments, the polypeptide is immunogenic.

[0036] In another aspect, the present invention relates to a nucleic acid encoding a polypeptide according to any one of the embodiments described herein. In some embodiments, the nucleic acid comprises a self-replicating RNA molecule. In some embodiments, the nucleic acid comprises a modified RNA molecule. In another aspect, the present invention relates to a composition comprising a nucleic acid according to any one of the embodiments described herein.

[0037] In another aspect, the present invention relates to a composition comprising a polypeptide according to any one of the embodiments described herein and further comprising a CMV antigen. In some embodiments, the composition further comprises any one of the following polypeptides: gO, gH, gL, pUL128, pUL130, pUL131, and any combination thereof. In some embodiments, the composition further comprises a diluent. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition is immunogenic. In some embodiments, the composition is for use in eliciting an immune response against cytomegalovirus.

[0038] In another aspect, the present invention relates to a method of raising an immune response in a mammal, the method comprising administering to the mammal an effective amount of a polypeptide according to any one of the embodiments described herein.

[0039] In another aspect, the present invention relates to a method for reducing viral shedding of cytomegalovirus in a mammal, the method comprising administering to the mammal an effective amount of a polypeptide according to any one of the embodiments described herein.

[0040] In another aspect, the present invention relates to compositions comprising polynucleotides capable of eliciting an immune response in a mammal. The polynucleotide encodes at least one polypeptide of interest, e.g., an antigen. The antigens disclosed herein may be wild-type (i.e., derived from an infectious agent) or, preferably, modified (e.g., genetically engineered, designed, or artificial). The nucleic acid molecules described herein, specifically polynucleotides, in some embodiments, encode one or more peptides or polypeptides of interest. Such peptides or polypeptides can serve as antigens or antigenic molecules. The term "nucleic acid" includes any compound comprising a polymer of nucleotides. These polymers are referred to as "polynucleotides." Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), including mRNA, and deoxyribonucleic acid (DNA).

[0041] In some embodiments, the composition comprises DNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition comprises RNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition comprises an mRNA polynucleotide encoding a polypeptide or fragment thereof described herein. Such a composition may result in the proper protein conformation upon translation.

[0042] In one aspect, the invention relates to a composition comprising at least one polynucleotide encoding a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB.

[0043] In some embodiments, the present invention relates to compositions comprising at least one polynucleotide encoding at least one hCMV gB polypeptide or an immunogenic fragment or epitope thereof.

[0044] In some embodiments, the composition comprises at least one polynucleotide encoding two or more additional polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the composition comprises two or more polynucleotides encoding two or more additional polypeptides, or immunogenic fragments or epitopes thereof. The one or more additional polypeptides can be encoded on a single polynucleotide or can be individually encoded on multiple (e.g., two or more) polynucleotides.

[0045] In another aspect, the present invention relates to a composition comprising (a) a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) an additional polypeptide, preferably an HCMV polypeptide, more preferably an HCMV antigenic polypeptide. The additional polypeptide can be selected from gH, gL, gB, gO, gN, and gM, and immunogenic fragments or epitopes thereof. In some embodiments, the additional polypeptide is pp65. In some embodiments, the additional polypeptide can be selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131A, and fragments thereof. In another aspect, the present invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) an additional polypeptide, preferably a polynucleotide encoding an HCMV antigenic polypeptide. The additional polypeptide can be selected from HCMV gH, gL, gB, gO, gN, and gM, and immunogenic fragments or epitopes thereof. In some embodiments, the additional polypeptide is HCMV pp65. In some embodiments, the additional polypeptide can be selected from HCMV gH, gL, gO, gM, gN, UL128, UL130, and UL131A, and fragments thereof.

[0046] In another aspect, the present invention relates to a method of inducing an immune response in a mammal, comprising administering to the mammal an effective amount of a composition for inducing an immune response, wherein the composition comprises a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV gB. The compositions disclosed herein can be formulated in an effective amount to generate an antigen-specific immune response in a mammal. [Brief explanation of the drawings]

[0047] [Figure 1A] Figure 1A depicts two-dimensional (2D) class averages of gB conformers. Figure 1A shows 2D projections from the post-fusion gB structure. Figure 1B shows a projection of the electron cryomicroscopy structure of post-fusion gB bound to antibody Fab. [Figure 1B] Figure 1B depicts two-dimensional (2D) class averages of gB conformers. Figure 1B shows the 2D class averages. Shown on the right are 2D class averages from electron cryomicroscopy images obtained from a preparation of gB extracted from CMV virions after treatment with fusion inhibitors and cross-linking agents and binding of antibody fragments. Class average images that do not resemble any of the reference post-fusion gB 2D projections are identified by circles. [Figure 2]Figure 1 lists glycoprotein B amino acids included in the pre-fusion and post-fusion gB-Fab complex models from our electron cryomicroscopy structures. Amino acids that can be modeled in the electron cryomicroscopy density map are highlighted using a domain color code (Domain I (italics only, i.e., top row sequence (pre-fusion) residues 133-344; bottom row sequence (post-fusion) residues 133-344), Domain II (bold and underlined, i.e., top row sequence (pre-fusion) residues 121-132 and 345-436; bottom row sequence (post-fusion) residues 121-132 and 345-439), Domain III (bold only, i.e., top row sequence (pre-fusion) residues 86-120 and 483-550; bottom row sequence ( (post-fusion) residues 86-120 and 474-550), domain IV (italic and underlined, i.e., upper row sequence (pre-fusion) residues 551-641, lower row sequence (post-fusion) residues 551-641), domain V (italic and bold, i.e., upper row sequence (pre-fusion) residues 642-724, lower row sequence (post-fusion) residues 642-697), MPR (underline only, i.e., upper row sequence (pre-fusion) residues 25-7507, lower row sequence (post-fusion) residues none), TM (italic, bold, and underlined, i.e., upper row sequence (pre-fusion) residues 751-769, lower row sequence (post-fusion) residues none). The upper and lower row sequences are from the pre-fusion and post-fusion structural models, respectively. [Figure 3A] (Figure 1) Fitting of a model to a density map. A model of the pre-fusion gB conformation stabilized with an inhibitor compound is fitted to a light gray density map. The gB component is in dark gray, and the SM5-1 fa b component is in black. The approximate location of the viral envelope, determined by the location of the TM region in the pre-fusion structure, is indicated by a black horizontal line. [Figure 3B] (Figure 1) Fitting of a model to a density map. A model of the post-fusion gB conformation stabilized with an inhibitor compound is fitted to a light gray density map. The gB component is in dark gray, and the SM5-1 fa b component is in black. The approximate location of the viral envelope, determined by the location of the TM region in the pre-fusion structure, is indicated by a black horizontal line. [Figure 4A]Figure 1 shows a comparison of the structures of gB in two conformations. The gB-stabilized pre-fusion structure is shown using one protomer, showing domains I, II, III, IV, V, MPR, and TM. The vertical black dashed lines extending from the top of the pre-fusion structure represent residues missing from the model due to a poorly defined density map. The overall dimensions of the buildable ectodomain portion of the structure are indicated by the dashed rectangle. The arrows indicate the direction pointed by the C-terminus of the central three-helix bundle in domain III in each conformation. The 115 Å dimension on the pre-fusion structure indicates the height of the modeled portion of the ectodomain. [Figure 4B] Figure 1 shows a comparison of the structures of gB in two conformations. The gB-stabilized post-fusion structure is shown using one protomer, showing domains I, II, III, IV, V, MPR, and TM. The vertical black dashed lines extending from the top of the pre-fusion structure represent residues missing from the model due to a poorly defined density map. The overall dimensions of the buildable ectodomain portion of the structure are indicated by the dashed rectangle. The arrows indicate the direction pointed by the C-terminus of the central three-helix bundle in domain III in each conformation. [Figure 5A] FIG. 1 shows the location of the fusion inhibitor compound N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide in a model of prefusion gB in black. [Figure 5B] Figure 1 shows a close-up of the electron density around the fusion inhibitor compound N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide (gray transparent surface). Nearby amino acid residues are indicated and domains are labeled. [Figure 5C] FIG. 1 shows interacting residues surrounding the fusion inhibitor compound N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide. [Figure 5D] FIG. 1 shows the chemical structure of the fusion inhibitor compound N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide. [Figure 6A] Figure 6A shows a model of the structural rearrangements of gB during membrane fusion. FL (and asterisk) - fusion loop. DI - domain 1. DII - domain 2. DV - domain 5. TM - transmembrane region. Light blue line indicates viral membrane. Lines indicate membrane. Figure 6A (pre-fusion) shows the pre-fusion conformation. [Figure 6B] Figure 6B shows a model of the structural rearrangements of gB during membrane fusion. FL (and asterisk) - fusion loop. DI - domain 1. DII - domain 2. DV - domain 5. TM - transmembrane region. Light blue lines indicate the viral membrane. Lines indicate membranes. Figure 6B (extended intermediate) shows the conformation of the extended intermediate. [Figure 6C] Figure 6B shows a model of the structural rearrangements of gB during membrane fusion. FL (and asterisk) - fusion loop. DI - domain 1. DII - domain 2. DV - domain 5. TM - transmembrane region. Light blue line indicates viral membrane. Lines indicate membrane. Figure 6C (post-fusion) shows the post-fusion conformation. [Figure 7A] 1 shows exemplary disulfide bond mutations to stabilize gB in the pre-fusion conformation. The positions of residues participating in disulfide bonds are shown as gray spheres in the pre-fusion conformation. [Figure 7B] 1 shows exemplary disulfide bond mutations to stabilize gB in the pre-fusion conformation. The positions of residues participating in disulfide bonds are shown as gray spheres in the post-fusion conformation. [Figure 8]Crystal structure of the extracellular domain of glycoprotein B from human cytomegalovirus (strain AD169), from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), file:5CXF, deposited on 2015-07-28, DOI:10.2210 / pdb5CXF / pdb. Unit cell:

[0048] [Table 1] [Figure 9]

[0023] Figures 110-111 show the sequences of gB from clinical and laboratory-adapted HCMV strains. Additional sequences can be found in the amino acid sequence alignment of gB from clinical and laboratory-adapted HCMV found in S4 Figure from Burke et al., PLoS Pathog., 2015 Oct. 20; 11(10):e1005227. According to Burke et al., 60 HCMV gB sequences from clinical and laboratory-adapted strains downloaded from NCBI's RefSeq database were aligned and analyzed using ClustalW2 and ESPript 3.x. In S4 Figure of Burke et al., identical residues are shown as white text on a red background, and similar residues are highlighted in yellow; said S4 Figure and its description are hereby incorporated by reference in their entirety. [Figure 10] FIG. 1 shows the amino acid sequences of SEQ ID NOs: 1 to 43 and SEQ ID NOs: 47 to 106. [Figure 11]

[0033] Figure 1 shows dose-dependent IgG responses in mice immunized with both gB1666 and wild-type gB (Towne). The graph shows that 10 of 10 mice immunized with wild-type gB DNA and 9 of 10 mice immunized with gB1666 DNA developed detectable anti-gB IgG titers. Mean ± SD, LLOQ = 25. [Figure 12]The structural model of the engineered gB1666 (light gray, structure code: P-GB-002) is overlaid with the structural model of wild-type HCMV gB (dark gray, structure code: P-GB-001). The new structure allows for modeling of additional residues 437–448 and 478–482 at the membrane-distal end of the molecule and residues 770–779 in the transmembrane domain. [Figure 13] Figure 1 shows an example of a combination of additional mutations on the pSB1666 background that can further stabilize pre-fusion gB. A disulfide bond at M371, W506 can link domains II and III. Disulfide bonds at N524, M684, and F541, E681 can link domains IV and V. Mutation of the negatively charged patch at E686 to hydrophobic residues can further stabilize gB in the pre-fusion conformation. The respective domains are identified. Abbreviations: membrane proximal region (MPR) and transmembrane domain (TMD). [Figure 14] This image shows an SDS-PAGE analysis documenting the expression and purification of recombinant gB2459 protein. The pSB2459 expression plasmid was transiently transfected into Expi293F cells. Cell pellets were harvested 68 h posttransfection, and the glycoprotein product, gB2459, was purified through a series of solubilization, affinity, and size-exclusion chromatography steps in 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% n-dodecyl β-D-maltoside (DDM), and 0.002% cholesteryl hemisuccinate (CHS). This figure shows the purified protein analyzed by stain-free 4-20% SDS-PAGE under reducing conditions. The smearing of the protein band is consistent with gB2459 being heavily glycosylated. Lane M: protein marker, lane 1: gB2457, and lane 2: gB2459. [Figure 15]This image shows the construct pSB2459, which contains the N524C and M684C mutations on a pSB1666 background. The protein product, gB2459, was purified through affinity tag analysis without any fusion inhibitors. These are the pre-fusion classes observed in the 2D class average images (two classes with clear pre-fusion features are numbered 1 and 2). Furthermore, pre-fusion gB2459 is stable over a period of several days. A sample solution of gB2459 was stored at 4°C, and aliquots of the sample were obtained on days 1 and 7 to prepare negatively stained grids. Image datasets were collected and processed on these two grids. For each dataset, the particle populations in the pre-fusion and post-fusion 2D classes were counted. The ratio of pre-fusion to post-fusion conformations was 5:1 for the day 1 sample and 3:1 for the day 7 sample. [Figure 16A] Figure 16B shows the design of a soluble, detergent-free gB ectodomain. The gB ectodomain (1-707) with the MPR, TM, and CT regions removed is shown. Legend: Domain I (residues 134-344) - dark gray 3D volume structure; Domain II (residues 121-133 and 345-436) - light gray 3D volume structure; Domain III (residues 97-111, 475-539, and 640-648) - light gray vertical coil in the top center; Domain V (residues 649-707) - dark gray internal coil in the bottom (see arrow in Figure 16B), and rectangle - position of trimerization. [Figure 16B] 16B shows the design of a soluble, detergent-free gB ectodomain. The gB ectodomain is stabilized with additional cysteine ​​mutations in domain V, e.g., D703C and P704C. Legend: Domain I (residues 134-344)—3D volume structure in dark gray; Domain II (residues 121-133 and 345-436)—3D volume structure in light gray; Domain III (residues 97-111, 475-539, and 640-648)—top center light gray vertical coil; Domain V (residues 649-707)—bottom interior dark gray coil (see arrow in FIG. 16B), and rectangle—position of trimerization. [Figure 16C]Figure 16B shows the design of a soluble, detergent-free gB ectodomain. The gB ectodomain is shown fused to a C-terminal GCN4 trimerization motif. Legend: Domain I (residues 134-344) - dark gray 3D volume structure; Domain II (residues 121-133 and 345-436) - light gray 3D volume structure; Domain III (residues 97-111, 475-539, and 640-648) - light gray vertical coil in the top center; Domain V (residues 649-707) - dark gray internal coil in the bottom (see arrow in Figure 16B), and rectangle - location of trimerization. [Figure 16D] Figure 16B shows the design of a soluble, detergent-free gB ectodomain. The gB ectodomain is shown fused to the C-terminal T4 fibritin foldon domain. Legend: Domain I (residues 134-344) - dark gray 3D volume structure; Domain II (residues 121-133 and 345-436) - light gray 3D volume structure; Domain III (residues 97-111, 475-539, and 640-648) - light gray vertical coil in the top center; Domain V (residues 649-707) - dark gray internal coil in the bottom (see arrow in Figure 16B), and rectangle - position of trimerization. [Figure 17] 1 is a graph showing the gel filtration profile of purified gB ectodomains gB2264-gB2269 analyzed on Superose 6 Increase 10 / 300 in 20 mM HEPES pH 7.5, 250 mM NaCl. [Figure 18A] Image showing negative stain EM of recombinant gB2555 without bound fusion inhibitors, demonstrating that the monodisperse gB protein is suitable for use as a framework to add mutations that make it more stabilizing relative to the pre-fusion form of gB in the absence of inhibitors and detergents. [Figure 18B] Representative 2D class average images from negative stain EM of recombinant gB2555 without bound fusion inhibitors, demonstrating that the monodisperse gB protein is suitable for use as a framework for adding mutations that make it more stabilizing to the pre-fusion form of gB in the absence of inhibitors and detergents. [Figure 19A] Image showing negative stain EM of recombinant gB2556 without bound fusion inhibitors, demonstrating that the monodisperse gB protein is suitable for use as a framework to add mutations that make it more stabilizing to the pre-fusion form of gB in the absence of inhibitors and detergents. [Figure 19B] Representative 2D class average images from negative stain EM of recombinant gB2556 without bound fusion inhibitors, demonstrating that the monodisperse gB protein is suitable for use as a framework for adding mutations that make it more stabilizing to the pre-fusion form of gB in the absence of inhibitors and detergents. [Figure 20A] Image showing purified proteins analyzed on a 4-20% Mini-PROTEAN® TGX STAIN-FREE® protein gel (Biorad) in Tris / glycine / SDS buffer under reducing conditions (arrows indicate proteins on the gel). [Figure 20B] 1 is an image showing negatively stained proteins by electron microscopy. [Figure 21] 1 is a graph showing the dose-dependent IgG response to gB in mice immunized with both gB2796 and Sanofi gB. [Figure 22A] 1 is a graph showing a comparison of the SEC profiles between gB1666 and gB1764 (wild-type CMVgB) (SEC performed on a Superose6 increase 5 / 150 column in a buffer of 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS, 3 μg / ml WAY-174865). [Figure 22B] 1 is a graph showing a comparison of thermal stability between gB1666 and gB1764 (assays performed on a Tycho NT.6 using a heating rate of 20° C. / min). [Figure 23A]1 is a graph showing the SEC profile of gB1666 with and without inhibitor WAY-174865 (SEC performed on a Superose6 increase 5 / 150 column in a buffer of 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS, 3 μg / ml WAY-174865). [Figure 23B] FIG. 1 shows a comparison of thermal stability between gB1666, gB1666 and inhibitors and gB1764 (assays performed on a Tycho NT.6 using a heating rate of 20° C. / min). [Figure 24A] 1 is a graph showing a comparison of the SEC profiles of GB2457 and gB2459 (SEC performed on a Superose6 increase 5 / 150 column in a buffer of 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS). [Figure 24B] 1 is a graph showing a comparison of thermal stability between gB2457 and gB2459 (assays performed on a Tycho NT.6 using a heating rate of 20° C. / min). [Figure 25A] 1 is a graph showing a comparison of the SEC profiles of GB2555 and gB2556 (SEC performed on a Superose6 increase 5 / 150 column in a buffer of 20 mM HEPES pH 7.5, 250 mM NaCl). [Figure 25B] 1 is a graph showing a comparison of thermal stability between gB2555 and gB2556 (assays performed on a Tycho NT.6 using a heating rate of 20° C. / min). [Figure 25C] This image shows 2D classes averaged from negative-stain EM images of gB2555. A representative class of pre-fusion gB (tapered shape) is shown as a circle, and post-fusion gB is shown as a square. [Figure 25D] This image shows 2D classes averaged from negative-stain EM images of gB2556. Representative classes of pre-fusion gB (tapered shape) are shown as circles, and post-fusion gB are shown as squares. [Figure 26A]1 is a graph showing a comparison of the SEC profile of GB2796 (SEC performed on a Superose6 increase 5 / 150 column in a buffer of 20 mM HEPES pH 7.5, 250 mM NaCl). [Figure 26B] 1 is a graph showing the thermal stability of gB2796 (assay performed on a Tycho NT.6 using a heating rate of 20° C. / min). DETAILED DESCRIPTION OF THE INVENTION

[0049] Sequence Identifier SEQ ID NO: 1 sets forth the amino acid sequence derived from native HCMV gB (Towne strain).

[0050] SEQ ID NO:2 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Q98C, G271C.

[0051] SEQ ID NO:3 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Q98C, I653C.

[0052] SEQ ID NO:4 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: G99C, A267C.

[0053] SEQ ID NO:5 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T100C, A267C.

[0054] SEQ ID NO:6 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T100C, S269C.

[0055] SEQ ID NO:7 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T100C, L651C.

[0056] SEQ ID NO:8 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: D217C, F584C.

[0057] SEQ ID NO:9 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Y218C, A585C.

[0058] SEQ ID NO: 10 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: S219C, D654C.

[0059] SEQ ID NO:11 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: N220C, D652C.

[0060] SEQ ID NO: 12 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: T221C, D652C.

[0061] SEQ ID NO: 13 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: W240C, G718C.

[0062] SEQ ID NO: 14 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: Y242C, K710C.

[0063] SEQ ID NO: 15 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: Y242C, D714C.

[0064] SEQ ID NO: 16 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: S269C, I653C.

[0065] SEQ ID NO: 17 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: G271C, P614C.

[0066] SEQ ID NO: 18 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: S367C, L499C.

[0067] SEQ ID NO: 19 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutations: T372C, W506C.

[0068] SEQ ID NO:20 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: F541C, Q669C.

[0069] SEQ ID NO:21 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: L548C, A650C.

[0070] SEQ ID NO:22 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: A549C, I653C.

[0071] SEQ ID NO:23 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S550C, D652C.

[0072] SEQ ID NO:24 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: G604C, F661C.

[0073] SEQ ID NO:25 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: N605C, E665C.

[0074] SEQ ID NO:26 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: R607C, S675C.

[0075] SEQ ID NO:27 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T608C, D679C.

[0076] SEQ ID NO:28 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: E609C, F678C.

[0077] SEQ ID NO:29 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: R673C, S674C.

[0078] SEQ ID NO:30 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: N676C, V677C.

[0079] SEQ ID NO:31 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: L680C, E681C.

[0080] SEQ ID NO:32 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: I683C, M684C.

[0081] SEQ ID NO:33 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: F687C, N688C.

[0082] SEQ ID NO:34 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Y690C, K691C.

[0083] SEQ ID NO:35 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: K695C, K724C.

[0084] SEQ ID NO:36 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T746C, F747C.

[0085] SEQ ID NO:37 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: K749C, N750C.

[0086] SEQ ID NO:38 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: K670L.

[0087] SEQ ID NO:39 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: K670F.

[0088] SEQ ID NO:40 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: R673L.

[0089] SEQ ID NO:41 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: R673F.

[0090] SEQ ID NO:42 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: K691L.

[0091] SEQ ID NO:43 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: K691F.

[0092] SEQ ID NO: 44 sets forth the amino acid sequence of native HCMV gB (AD169, PDB:5CXF) that folds into the post-fusion conformation when expressed.

[0093] SEQ ID NO: 45 sets forth the amino acid sequence of an HCMV gB variant (gB705) that folds into the post-fusion conformation when expressed.

[0094] SEQ ID NO: 46 sets forth the amino acid sequence of native HCMV gB (Merlin strain) that folds into the post-fusion conformation when expressed.

[0095] SEQ ID NO:47 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: M96C and D660C.

[0096] SEQ ID NO:48 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Q98C and N658C.

[0097] SEQ ID NO:49 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T100C and R258C.

[0098] SEQ ID NO:50 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T100C and L656C.

[0099] SEQ ID NO:51 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: T100C and N658C.

[0100] SEQ ID NO:52 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: I117C and T406C.

[0101] SEQ ID NO:53 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: I117C and S407C.

[0102] SEQ ID NO:54 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: Y153C and L712C.

[0103] SEQ ID NO:55 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: L162C and M716C.

[0104] SEQ ID NO:56 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: D217C and S587C.

[0105] SEQ ID NO:57 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: D217C and Y589C.

[0106] SEQ ID NO:58 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S219C and F584C.

[0107] SEQ ID NO:59 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S219C and A585C.

[0108] SEQ ID NO:60 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S219C and N586C.

[0109] SEQ ID NO:61 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: N220C and T659C.

[0110] SEQ ID NO:62 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S223C and T659C.

[0111] SEQ ID NO:63 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: W240C and A732A.

[0112] SEQ ID NO:64 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: W240C and G735C.

[0113] SEQ ID NO:65 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: Y242C and V728C.

[0114] SEQ ID NO:66 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: Y242C and G731C.

[0115] SEQ ID NO:67 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: R258C and L656C.

[0116] SEQ ID NO:68 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S269C and L656C.

[0117] SEQ ID NO:69 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S269C and N658C.

[0118] SEQ ID NO:70 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: D272C and P614C.

[0119] SEQ ID NO:71 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: V273C and V629C.

[0120] SEQ ID NO:72 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: W349C and A650C.

[0121] SEQ ID NO:73 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S367C and A500C.

[0122] SEQ ID NO:74 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S367C and A503C.

[0123] SEQ ID NO:75 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: K370C and Q501C.

[0124] SEQ ID NO:76 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: K522C and I683C.

[0125] SEQ ID NO:77 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: I523C and I683C.

[0126] SEQ ID NO:78 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: I523C and M684C.

[0127] SEQ ID NO:79 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: N524C and M684C.

[0128] SEQ ID NO:80 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: P525C and E681C.

[0129] SEQ ID NO:81 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: R540C and L680C.

[0130] SEQ ID NO:82 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: F541C and L680C.

[0131] SEQ ID NO:83 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: L548C and P655C.

[0132] SEQ ID NO:84 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: A549C and N658C.

[0133] SEQ ID NO:85 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S550C and P655C.

[0134] SEQ ID NO:86 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: S550C and E657C.

[0135] SEQ ID NO:87 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Q591C and S668C.

[0136] SEQ ID NO:88 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: L603C and Y667C.

[0137] SEQ ID NO:89 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: G604C and L672C.

[0138] SEQ ID NO:90 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: R607C and N688C.

[0139] SEQ ID NO:91 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: T608C and Q692C.

[0140] SEQ ID NO:92 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: E609C and K691C.

[0141] SEQ ID NO:93 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: E610C and S674C.

[0142] SEQ ID NO:94 sets forth the amino acids of SEQ ID NO:1 containing the following mutations: E610C and S675C.

[0143] SEQ ID NO:95 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: Q612C and V663C.

[0144] SEQ ID NO:96 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: V737C and F755C.

[0145] SEQ ID NO:97 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: V741C and A754C.

[0146] SEQ ID NO:98 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutations: V741C and F755C.

[0147] SEQ ID NO:99 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: D679S.

[0148] SEQ ID NO: 100 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutation: D679N.

[0149] SEQ ID NO:101 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: E682S.

[0150] SEQ ID NO: 102 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutation: E682Q.

[0151] SEQ ID NO: 103 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutation: E686S.

[0152] SEQ ID NO: 104 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutation: E686Q.

[0153] SEQ ID NO:105 sets forth the amino acid sequence of SEQ ID NO:1 containing the following mutation: N118P.

[0154] SEQ ID NO: 106 sets forth the amino acid sequence of SEQ ID NO: 1 containing the following mutation: D646P.

[0155] SEQ ID NO: 107 sets forth the amino acid sequence of >5CXF:A|PDBID|strand|sequence in Figure 8.

[0156] SEQ ID NO: 108 sets forth the amino acid sequence of >5CXF:B|PDBID|strand|sequence in Figure 8.

[0157] SEQ ID NO: 109 sets forth the amino acid sequence of >5CXF:C|PDBID|chain|sequence in Figure 8.

[0158] SEQ ID NO: 110 sets forth the amino acid sequence of the gB polypeptide derived from the complete genome reverse complement sequence of human herpesvirus 5 strain HAN13, HAN13 gi|242345614|gb|GQ221973.1|:81988-84705, referred to in the description of Figure 9 .

[0159] SEQ ID NO: 111 sets forth the amino acid sequence of the gB polypeptide derived from the complete reverse complement sequence of human herpesvirus 5 strain VR1814 genome, VR1814 gi|270355759|gb|GU179289.1|:81925-84642, referred to in the description of Figure 9 .

[0160] SEQ ID NOs: 112-140 set forth the amino acid sequences of gB polypeptides from various CMV gB strains listed in FIG.

[0161] SEQ ID NO: 141 to SEQ ID NO: 210 set forth polynucleotide sequences encoding polypeptides derived from HCMV, such as gH, gL, UL128, UL130, UL131, gB, or pp65.

[0162] SEQ ID NO: 211 to SEQ ID NO: 223 set forth the amino acid sequences of polypeptides derived from HCMV, such as gH, gL, UL128, UL130, UL131, gB, or pp65.

[0163] SEQ ID NO: 224 sets forth the amino acid sequence of a polypolypeptide derived from HCMV.

[0164] SEQ ID NO: 225 to SEQ ID NO: 254 set forth polynucleotide sequences encoding polypolypeptides derived from HCMV.

[0165] SEQ ID NO:255 sets forth the amino acid sequence of CMV gB1666, residues V23 to V907 of SEQ ID NO:1 (Towne), with the following mutations: D217C, Y589C, and I675S, without the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0166] SEQ ID NO:256 sets forth the nucleic acid sequence of CMV gB1666, which encodes residues V23 to V907 of SEQ ID NO:1 (Towne) with the following mutations: D217C, Y589C, and I675S, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0167] SEQ ID NO:257 sets forth the amino acid sequence of CMV gB2457 (pre-fusion, full-length), residues V23 to V907 of SEQ ID NO:1 (Towne), with the following mutations: D217C, M371C, W506C, Y589C, and I675S, without the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0168] SEQ ID NO:258 sets forth the nucleic acid sequence of CMV gB2457 (pre-fusion, full-length), encoding residues V23 to V907 of SEQ ID NO:1 (Towne) with the following mutations: D217C, M371C, W506C, Y589C, and I675S, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0169] SEQ ID NO:259 sets forth the amino acid sequence of CMV gB2459 (pre-fusion, full-length), residues V23 to V907 of SEQ ID NO:1 (Towne), with the following mutations: D217C, N524C, Y589C, M684C, and I675S, without the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0170] SEQ ID NO:260 sets forth the nucleic acid sequence of CMV gB2459 (pre-fusion, full-length) encoding residues V23 to V907 of SEQ ID NO:1 (Towne) with the following mutations: D217C, N524C, Y589C, M684C, and I675S, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0171] SEQ ID NO: 261 sets forth the amino acid sequence of CMV gB2555 (pre-fusion, including the trimerization domain (GCN4 CC tri2)), residues V23 to V702 of SEQ ID NO: 1 (Towne), with the following mutations: YIH to GHR(155-157), D217C, W240A, M371C, C246S, W506C, Y589C, and I675S, but excluding the signal sequence (residues M1 to A22 of SEQ ID NO: 1).

[0172] SEQ ID NO:262 sets forth the nucleic acid sequence of CMV gB2555 (pre-fusion, including the trimerization domain (GCN4 CC tri2)) encoding residues V23 to V702 of SEQ ID NO:1 (Towne) with the following mutations: YIH to GHR(155-157), D217C, W240A, M371C, C246S, W506C, Y589C, and I675S, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0173] SEQ ID NO: 263 sets forth the amino acid sequence of CMV gB2556 (pre-fusion, including the trimerization domain (GCN4 CC tri2)), residues V23 to V702 of SEQ ID NO: 1 (Towne), with the following mutations: YIH to GHR(155-157), D217C, W240A, C246S, N524C, Y589C, I675S, and M684C, but excluding the signal sequence (residues M1 to A22 of SEQ ID NO: 1).

[0174] SEQ ID NO:264 sets forth the nucleic acid sequence of CMV gB2556 (pre-fusion, including the trimerization domain (GCN4 CC tri2)), encoding residues V23 to V702 of SEQ ID NO:1 (Towne) with the following mutations: YIH to GHR(155-157), D217C, W240A, C246S, N524C, Y589C, I675S, and M684C, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0175] SEQ ID NO: 265 sets forth the amino acid sequence of CMV gB2796 (pre-fusion, ectodomain), residues V23 to D646 of SEQ ID NO: 1 (Towne), with the following mutations: YIH to GHR(155-157), D217C, W240A, C246S, M371C, W506C, and Y589C, excluding the signal sequence (residues M1 to A22 of SEQ ID NO: 1).

[0176] SEQ ID NO:266 sets forth the nucleic acid sequence of CMV gB2796 (pre-fusion, ectodomain) encoding amino acids V23 to D646 of SEQ ID NO:1 (Towne) with the following amino acid mutations: YIH to GHR(155-157), D217C, W240A, C246S, M371C, W506C, and Y589C, excluding nucleotides encoding the signal sequence (residues M1 to A22 of SEQ ID NO:1).

[0177] SEQ ID NO: 267 sets forth the CMV gB ectodomain, V23 to P707 of SEQ ID NO: 1 (Towne strain).

[0178] SEQ ID NO: 268 sets forth the amino acid sequence of the signal sequence of wt HCMV gB (Towne).

[0179] SEQ ID NO: 269 sets forth the amino acid sequence of the GCN4 CC tri2 trimerization domain (see Table 9).

[0180] SEQ ID NO: 270 sets forth the nucleic acid sequence encoding the GCN4 CC tri2 trimerization domain.

[0181] SEQ ID NO: 271 sets forth the amino acid sequence of the T4 fibritin foldon domain (see Table 9).

[0182] SEQ ID NOs: 272-273 set forth the amino acid sequences of various GCN4 trimerization domains.

[0183] SEQ ID NO: 274 sets forth the amino acid sequence of the C-terminal fusion sequence set forth in Table 9.

[0184] Detailed Description As described herein, the inventors have solved the three-dimensional structure of the HCMV glycoprotein B (gB) polypeptide in a conformation they refer to as the pre-fusion conformation, as distinct from the post-fusion conformation. Mutations have also been discovered that stabilize the polypeptide in the pre-fusion conformation. This structure can be used to generate a higher HCMV neutralizing antibody response than that achieved with previous HCMV gB-based immunogens. The polypeptides described herein, and nucleic acids encoding the polypeptides, can be used, for example, as potential immunogens in vaccines against HCMV, and as diagnostic tools, among other uses.

[0185] The inventors have further discovered mutations that can be introduced into cytomegalovirus (CMV) gB polypeptides that, inter alia, can greatly facilitate the production and subsequent purification of gB antigens stabilized in the pre-fusion conformation; can significantly improve the efficiency of production of gB polypeptides in the pre-fusion conformation; can alter the antigenicity of gB polypeptides compared to wild-type gB polypeptides; can promote a focused immune response against pre-fusion gB; and can reduce and / or eliminate steric blockage of neutralizing epitopes of gB.

[0186] definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes singular or multiple antigens and can be considered equivalent to the phrase "at least one antigen."

[0187] As used herein, the term "additional mutations" includes, but is not limited to, amino acid substitutions, including conservative substitutions, that introduce electrostatic mutations, that fill cavities, that alter residue packing, that introduce N-linked glycosylation sites, that introduce interprotomer disulfide bonds, and combinations thereof, relative to native HCMV gB. Examples of "additional mutations" can be found throughout this specification, but are most specifically described in Tables 3 and 4 and in the Examples.

[0188] The term "adjuvant" refers to a substance that can enhance, promote, or prolong the body's immune response to an immunogen or immunogenic composition, such as a vaccine (but is not itself immunogenic). Adjuvants can be included in immunogenic compositions, such as vaccines, or administered separately from the immunogenic composition.

[0189] The term "administration" refers to the introduction of a substance or composition into a subject by a selected route. Administration may be local or systemic. For example, if the selected route is intramuscular, a composition (such as a composition comprising a disclosed immunogen) is administered by introducing the composition into the muscle of the subject.

[0190] The term "antigen" refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants, such as those recognized by immune cells.

[0191] The term "conservative substitution" refers to the replacement of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0192] The term "degenerate variant" of a reference polynucleotide refers to a polynucleotide that differs in nucleotide sequence from the reference polynucleotide but encodes the same polypeptide sequence as that encoded by the reference polynucleotide. There are 20 naturally occurring amino acids, many of which are specified by more than one codon. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at all positions where arginine is specified in a protein-coding sequence, the codon can be changed to any of the corresponding codons listed without changing the encoded protein. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide.

[0193] The term "effective amount" refers to the amount of an agent sufficient to produce a desired response, for example, the amount necessary to inhibit viral replication or measurably alter the outward symptoms of a viral infection.

[0194] The term "epitope" (or "antigenic determinant" or "antigenic site") refers to the region of an antigen to which an antibody, B-cell receptor, or T-cell receptor binds or responds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by secondary, tertiary, or quaternary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by higher order folding are typically lost upon treatment with denaturing solvents.

[0195] The term "subject" refers to either human or non-human mammal. The term "mammal" refers to any animal species of the mammalian class. Examples of mammals include humans; non-human primates such as monkeys; laboratory animals such as rats, mice, and guinea pigs; livestock animals such as cats, dogs, rabbits, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0196] The term "glycoprotein" refers to a protein containing oligosaccharide chains (glycans) covalently attached to polypeptide side chains. Carbohydrates are attached to proteins in a co- or post-translational modification known as glycosylation. The term "glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, that provides for the attachment of a glycan. An N-linked glycosylation site is a triplet sequence, N-X(S / T), where N is asparagine, X is any residue except proline, and (S / T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan can also be used to refer to the carbohydrate moiety of a glycoconjugate, such as a glycoprotein, glycolipid, or proteoglycan.

[0197] The term "host cell" refers to a cell in which a vector can be propagated and its DNA or RNA expressed. The cells may be prokaryotic or eukaryotic.

[0198] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum identity, are the same or have a specified percentage of identical amino acid residues or nucleotides. Methods for aligning sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence described in the identified sequence or by the combined length (such as 100 consecutive nucleotides or amino acid residues from the sequence described in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166 ÷ 1554 × 100 = 75.0).

[0199] Optimal alignment of sequences for comparison can be determined, for example, by the partial homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443, 1970, by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, Supplement 104, 2013).

[0200] The term "immunogen" refers to a compound, composition, or substance that is immunogenic, as defined herein below.

[0201] The term "immunogenicity" refers to the ability of a substance, with or without an adjuvant, to provoke, elicit, stimulate, or induce an immune response in a subject against a particular antigen.

[0202] The term "immune response" refers to any detectable response of a cell or cells of the immune system of a host mammal to a stimulus (such as an immunogen), including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as the production and secretion of antibodies into plasma, lymph, and / or tissue fluids). Examples of immune responses include Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th2, or Th17-type cytokine) or chemocin) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody production and / or secretion), binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte ("CTL") response, altered (e.g., increased) induction of a B cell response (e.g., antibody production), and expansion of cells of the immune system (e.g., T cells and B cells) (e.g., proliferation of cell populations), and increased antigen processing and presentation by antigen-presenting cells. The term "immune response" also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of a vertebrate's immune system in vitro.

[0203] The term "immunogenic composition" refers to a composition that includes an immunogen.

[0204] The term "mutation" refers to the deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout this specification and claims, an amino acid substitution at one specific position in a protein sequence is referred to using the notation "(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)." For example, the notation Y75A refers to the substitution of an alanine (A) residue for a tyrosine (Y) residue at position 75 of the amino acid sequence of the reference protein (in a mutant of the reference protein). If there is a variation in the amino acid residue at the same position between different wild-type sequences, the amino acid code before the position number, such as "75A," may be omitted in the notation.

[0205] The term "native" or "wild-type" protein, sequence, or polypeptide refers to a naturally occurring protein, sequence, or polypeptide that has not been artificially modified by selective mutation.

[0206] The term "pharmaceutically acceptable carrier" refers to a material or composition that, when combined with an active ingredient, is compatible with the active ingredient and does not cause toxic or otherwise undesirable reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffers, lubricants, emulsifiers, absorbents, dispersion media, coatings, and stabilizers.

[0207] The term "pre-fusion specific antibody" refers to an antibody that specifically binds to the CMV gB glycoprotein in the pre-fusion conformation, but does not bind to the CMV gB protein in the post-fusion conformation.

[0208] The term "pre-fusion trimer-specific antibody" refers to an antibody that specifically binds to the CMV gB glycoprotein in the pre-fusion trimer conformation, but does not bind to the CMV gB protein in the post-fusion conformation or in a pre-fusion conformation that is not trimeric. "Pre-fusion trimer-specific antibodies" are a subset of "pre-fusion specific antibodies."

[0209] The term "prime-boost vaccination" refers to an immunotherapy regimen that includes administering a first immunogenic composition (primer vaccine) to a subject to induce an immune response, followed by administering a second immunogenic composition (booster vaccine).The primer vaccine and the booster vaccine typically contain the same immunogen and are provided in the same or similar format.However, they can also be provided in different formats, for example, one in the form of a vector and the other in the form of a naked DNA plasmid.Those skilled in the art will understand the appropriate time interval between the administration of the primer vaccine and the booster vaccine.In addition, the primer vaccine, the booster vaccine, or both the primer vaccine and the booster vaccine further comprise an adjuvant.

[0210] The term "soluble protein" refers to a protein that can dissolve and remain dissolved in an aqueous liquid. The solubility of a protein can vary depending on the concentration of the protein in the water-based liquid, the buffering state of the liquid, the concentration of other solutes in the liquid, such as salt and protein concentrations, and the temperature of the liquid.

[0211] The term "specifically binds" in the context of antibody binding to a given target molecule refers to the antibody binding to the target molecule with higher affinity than its binding to other test substances. For example, an antibody that specifically binds to CMV gB protein in the pre-fusion conformation is an antibody that binds to CMV gB protein in the pre-fusion conformation with higher affinity than it binds to CMV gB protein in the post-fusion conformation.

[0212] The term "therapeutically effective amount" refers to an amount of an agent sufficient to prevent, treat (including prophylaxis), reduce, and / or ameliorate the symptoms and / or underlying causes of a disorder.

[0213] The term "vaccine" refers to a pharmaceutical composition comprising an immunogen capable of eliciting a prophylactic or therapeutic immune response in a subject. Typically, a vaccine elicits an antigen-specific immune response against an antigen of a pathogen, e.g., a viral pathogen.

[0214] The term "vector" refers to a nucleic acid molecule capable of transporting or transferring a foreign nucleic acid molecule. This term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector capable of expressing an insert in a target cell and generally contains control sequences, such as enhancer, promoter, and terminator sequences, that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. The foreign nucleic acid molecule is referred to as an "insert" or "transgene." A vector generally consists of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or origin of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors, and artificial chromosomes.

[0215] Natural HCMV gB Native HCMV gB is synthesized as a 906 or 907 amino acid polypeptide (depending on the CMV strain) that undergoes extensive post-translational modifications, including glycosylation at N- and O-linked sites and cleavage by ubiquitous cellular endoproteases into amino- and carboxy-terminal fragments. The N- and C-terminal fragments of gB, gp116, and gp55, respectively, are covalently linked by disulfide bonds, and mature glycosylated gB adopts a trimeric configuration. The gB polypeptide contains a large extracellular domain (cleaved into the extracellular domains of gp116 and gp55), a transmembrane domain (TM), and an intraviral (or cytoplasmic) domain (cytoplasmic domain).

[0216] Native HCMV gB sequences from various strains are known. For example, at least 60 HCMV gB sequences from clinical and laboratory-adapted strains are available from NCBI's RefSeq database, as described in Burke et al., "Crystal Structure of the Human Cytomegalovirus Glycoprotein B," PLoS Pathog. 2015 Oct 20;11(10):e1005227, S4 Fig, which is incorporated herein by reference in its entirety.

[0217] Thus, as used herein, the term "CMV gB" polypeptide or "HCMV gB" polypeptide should be understood to refer to a native HCMV gB polypeptide derived from any human HCMV strain, including but not limited to the Towne strain. Depending on the actual sequence alignment, it may be necessary to adjust the actual residue position numbers for gB derived from other human CMV strains. However, one of skill in the art would understand how to align sequences from different strains from one strain to another to identify corresponding residue positions.

[0218] HCMV gB is encoded by the UL55 gene of the HCMV genome. It is an envelope glycoprotein that mediates fusion of the HCMV viral membrane with the host cell membrane. The protein undergoes a series of conformational changes from a pre-fusion to a post-fusion form. A crystal structure of gB in the post-fusion form is available (PDB accession code 5CXF), and the pre-fusion conformation is described herein.

[0219] Conformation The HCMV gB post-fusion conformation refers to the structural conformation adopted by HCMV gB after the fusion of the viral envelope with the host cell membrane. Native HCMV gB can also adopt a post-fusion conformation outside the context of the fusion event, for example, under stress conditions such as exposure to heat, extraction from the membrane, expression as an extracellular domain, or storage. More specifically, the gB post-fusion conformation is described, for example, in Burke et al., Crystal Structure of the Human Cytomegalovirus Glycoprotein B, PLoS Pathog. 2015 Oct. 20; 11(10): e1005227. See also Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB):5CXF, Crystal structure of the extracellular domain of glycoprotein B from Human Cytomegalovirus, from Human cytomegalovirus (strain AD169), deposited 2015-07-28; DOI:10.2210 / pdb5CXF / pdb; and Burke et al., PLoS Pathog. 2015 Oct 20;11(10):e1005227. The sequence of a protein that can fold into a post-fusion conformation when expressed is provided as SEQ ID NO:44. Another example of a protein that folds into a post-fusion conformation when expressed is provided as SEQ ID NO:45. The post-fusion conformation is approximately 165 Å high and 65 Å wide.

[0220] As used herein, "pre-fusion conformation" refers to a structural conformation adopted by a polypeptide that differs from the HCMV gB post-fusion conformation, at least in terms of molecular dimensions or three-dimensional coordinates. The pre-fusion conformation refers to the structural conformation adopted by HCMV gB before the induction of a fusion event that results in the transition of gB to the post-fusion conformation. Isolation of HCMV gB in a stable pre-fusion conformation may be useful in informing and directing the improvement of vaccines and immunogenic compositions to address the important public health problem of cytomegalovirus infection. In some embodiments, the pre-fusion conformation comprises a conformation capable of binding to a pre-fusion-specific antibody. In some embodiments, the pre-fusion conformation comprises a conformation characterized by the coordinates set forth in Table 1A, the entire contents of which are incorporated herein by reference. In some embodiments, the polypeptide is characterized by structural coordinates that include the root mean square deviation (RMSD) of the backbone atoms of conserved residues when superimposed onto the backbone atoms described by the structural coordinates set forth in Table 1A. In some embodiments, the pre-fusion conformation comprises a conformation characterized by the coordinates set forth in Table 1B, which is incorporated herein by reference in its entirety. In some embodiments, the polypeptide is characterized by structural coordinates comprising the root mean square deviation (RMSD) of the backbone atoms of conserved residues when superimposed onto the backbone atoms described by the structural coordinates set forth in Table 1B. In some embodiments, a polypeptide having an HCMV gB pre-fusion conformation refers to a polypeptide comprising a trimeric helix bundle centered on the three-fold axis of the trimer and comprising residues L479 to K522 of each protomer, with the direction of the bundle from the N-terminus to the C-terminus along the three-fold axis (indicated by the arrow in Figures 4A and 4B) directed toward a point on the three-fold axis that intersects with a plane defined by residue W240 of each protomer, located in the fusion loop near the tip of each domain I of the trimer. In some embodiments, the helix bundle comprises residues between L479 and K522 according to the numbering of SEQ ID NO: 1.

[0221] Polypeptides of the Invention The present invention relates to polypeptides that contain amino acid mutations compared to the corresponding amino acid sequence of wild-type HCMV gB. The amino acid mutations include amino acid substitutions, deletions, or additions compared to wild-type HCMV gB. Thus, the polypeptides are mutants of wild-type HCMV gB.

[0222] In some embodiments, the polypeptide has certain beneficial characteristics, such as being immunogenic. In some embodiments, the polypeptide has increased immunogenic properties or improved stability in the pre-fusion conformation compared to the corresponding wild-type HCMV gB. Stability refers to the degree to which the transition from pre-fusion to post-fusion HCMV gB conformation is inhibited or prevented. In still other embodiments, the present disclosure provides polypeptides exhibiting one or more introduced mutations described herein, which may result in improved stability in the pre-fusion conformation. The introduced amino acid mutations in HCMV gB include amino acid substitutions, deletions, or additions. In some embodiments, the only mutation in the amino acid sequence of the mutant is an amino acid substitution compared to wild-type HCMV gB.

[0223] Some ways to stabilize the polypeptide conformation include amino acid substitutions that introduce disulfide bonds, introduce electrostatic mutations, fill cavities, alter residue packing, introduce N-linked glycosylation sites, and combinations thereof, relative to native HCMV gB.

[0224] In one aspect, the present invention relates to a polypeptide that exhibits a conformation that is not a post-fusion conformation. That is, the polypeptide exhibits the pre-fusion conformation described above, but does not exhibit a post-fusion conformation. See, for example, the pre-fusion conformation shown in Figure 3A compared to the post-fusion conformation shown in Figure 3B; Figure 4A compared to the post-fusion conformation shown in Figure 4B; and Figure 6A compared to the post-fusion conformation shown in Figure 6C. In some embodiments, the polypeptide is characterized by structural coordinates that include root mean square deviations (RMSDs) of backbone atoms of conserved residues when superimposed on the backbone atoms described by the structural coordinates in Table 1A. In some embodiments, the polypeptide is characterized by structural coordinates that include root mean square deviations (RMSDs) of backbone atoms of conserved residues when superimposed on the backbone atoms described by the structural coordinates in Table 1B.

[0225] In some embodiments, the polypeptide is isolated, i.e., separated from HCMV gB polypeptides having a post-fusion conformation. Thus, the polypeptide may be, for example, at least 80% isolated, at least 90%, 95%, 98%, 99%, or even 99.9% isolated from HCMV gB polypeptides in a post-fusion conformation. In one aspect, the invention relates to polypeptides that specifically bind to HCMV gB pre-fusion-specific antibodies.

[0226] It will be understood that a homogeneous population of polypeptides in a particular conformation may include variations that do not alter the conformational state of the polypeptide (such as polypeptide-modifying variations, e.g., glycosylation state, etc.). In some embodiments, the population of polypeptides remains homogeneous for extended periods of time. For example, in some embodiments, the polypeptides, when dissolved in aqueous solution, form a population of polypeptides stabilized in a pre-fusion conformation for at least 12 hours, e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, or more.

[0227] Without being bound by theory, it is believed that the polypeptides disclosed herein promote a stabilized pre-fusion conformation of the HCMV gB polypeptide. The polypeptides contain at least one mutation compared to the corresponding native HCMV gB polypeptide. Those skilled in the art will understand that the polypeptides are useful for eliciting an immune response against CMV in mammals.

[0228] Native HCMV gB is conserved among HCMV entry glycoproteins and is required for entry into all cell types. Given the substantial conservation of the HCMV gB sequence, amino acid positions between different native HCMV gB sequences can be compared to identify corresponding HCMV gB amino acid positions between different HCMV strains. Thus, the conservation of native HCMV gB sequences across strains allows the use of a reference HCMV gB sequence for comparison of amino acids at specific positions in the HCMV gB polypeptide. Therefore, unless otherwise explicitly stated, the polypeptide amino acid positions provided herein refer to the reference sequence of the HCMV gB polypeptide set forth in SEQ ID NO: 1.

[0229] It should be noted, however, that different native HCMV gB sequences may have a different numbering system than SEQ ID NO: 1; for example, there may be additional amino acid residues added or removed in native HCMV gB sequences from strains other than Towne compared to SEQ ID NO: 1. Thus, when a particular amino acid residue is referenced by its number, it should be understood that the reference is not limited to only the amino acid located at that exact numbered position when counting from the beginning of a given amino acid sequence, but rather, the equivalent or corresponding amino acid residue in any and all HCMV gB sequences is intended, even if the residue is not at the same exact numbered position, for example, if the HCMV sequence is shorter (e.g., a fragment) or longer than SEQ ID NO: 1, or if it has an insertion or deletion compared to SEQ ID NO: 1.

[0230] In some embodiments, a polypeptide is full-length if it contains the same number of amino acid residues as the mature, full-length wild-type HCMV gB. In some embodiments, a polypeptide is a fragment if it contains fewer amino acid residues than the total number of mature, full-length wild-type HCMV gB. As used herein, the terms "fragment" and "truncated" are interchangeable. In some embodiments, a truncated gB polypeptide contains only the extracellular domain sequence.

[0231] 1. Cysteine ​​(C) substitution In some embodiments, the polypeptide contains an introduced cysteine ​​substitution compared to native HCMV gB. In some embodiments, the polypeptide contains any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine ​​substitutions. Without being bound by theory or mechanism, it is believed that the cysteine ​​substitutions described herein promote the stability of the polypeptide in a conformation other than the HCMV gB post-fusion conformation. The introduced cysteine ​​substitutions can be introduced by protein engineering, for example, by including one or more substituted cysteine ​​residues that form disulfide bonds. In some embodiments, the amino acid positions of the cysteines are within a sufficiently close distance for disulfide bond formation in the pre-fusion conformation of HCMV gB, but not in the post-fusion conformation.

[0232] Disulfide bond-forming cysteine ​​residues can be introduced into the native HCMV gB sequence by two or more amino acid substitutions, for example, in some embodiments, two cysteine ​​residues are introduced into the native HCMV gB sequence to form disulfide bonds.

[0233] In some embodiments, the polypeptide comprises recombinant HCMV gB stabilized in the pre-fusion conformation by a disulfide bond between cysteines introduced at a pair of amino acid positions that are close to each other in the pre-fusion conformation and farther apart in the post-fusion conformation.

[0234] Exemplary cysteine ​​substitutions relative to native HCMV gB include any mutation selected from Table 2, the numbering of which is based on the numbering of SEQ ID NO:1.

[0235] [Table 2-1]

[0236] [Table 2-2]

[0237] [Table 2-3]

[0238] [Table 2-4]

[0239] [Table 2-5]

[0240] In some embodiments, the polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22 and one or more (such as two, three, four, five, six, seven, eight, nine or ten) cysteine ​​substitutions at any one of the positions listed in one or more of lines 6, 67, 68, 69, 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, 99, 100 or 101, wherein the resulting polypeptide does not exhibit an HCMV post-fusion conformation.

[0241] In some embodiments, the polypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) cysteine ​​substitutions at any one of the positions listed at one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 47, 69, or 91 of column (ii) of Table 2, and the resulting polypeptide does not exhibit an HCMV post-fusion conformation.

[0242] In some embodiments, the polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, or 101. In some embodiments, the resulting polypeptide does not exhibit an HCMV post-fusion conformation. In some embodiments, the resulting polypeptide exhibits an HCMV pre-fusion conformation.

[0243] In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 98 and 653 according to the numbering of SEQ ID NO: 1 (listed in line 2, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 100 and 269 according to the numbering of SEQ ID NO: 1 (listed in line 5, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 217 and 584 according to the numbering of SEQ ID NO: 1 (listed in line 7, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 242 and 710 according to the numbering of SEQ ID NO: 1 (listed in line 13, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 242 and 714 according to the numbering of SEQ ID NO: 1 (listed in line 14, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 367 and 499 according to the numbering of SEQ ID NO: 1 (listed in line 17, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 372 and 506 according to the numbering of SEQ ID NO: 1 (listed in line 18, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 550 and 652 according to the numbering of SEQ ID NO: 1 (listed in line 22, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 608 and 679 according to the numbering of SEQ ID NO: 1 (listed in line 26, column (ii) of Table 2) compared to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions 695 and 724 according to the numbering of SEQ ID NO: 1 (listed in line 34, column (ii) of Table 2) compared to the amino acid sequence of wild-type HCMV gB.

[0244] In some embodiments, the polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 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, 99, 100 and 101. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 98 and 653 according to the numbering of SEQ ID NO: 1 (listed in row 2, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 100 and 269 according to the numbering of SEQ ID NO: 1 (listed in line 5, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 217 and 584 according to the numbering of SEQ ID NO: 1 (listed in line 7, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 242 and 710 according to the numbering of SEQ ID NO: 1 (listed in line 13, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB.In another preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 242 and 714 according to the numbering of SEQ ID NO: 1 (listed in line 14, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 367 and 499 according to the numbering of SEQ ID NO: 1 (listed in line 17, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 372 and 506 according to the numbering of SEQ ID NO: 1 (listed in line 18, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 550 and 652 according to the numbering of SEQ ID NO: 1 (listed in line 22, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 608 and 679 according to the numbering of SEQ ID NO: 1 (listed in line 26, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 695 and 724 according to the numbering of SEQ ID NO: 1 (listed in line 34, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 217 and 589 according to the numbering of SEQ ID NO: 1 (listed in line 47, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 524 and 684 according to the numbering of SEQ ID NO: 1 (listed in line 69, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 371 and 506 according to the numbering of SEQ ID NO: 1 (listed in line 91, column (ii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB.

[0245] In further embodiments, the polypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) disulfide bonds between pairs of cysteine ​​residues introduced by cysteine ​​amino acid substitutions at any one of the pairs of positions listed at one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of column (iii) of Table 2, and the polypeptide does not exhibit an HCMV post-fusion conformation.

[0246] In further embodiments, the polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) disulfide bonds between pairs of cysteine ​​residues introduced by cysteine ​​amino acid substitutions at any one of the pairs of positions listed in one or more of lines 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, 99, 100, or 101, wherein the polypeptide does not exhibit an HCMV post-fusion conformation.

[0247] In some embodiments, the polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 2 The polypeptides include a disulfide bond between a pair of cysteine ​​residues substituted at any one of the pairs of positions listed in any one of lines 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, or 101. In some embodiments, the resulting polypeptide does not exhibit an HCMV post-fusion conformation. In some embodiments, the resulting polypeptide exhibits an HCMV pre-fusion conformation.

[0248] In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions Q98C and I653C according to the numbering of SEQ ID NO: 1 (listed in line 2, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions T100C and S269C according to the numbering of SEQ ID NO: 1 (listed in line 5, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions D217C and F584C according to the numbering of SEQ ID NO: 1 (listed in line 7, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions Y242C and K710C according to the numbering of SEQ ID NO: 1 (listed in line 13, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions Y242C and D714C according to the numbering of SEQ ID NO: 1 (listed in line 14, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions S367C and L499C according to the numbering of SEQ ID NO: 1 (listed in line 17, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions T372C and W506C according to the numbering of SEQ ID NO: 1 (listed in line 18, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions S550C and D652C according to the numbering of SEQ ID NO: 1 (listed in line 22, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions T608C and D679C (listed in line 26, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, compared to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions K695C and K724C according to the numbering of SEQ ID NO: 1 (listed in line 34, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions D217C and Y589C according to the numbering of SEQ ID NO: 1 (listed in line 47, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions N524C and M684C according to the numbering of SEQ ID NO: 1 (listed in line 69, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises cysteine ​​substitutions at positions M371C and W506C according to the numbering of SEQ ID NO: 1 (listed in line 91, column (iii) of Table 2) relative to the amino acid sequence of wild-type HCMV gB.

[0249] In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 96 and 660, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 98 and 658, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 100 and 258, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 100 and 656, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In another preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 100 and 658, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a further preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 117 and 406, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 117 and 407, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 153 and 712, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 162 and 716, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 217 and 587, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 217 and 589, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 219 and 584, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 219 and 585, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 219 and 586, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 220 and 659, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 223 and 659, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 240 and 732, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 240 and 735, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 242 and 728, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 242 and 731, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 258 and 656, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 269 and 656, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 269 and 658, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 272 and 614, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 273 and 629, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 349 and 650, according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 367 and 500, according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 367 and 503, according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 370 and 501, according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 522 and 683, according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 523 and 683, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 523 and 684, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 524 and 684, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 525 and 681, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 540 and 680, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 541 and 680, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 548 and 655, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 549 and 658, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 550 and 655, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 550 and 657, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 591 and 668, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 603 and 667, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 604 and 672, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 607 and 688, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 608 and 692, as numbered in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 609 and 691, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 610 and 674, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 610 and 675, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 612 and 663, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 737 and 755, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 741 and 754, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB. In a preferred embodiment, the polypeptide comprises a disulfide bond between a pair of cysteine ​​residues substituted at positions 741 and 755, numbered as in SEQ ID NO: 1, relative to the amino acid sequence of wild-type HCMV gB.

[0250] In some embodiments, the polypeptide comprises a combination of two or more disulfide bonds between cysteine ​​residues listed in Table 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0251] In some embodiments, the polypeptide is SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, The amino acid sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0252] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably 99%, or 100% identity to any sequence selected from SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:60.

[0253] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably 99%, or 100% identity to any sequence selected from SEQ ID NO:51, SEQ ID NO:73, SEQ ID NO:70, and SEQ ID NO:78.

[0254] In some embodiments, the composition preferably does not include a polypeptide having a sequence set forth in any one of SEQ ID NO:59, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:71, SEQ ID NO:52, SEQ ID NO:96, and SEQ ID NO:50.

[0255] In further embodiments, the polypeptide comprises the amino acid sequence set forth in any one of the SEQ ID NOs listed in column (iv) of Table 2. That is, exemplary polypeptides include polypeptides having an amino acid sequence selected from any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0256] In some embodiments, the polypeptide has an amino acid sequence selected from any one of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0257] In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:35.

[0258] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23-907 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23-907 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23-907 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23-907 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 98% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99.5% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99.6% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99.7% identical to amino acids 23-907 of SEQ ID NO:1. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99.8% identical to amino acids 23-907 of SEQ ID NO:1. In one embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23-907 of SEQ ID NO:1.

[0259] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 98% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.5% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.6% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.7% identical to amino acids 23-707 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.8% identical to amino acids 23-707 of SEQ ID NO:1. In one embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23-707 of SEQ ID NO:1.

[0260] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 97% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 98% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.5% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.6% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.7% identical to amino acids 23-646 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence at least 99.8% identical to amino acids 23-646 of SEQ ID NO:1. In one embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23-646 of SEQ ID NO:1.

[0261] In some embodiments, amino acids can be inserted (or deleted) from the native HCMV gB sequence to adjust the alignment of residues in the polypeptide structure so that particular pairs of residues are within close enough distance to form disulfide bonds in the pre-fusion, but not the post-fusion, conformation. In some such embodiments, the polypeptide, in addition to comprising at least one amino acid insertion, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 1 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100 or 101.

[0262] In some embodiments, the polypeptide comprises a phenylalanine substitution compared to native HCMV gB. In some embodiments, the polypeptide comprises a leucine substitution compared to native HCMV gB. In some embodiments, the polypeptide can be stabilized by amino acid mutations (e.g., phenylalanine (F) and leucine (L) substitutions) that reduce ionic repulsion between residues that are adjacent to each other in the folded structure of the polypeptide compared to the HCMV gB polypeptide in the post-fusion conformation. In some embodiments, the polypeptide can be stabilized by amino acid mutations that increase ionic attraction between residues that are adjacent to each other in the folded structure of the polypeptide compared to the HCMV gB in the post-fusion conformation.

[0263] Exemplary mutations include any mutation selected from Table 3, according to the numbering of SEQ ID NO:1, relative to native HCMV gB.

[0264] [Table 3]

[0265] [Table 4-1]

[0266] [Table 4-2]

[0267] In some embodiments, the polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) residues substituted at any one of the positions listed at one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of column (ii) of Table 3, and the polypeptide does not exhibit an HCMV gB post-fusion conformation. In certain embodiments, the resulting polypeptide exhibits an HCMV gB pre-fusion conformation.

[0268] In some embodiments, the polypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) residues substituted at any one of the positions listed at one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 of column (ii) of Table 4, and the polypeptide does not exhibit an HCMV gB post-fusion conformation. In certain embodiments, the resulting polypeptide exhibits an HCMV gB pre-fusion conformation.

[0269] In some embodiments, the polypeptide comprises a mutation at position 670 according to the numbering of SEQ ID NO: 1 (listed in column (ii), lines 1 and 2 of Table 3). In some embodiments, the polypeptide comprises a mutation at position 673 according to the numbering of SEQ ID NO: 1 (listed in column (ii), lines 3 and 4 of Table 3). In some embodiments, the polypeptide comprises a mutation at position 691 according to the numbering of SEQ ID NO: 1 (listed in column (ii), lines 5 and 6 of Table 3).

[0270] In some embodiments, the polypeptide comprises a mutation at position 670 according to the numbering of SEQ ID NO:1. In some embodiments, the polypeptide comprises a mutation at position 682 according to the numbering of SEQ ID NO:1. In some embodiments, the polypeptide comprises a mutation at position 686 according to the numbering of SEQ ID NO:1. In some embodiments, the polypeptide comprises a mutation at position 118 according to the numbering of SEQ ID NO:1. In some embodiments, the polypeptide comprises a mutation at position 646 according to the numbering of SEQ ID NO:1.

[0271] In a further embodiment, the polypeptide comprises an electrostatic mutation introduced by substitution at any one of the positions listed in one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of column (iii) of Table 3, and the polypeptide does not exhibit an HCMV post-fusion conformation.

[0272] In a preferred embodiment, the polypeptide comprises a substitution K670L according to the numbering of SEQ ID NO: 1 (listed in line 1, column (iii) of Table 3). In another preferred embodiment, the polypeptide comprises a substitution K670F according to the numbering of SEQ ID NO: 1 (listed in line 2, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution R673L according to the numbering of SEQ ID NO: 1 (listed in line 3, column (iii) of Table 3). In a preferred embodiment, the polypeptide comprises a substitution R673F according to the numbering of SEQ ID NO: 1 (listed in line 4, column (iii) of Table 3). In another preferred embodiment, the polypeptide comprises a substitution K691L according to the numbering of SEQ ID NO: 1 (listed in line 5, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 6, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 7, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 8, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 9, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 10, column (iii) of Table 3). In a further preferred embodiment, the polypeptide comprises a substitution K691F according to the numbering of SEQ ID NO: 1 (listed in line 11, column (iii) of Table 3).

[0273] In some embodiments, the polypeptide comprises a combination of two or more phenylalanine (F) and leucine (L) substitutions listed in Table 3.

[0274] In a preferred embodiment, the polypeptide comprises a substitution D679S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution D679N according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution E682S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution E682Q according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution E686S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution E686Q according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution N118P according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide comprises a substitution D646P according to the numbering of SEQ ID NO: 1.

[0275] In some embodiments, the polypeptide comprises a combination of two or more phenylalanine (F) and leucine (L) substitutions listed in Table 3. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.

[0276] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106.

[0277] In further embodiments, the polypeptide comprises the amino acid sequence set forth in any one of the SEQ ID NOs listed in column (iv) of Table 3. That is, exemplary polypeptides include polypeptides having an amino acid sequence selected from any one of SEQ ID NOs: 38, 39, 40, 41, 42, and 43. In some embodiments, the polypeptide has an amino acid sequence selected from any one of SEQ ID NOs: 99, 100, 102, 103, 104, 105, and 106.

[0278] In some embodiments, the polypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) residues substituted at any one of the positions listed at one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 of column (iii) of Table 4, and the polypeptide does not exhibit an HCMV gB post-fusion conformation. In certain embodiments, the resulting polypeptide exhibits an HCMV gB pre-fusion conformation.

[0279] In some embodiments, amino acids can be inserted (or deleted) from the native HCMV gB sequence to adjust the alignment of residues in the polypeptide structure so that particular pairs of residues are within close enough distance to form the desired electrostatic interaction in the pre-fusion, but not post-fusion, conformation. In some such embodiments, the polypeptide contains the desired electrostatic interaction at any of the positions listed in one or more of lines 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of column (ii) of Table 3, and the polypeptide does not exhibit the HCMV post-fusion conformation.

[0280] The present invention provides a mutant of a wild-type cytomegalovirus (CMV) glycoprotein B (gB) protein, comprising at least two amino acid mutations compared to the amino acid sequence of the wild-type CMV gB protein, wherein the amino acid mutations are: (1) genetically engineered disulfide bond mutations; (2) additional mutations; and (3) a combination of at least one engineered disulfide mutation and at least one additional mutation; The present invention provides a mutant selected from the group consisting of:

[0281] In one embodiment, the amino acid mutations comprise a combination of at least two engineered disulfide mutations and at least one additional mutation. In another embodiment, the mutant wild-type CMV gB protein is in the form of a trimer.

[0282] In another embodiment, the mutant of the wild-type CMV gB protein has increased stability in the pre-fusion form compared to the corresponding wild-type CMV gB protein, where the stability is measured by pre-fusion-specific antibody binding, thermal shift assay, or EM imaging.

[0283] In another embodiment, the mutant of wild-type CMV gB is the Towne strain.

[0284] In another embodiment of the invention, the engineered disulfide mutations are selected from the group consisting of D217C and Y589C; M371C and W506C; and N524C and M684C.

[0285] In a further aspect of the invention, the additional mutation is (1) Substitution of YIH at positions 155–157 by GHR; (2) substitution of W at position 240 by A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; and (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648–653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) Substitution of Y at position 696 and V at position 697 with C is selected from the group consisting of:

[0286] In another aspect of the invention, the additional mutation is (1) Substitution of YIH at positions 155–157 by GHR; (2) substitution of W at position 240 by A; (3) substitution of C at position 246 with S; and (4) Substitution of I at position 675 with S is selected from the group consisting of:

[0287] In another embodiment of the invention, the amino acid mutations are a combination of at least two engineered disulfide mutations and at least one additional mutation, (i) the engineered disulfide mutation is selected from the group consisting of D217C and Y589C; M371C and W506C; and N524C and M684C; and (ii) Additional mutations are (1) Substitution of YIH at positions 155–157 by GHR; (2) substitution of W at position 240 by A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; and (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648–653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) Substitution of Y at position 696 and V at position 697 with C is selected from the group consisting of:

[0288] In another aspect of the invention, the amino acid mutation is (1) Combinations of D217C and Y589C, M371C and W506C, and I675S; (2) combinations of D217C and Y589C, N524C and M684C, and I675S; (3) combinations of D217C and Y589C, M371C and W506C, Y155G, I156H, H157R, W240A, C246S, and I675S; and (4) Combinations of D217C and Y589C, N524C and M684C, Y155G, I156H, H157R, W240A, C246S and I675S and a combination of mutations selected from the group consisting of:

[0289] In another embodiment of the invention, the mutant comprises a cysteine ​​(C) at position 217 (217C) and a cysteine ​​at position 589 (589C), a cysteine ​​(C) at position 371 (371C) and a cysteine ​​at position 506 (506C), and a serine (S) at position 675 (675S), (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 257; and (2) A mutant comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 257. is selected from the group consisting of:

[0290] In another embodiment of the invention, the mutant comprises a cysteine ​​(C) at position 217 (217C) and a cysteine ​​at position 589 (589C), a cysteine ​​(C) at position 524 (524C) and a cysteine ​​at position 684 (684C), and a serine (S) at position 675 (675S), (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 259; and (2) A mutant comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 259. is selected from the group consisting of:

[0291] In another embodiment of the invention, the mutant comprises a cysteine ​​(C) at position 217 (217C) and a cysteine ​​at position 589 (589C), a cysteine ​​(C) at position 371 (371C) and a cysteine ​​at position 506 (506C), a serine (S) at position 675 (675S), a glycine (G) at position 155 (155G), a histidine (156H) at position 156, an arginine (157R) at position 157, an alanine (240A) at position 240, and a serine (246S) at position 246, wherein the mutant is (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 261; and (2) a mutant comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 261; (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 261; (3) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 265; and (4) A mutant comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 265. is selected from the group consisting of:

[0292] In another embodiment of the invention, the mutant comprises a cysteine ​​(C) at position 217 (217C) and a cysteine ​​at position 589 (589C), a cysteine ​​(C) at position 524 (524C) and a cysteine ​​at position 684 (684C), a serine (S) at position 675 (675S), a glycine (G) at position 155 (155G), a histidine (156H) at position 156, an arginine (157R) at position 157, an alanine (240A) at position 240, and a serine (246S) at position 246, wherein the mutant is (1) a mutant comprising the amino acid sequence of SEQ ID NO: 263; and (2) A mutant comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 263. is selected from the group consisting of:

[0293] In another embodiment of the invention, the amino acid sequence of a wild-type CMV gB polypeptide is set forth in SEQ ID NO:1.

[0294] In another embodiment of the invention, the amino acid sequence of the mutant does not include a signal sequence. In another embodiment of the invention, the mutant comprises residues 23 to 907 of SEQ ID NO:1.

[0295] In another embodiment of the invention, the amino acid sequence of the mutant does not include the MPR, TM or CT domains. In another embodiment of the invention, the mutant comprises residues 23 to 707 of SEQ ID NO:1.

[0296] In another embodiment of the invention, the amino acid sequence of the mutant comprises a truncated domain V region. In another embodiment of the invention, the mutant comprises residues 23-702 or 23-703 of SEQ ID NO:1.

[0297] In another embodiment of the invention, the amino acid sequence of the mutant does not include the domain V region. In another embodiment of the invention, the mutant comprises residues 23 to 646 of SEQ ID NO:1.

[0298] In another embodiment of the invention, the mutant further comprises a trimerization motif linked to the C-terminus of the mutant. (i) Interprotomer disulfide rings; (ii) GCN4; (iii) T4 fibritinfoldon; and (iv) C-terminal fusion sequence is selected from the group consisting of:

[0299] In another aspect of the invention, (i) GCN4 comprises the amino acid sequence set forth in SEQ ID NO: 269, 272, or 273; (ii) the T4 fibritin foldon comprises the amino acid sequence set forth in SEQ ID NO: 271; or (iii) The C-terminal fusion sequence comprises the amino acid sequence set forth in SEQ ID NO: 274.

[0300] In another aspect of the invention, the interprotomer disulfide ring is (i) 696C and 697C; (ii) 703C and 704C; or (iii) 767C and 768C The compound comprises at least two engineered cysteine ​​mutations selected from:

[0301] Several exogenous multimerization domains that promote the formation of stable trimers of soluble proteins are known in the art. Examples of such multimerization domains that can be linked to the mutants provided by the present disclosure include, but are not limited to, (1) the GCN4 leucine zipper (Harbury et al., 1993 Science 262:1401-1407); (2) the trimerization motif from pulmonary surfactant protein (Hoppe et al., 1994 FEB S Lett 344:191-195); (3) collagen (McAlinden et al., 2003 Biol Chem 278:42200-42207); and (4) the phage T4 fibritin foldon (Miroshnikov et al., 1998 Protein Eng 11:329-414). In some embodiments, the multimerization domain is linked to the CMV gB mutant at the C-terminus. In certain embodiments, the trimerization domain is set forth in SEQ ID NOs: 269 to 274. Methods for attaching a multimerization domain to a gB polypeptide are well known in the art.

[0302] As used herein, "interprotomer disulfide ring" refers to a covalent ring formation between three helices achieved by three interhelix disulfide bonds formed by a ring system containing pairs of adjacent cysteine ​​residues, which establishes the functional topology and stabilization of the multimer (e.g., trimer). See Stewart-Jones GBE et al. (2015) A Cysteine ​​Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus. PLoS ONE 10(6):e0128779. doi:10.1371 / journal.pone.0128779.

[0303] In another embodiment of the invention, the mutant is secreted. In another embodiment of the invention, the mutant is soluble.

[0304] In another embodiment of the invention, the wild-type CMV gB polypeptide sequence is selected from SEQ ID NOs: 107-140 or 224.

[0305] In another embodiment of the invention, the wild-type CMV gB polypeptide sequence is encoded by the polynucleotide sequence set forth in SEQ ID NOs:225-254.

[0306] The present invention also provides nucleic acid molecules comprising nucleotides encoding the amino acid sequence of a CMV gB protein mutant according to the embodiments and aspects described herein.

[0307] In one embodiment, the nucleic acid comprises nucleotides having the sequences set forth in SEQ ID NOs: 225-254.

[0308] The present invention also provides pharmaceutical compositions comprising (i) a CMV gB protein mutant according to the embodiments and aspects described herein and (ii) a pharmaceutically acceptable carrier.

[0309] In one embodiment, the pharmaceutical composition is a vaccine.

[0310] The present invention also provides a method of reducing a CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0311] The present invention also provides a method of eliciting an immune response against CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0312] The present invention also provides a method for preventing CMV infection in a subject, comprising administering to the subject an effective amount of a vaccine described in the embodiments herein.

[0313] In one aspect, the subject is a human.

[0314] 2. Further Polypeptide Embodiments In some embodiments, the polypeptide does not comprise a mutation at any one of the following amino acid positions, according to the numbering of the reference sequence SEQ ID NO: 46: 280, 281, 283, 284, 285, 286, 290, 292, 295, 297, 298, 299, or any combination thereof. In some exemplary embodiments, the polypeptide does not comprise a substitution of any one of the following residues, according to the numbering of the reference sequence SEQ ID NO: 46: Y280; N281; T283; N284; R285; N286; F290; E292; N293; F297; F298; I299; F298; and any combination thereof. Without being bound by theory or mechanism, residues important for neutralizing antibodies may include Y280 / N284 and Y280 / N293 / D295. Thus, in a preferred embodiment, the polypeptide does not contain mutations at Y280, N293, N284, and D295 compared to the reference sequence SEQ ID NO:46.

[0315] In some embodiments, the polypeptide does not comprise a mutation at any one of the following amino acid positions, and any combination thereof, according to the numbering of the reference sequence SEQ ID NO: 44: R562, P577, S587, Y588, G592, G595, L601 / H605, C610, L612, P613, Y625, Y627, F632, and K633. In some embodiments, the polypeptide does not comprise any one of the following amino acid mutations, and any combination thereof, according to the numbering of the reference sequence SEQ ID NO: 44: R562C, P577L, S587L, Y588C, G592S, G595D, L601P / H605N, C610Y, L612F, P613Y, Y625C, Y627C, F632L, and K633T. Without being bound by theory or mechanism, it is believed that P577 and Y627 are located next to each other in the domain IV core, while C610 is involved in a conserved disulfide bond. Thus, all three residues may help maintain the position of domain IV in the pre-fusion structure and thus maintain the stability of the entire antigenic site AD-1. Furthermore, without being bound by theory or mechanism, it is believed that F632 and G595 are exposed on the surface of the pre-fusion form of gB. Therefore, in a preferred embodiment, the polypeptide does not contain mutations in P577, Y627, C610, F632, and G595, or any combination thereof, according to the numbering of the reference sequence SEQ ID NO: 44.

[0316] 3. Void-filling mutations In yet other embodiments, the polypeptide comprises one or more amino acid mutations that are gap-filling mutations. Examples of amino acids that can be substituted for gap-filling include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr), as well as amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. Examples of replacement amino acids include large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp).

[0317] 4. Combining Mutations In another aspect, the present invention relates to a polypeptide comprising at least two engineered disulfide bond mutations and at least one additional mutation, e.g., a combination of two or more different types of mutations selected from void-filling mutations, electrostatic mutations, interprotomer disulfide rings, etc., each of which is described herein. In some embodiments, the polypeptide comprises at least two disulfide bond mutations and at least one electrostatic mutation. More specifically, in some embodiments, the polypeptide comprises at least two cysteine ​​substitutions and at least one phenylalanine substitution. In some embodiments, the polypeptide comprises at least two cysteine ​​substitutions and at least one leucine substitution.

[0318] In some further embodiments, the polypeptide comprises at least two mutations selected from any one of the mutations in Table 2 and at least one mutation selected from any one of the mutations in Table 3. In some further embodiments, the polypeptide comprises at least two mutations selected from any one of the mutations in Table 2 and at least one mutation selected from any one of the mutations in Table 4. In some further embodiments, the polypeptide comprises at least two mutations selected from any one of the mutations in Table 2, at least one mutation selected from any one of the mutations in Table 3, and at least one mutation selected from any one of the mutations in Table 4.

[0319] Preparation of Polypeptides The polypeptides described herein can be prepared by routine methods known in the art, such as expression in recombinant host systems using suitable vectors. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and HIGH FIVE cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (typically HEK293 or Expi293 cells transformed with fragmented adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art. Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form. Avian cell expression systems are also known to those skilled in the art. Similarly, bacterial and mammalian cell expression systems are also known in the art.

[0320] Some suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known and commonly used in the art.Suitable vectors may contain several components, including, but not limited to, an origin of replication; a selectable marker gene; one or more expression control elements, such as transcription control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals; and one or more signal or leader sequences (e.g., mammalian origin, or from heterologous mammalian or non-mammalian species) for targeting to the secretory pathway in selected host cells.For example, for expression in insect cells, a suitable baculovirus expression vector such as PFASTBAC is used to produce recombinant baculovirus particles.The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins.For expression in mammalian cells, a vector that will drive the expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cell) is used.

[0321] Polypeptides can be purified using any suitable method. For example, methods for purifying polypeptides by immunoaffinity chromatography are known in the art. Suitable methods for purifying desired polypeptides are known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion. A suitable purification scheme can be created using two or more of these or other suitable methods. If necessary, the polypeptide can contain a "tag" that facilitates purification, such as an epitope tag or a histidine tag. Such tagged polypeptides can be purified from conditioned media, for example, by chelating or affinity chromatography.

[0322] Nucleic acids encoding polypeptides In another aspect, the present invention relates to nucleic acid molecules encoding the polypeptides described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules encoding only the extracellular domain of a polypeptide are also encompassed by the present invention. The nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0323] In some embodiments, the nucleic acid comprises a self-replicating RNA molecule. In some embodiments, the nucleic acid comprises a modified RNA molecule. In another aspect, the invention relates to a composition comprising a nucleic acid according to any one of the embodiments described herein.

[0324] Compound-stabilizing polypeptides The present inventors have discovered polypeptides stabilized in the pre-fusion conformation that can be identified, for example, by the binding of bis(aryl)thiourea compounds to HCMV gB. Bis(aryl)thiourea compounds, as exemplified by structural formula 1a, b (Formula I), are highly potent and specific inhibitors of CMV. In one aspect, the present invention relates to polypeptides capable of binding to bis(aryl)thiourea compounds. In a preferred embodiment, the compounds do not bind to the post-fusion conformation of the HCMV gB polypeptide.

[0325] [ka]

[0326] In preferred embodiments, the compound is a bis(aryl)thiourea thiodiol analog thereof. Most preferably, in some embodiments, the compound has the following structure:

[0327] [ka] N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide having the formula:

[0328] In another embodiment, the compound has the following structure:

[0329] [ka] It has.

[0330] In some embodiments, the polypeptide comprises an HCMV gB pre-fusion epitope that is not present in the post-fusion conformation of native HCMV gB.

[0331] In some embodiments, at least about 90% of the polypeptides in the homogeneous population (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% of the polypeptides) are conjugated to a bis(aryl)thiourea compound (e.g., a thiazole analog of a bis(aryl)thiourea compound, more preferably N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide). In some embodiments, the polypeptide capable of binding to the bis(aryl)thiourea compound does not have a post-fusion conformation. Rather, the polypeptide has a pre-fusion conformation, such as the HCMV gN pre-fusion conformation.

[0332] In another embodiment, the polypeptide may be at least 80% isolated, at least 90%, 95%, 98%, 99%, or preferably 99.9% isolated from HCMV gB polypeptides to which the bis(aryl)thiourea compound is not specifically bound.

[0333] Compositions Comprising Polypeptides and Methods of Use Thereof The present invention relates to compositions and methods that use the polypeptides described herein or nucleic acids encoding such polypeptides described herein. For example, the polypeptides of the present invention can be directly delivered as a component of an immunogenic composition. Alternatively, nucleic acids encoding the polypeptides of the present invention can be administered to produce the polypeptides or immunogenic fragments in vivo. Certain preferred embodiments, such as protein formulations, recombinant nucleic acids (e.g., DNA, RNA, self-replicating RNA, or any variant thereof) and viral vectors (e.g., live, single-round, non-replicating assembly virions, or otherwise virus-like particles, or alphavirus VRPs) containing sequences encoding the polypeptides, are further described herein and can be included in the compositions.

[0334] In one aspect, the present invention provides immunogenic compositions comprising the polypeptides described herein. The immunogenic compositions may include additional CMV proteins, such as gO, gH, gL, pUL128, pUL130, puL131, pp65, immunogenic fragments thereof, or combinations thereof. For example, the polypeptides can be combined with a CMV pentameric complex comprising gH or a pentameric fragment thereof, gL or a pentameric fragment thereof, pUL128 or a pentameric fragment thereof, pUL130 or a pentameric fragment thereof, and pUL131 or a pentameric fragment thereof. The polypeptides of the present invention can also be combined with a CMV trimeric complex comprising gH or a trimerizable fragment thereof, gL or a trimerizable fragment thereof, and gO or a trimerizable fragment thereof.

[0335] In another aspect, the present invention relates to compositions comprising polynucleotides capable of eliciting an immune response in a mammal. The polynucleotide encodes at least one polypeptide of interest, e.g., an antigen. The antigens disclosed herein may be wild-type (i.e., derived from an infectious agent) or, preferably, modified (e.g., genetically engineered, designed, or artificial). The nucleic acid molecules described herein, specifically polynucleotides, in some embodiments, encode one or more peptides or polypeptides of interest. Such peptides or polypeptides can serve as antigens or antigenic molecules. The term "nucleic acid" includes any compound comprising a polymer of nucleotides. These polymers are referred to as "polynucleotides." Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), including mRNA, and deoxyribonucleic acid (DNA).

[0336] In some embodiments, the composition comprises DNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition comprises RNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition comprises an mRNA polynucleotide encoding a polypeptide or fragment thereof described herein. Such a composition may result in the proper protein conformation upon translation.

[0337] In one aspect, the invention relates to a composition comprising at least one polynucleotide encoding a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB.

[0338] In one aspect, the present invention relates to a composition comprising at least one DNA polynucleotide encoding a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB.

[0339] In one aspect, the invention relates to a composition comprising at least one RNA polynucleotide encoding a polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB.

[0340] In some embodiments, the present invention relates to compositions comprising at least one polynucleotide encoding at least one hCMV gB polypeptide or an immunogenic fragment or epitope thereof.

[0341] In some embodiments, a composition comprises at least one polynucleotide encoding two or more antigenic polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, a composition comprises two or more polynucleotides encoding two or more antigenic polypeptides, or immunogenic fragments or epitopes thereof. One or more antigenic polypeptides can be encoded on a single polynucleotide or individually encoded on multiple (e.g., two or more) polynucleotides.

[0342] In another aspect, the invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) a polynucleotide encoding an additional polypeptide.

[0343] In another aspect, the present invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) an additional polypeptide, preferably a polynucleotide encoding an HCMV antigenic polypeptide. The additional polypeptide can be selected from HCMV gH, gL, gB, gO, gN, and gM, and immunogenic fragments or epitopes thereof. In some embodiments, the additional polypeptide is HCMV pp65. In some embodiments, the additional polypeptide can be selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131A, and fragments thereof. In some embodiments, the additional polypeptide is an HCMV gH polypeptide. In some embodiments, the additional polypeptide is an HCMV gL polypeptide. In some embodiments, the additional polypeptide is an HCMV gB polypeptide. In some embodiments, the additional polypeptide is an HCMV gO polypeptide. In some embodiments, the additional polypeptide is an HCMV gN polypeptide. In some embodiments, the additional polypeptide is an HCMV gM polypeptide. In some embodiments, the additional polypeptide is a variant gH polypeptide, a variant gL polypeptide, or a variant gB polypeptide. In some embodiments, the variant HCMV gH, gL, or gB polypeptide is a truncated polypeptide lacking one or more of the following domain sequences: (1) a hydrophobic membrane proximal domain, (2) a transmembrane domain, and (3) a cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide lacks the hydrophobic membrane proximal domain, the transmembrane domain, and the cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide comprises only the extracellular domain sequence. In some embodiments, the antigenic polypeptide is an HCMV protein selected from UL83, UL123, UL128, UL130, and UL131A, or an immunogenic fragment or epitope thereof.In some embodiments, the antigenic polypeptide is an HCMV UL83 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL123 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL128 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL130 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL131 polypeptide.

[0344] In another aspect, the present invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least two introduced amino acid mutations compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) a polynucleotide encoding an additional polypeptide having any one of the amino acid sequences set forth in SEQ ID NOs: 211-223. In another aspect, the present invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least two introduced amino acid mutations compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) a polynucleotide having any one of the sequences selected from SEQ ID NOs: 141-210. In another aspect, the present invention relates to a composition comprising (a) a polynucleotide encoding a polypeptide comprising at least two introduced amino acid mutations compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), and (b) an additional polypeptide having any one of the amino acid sequences selected from SEQ ID NOs: 211-223. In some embodiments, the polynucleotide encoding the additional polypeptide comprises at least one nucleic acid sequence selected from any of SEQ ID NOs: 225-254. In some embodiments, the polynucleotide encoding the additional polypeptide comprises at least one nucleic acid sequence selected from any of SEQ ID NOs: 141-147. In some embodiments, the polynucleotide encoding the additional polypeptide has at least one sequence selected from any of SEQ ID NOs: 220-223.

[0345] In some embodiments, the antigenic polypeptide comprises two or more HCMV proteins, fragments thereof, or epitopes. In some embodiments, the antigenic polypeptide comprises two or more glycoproteins, fragments thereof, or epitopes. In some embodiments, the antigenic polypeptide comprises at least one HCMV polypeptide, fragment thereof, or epitope and at least one other HCMV protein, fragment thereof, or epitope. In some embodiments, the two or more HCMV polypeptides are encoded by a single RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides are encoded by two or more RNA polynucleotides, e.g., each HCMV polypeptide is encoded by a separate RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins comprise pp65, or an immunogenic fragment or epitope thereof; and any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gB and one or more HCMV polypeptides selected from gH, gL, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gL and one or more HCMV polypeptides selected from gB, gH, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are gB and gH. In some embodiments, the two or more HCMV polypeptides are gB and gL.In some embodiments, the two or more HCMV polypeptides are gH and gL. In some embodiments, the two or more HCMV polypeptides are gB, gL, and gH. In some embodiments, the two or more HCMV proteins may be any combination of HCMV UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are UL123 and UL130. In some embodiments, the two or more HCMV polypeptides are UL123 and 131A. In some embodiments, the two or more HCMV polypeptides are UL130 and 131A. In some embodiments, the two or more HCMV polypeptides are UL128, UL130, and 131A. In some embodiments, the two or more HCMV proteins may be any combination of HCMV gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gL and one or more HCMV polypeptides selected from gH, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are gL, gH, UL128, UL130, and 131A. In any of these embodiments in which the composition comprises two or more HCMV proteins, the HCMV gH may be a variant gH such as any of the variant HCMV gH glycoproteins disclosed herein, e.g., any of the variant HCMV gH disclosed herein.In any of these embodiments in which the composition comprises two or more HCMV proteins, the HCMV gB can be a variant gB, such as any of the variant HCMV gB glycoproteins disclosed herein, e.g., any of the variant HCMV gBs disclosed herein. In any of these embodiments in which the composition comprises two or more HCMV gL proteins, the HCMV gL can be a variant gL, such as any of the variant HCMV gL glycoproteins disclosed herein, e.g., any of the variant HCMV gLs disclosed herein.

[0346] In certain embodiments, wherein the composition comprises two or more RNA polynucleotides encoding two or more HCMV antigenic polypeptides or immunogenic fragments or epitopes thereof (encoded by a single RNA polynucleotide or by two or more RNA polynucleotides, e.g., each protein is encoded by a separate RNA polynucleotide), the two or more HCMV proteins are an HCMV protein selected from variant gB, e.g., any of the variant gB polypeptides disclosed herein, and gH, gL, gO, gM, gN, UL128, UL130, and UL131 polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are an HCMV protein selected from variant gH, e.g., any of the variant gH polypeptides disclosed herein, and gH, gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are variant gH, such as any of the variant gH polypeptides disclosed herein, and an HCMV protein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131 polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the variant HCMV proteins are variant HCMV gB, variant HCMV gL, and variant HCMV gH, the variant HCMV polypeptide is a truncated polypeptide selected from the following: a truncated polypeptide lacking the hydrophobic membrane-proximal domain; a truncated polypeptide lacking the transmembrane domain; a truncated polypeptide lacking the cytoplasmic domain; a truncated polypeptide lacking two or more of the hydrophobic membrane-proximal domain, the transmembrane domain, and the cytoplasmic domain; and a truncated polypeptide containing only the extracellular domain. In some embodiments, the composition comprises a multimeric RNA polynucleotide encoding at least one HCMV antigenic polypeptide, or an immunogenic fragment or epitope thereof.In some embodiments, the composition comprises at least one RNA polynucleotide encoding at least one HCMV antigenic polypeptide or immunogenic fragment or epitope thereof, wherein the 5' UTR of the RNA polynucleotide comprises a patterned UTR. In some embodiments, the patterned UTR has a repeating or alternating pattern, such as ABABAB, or AABBAABBAABB, or ABCABCABC, or variants thereof repeated once, twice, or more than three times. In these patterns, each letter A, B, or C represents a UTR that differs at the nucleotide level. In some embodiments, the 5' UTR of an RNA polynucleotide (e.g., a first nucleic acid) has a region complementary to a UTR of another RNA polynucleotide (a second nucleic acid). For example, the UTR nucleotide sequences of two polynucleotides to be linked (e.g., in a multimeric molecule) can be modified to contain complementary regions such that the two UTRs hybridize to form a multimeric molecule. In some embodiments, the 5' UTR of an RNA polynucleotide encoding an HCMV antigenic polypeptide is modified to allow the formation of a multimeric sequence. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from UL128, UL130, and UL131 is modified to allow for the formation of multimeric sequences. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV polypeptide is modified to allow for the formation of multimeric sequences. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV polypeptide selected from gH, gL, gB, gO, gM, and gN is modified to allow for the formation of multimeric sequences. In any of these embodiments, the multimer may be a dimer, trimer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Thus, in some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131 is modified to allow for the formation of dimers.In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A is modified to allow trimer formation. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131 is modified to allow pentamer formation. In some embodiments, a composition comprises at least one RNA polynucleotide having a single open reading frame encoding two or more (e.g., 2, 3, 4, 5 or more) HCMV antigenic polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, a composition comprises at least one RNA polynucleotide having more than one open reading frame, e.g., 2, 3, 4, 5 or more open reading frames, encoding two, three, four, five or more HCMV antigenic polypeptides. In any of these embodiments, the at least one RNA polynucleotide may encode two or more HCMV antigenic polypeptides selected from gH, gB, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In some embodiments, the at least one RNA polynucleotide encodes UL83 and UL123. In some embodiments, the at least one RNA polynucleotide encodes gH and gL. In some embodiments, the at least one RNA polynucleotide encodes UL128, UL130, and UL131. In some embodiments, the at least one RNA polynucleotide encodes gH, gL, UL128, UL130, and UL131. In some embodiments, in which at least one RNA polynucleotide has a single open reading frame encoding two or more (e.g., 2, 3, 4, 5 or more) HCMV antigenic polypeptides, the RNA polynucleotide further comprises additional sequences, e.g., linker sequences or sequences that assist in processing of the HCMV RNA transcript or polypeptide, e.g., cleavage site sequences.In some embodiments, the additional sequence may be a protease sequence, such as a furin sequence. In some embodiments, the additional sequence may be a self-cleaving 2A peptide, such as a P2A, E2A, F2A, or T2A sequence. In some embodiments, linker and cleavage site sequences are interspersed between sequences encoding HCMV polypeptides.

[0347] In some embodiments, the at least one RNA polynucleotide comprises any nucleic acid sequence selected from any one of the nucleic acid sequences disclosed herein, or a homolog thereof having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to a nucleic acid sequence disclosed herein. In some embodiments, the open reading frame is encoded and codon-optimized. Some embodiments include compositions comprising at least one RNA polynucleotide encoding at least one HCMV antigenic polypeptide or immunogenic fragment thereof and at least one 5'-end cap. In some embodiments, the 5'-end cap is 7mG(5')ppp(5')NlmpNp.

[0348] In some embodiments, at least one polynucleotide comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 141-210. In some embodiments, at least one polynucleotide encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 211-223. In some preferred embodiments, the composition does not comprise a polypeptide having the amino acid sequence of SEQ ID NO: 216. In some preferred embodiments, the composition does not comprise a polynucleotide encoding the amino acid sequence of SEQ ID NO: 216. In some preferred embodiments, the composition does not comprise a polynucleotide having the sequence of SEQ ID NO: 152.

[0349] In some embodiments, the composition comprises at least one polynucleotide, and at least one polynucleotide has at least one chemical modification. In some embodiments, at least one polynucleotide further comprises a second chemical modification. Preferably, the polynucleotide is RAN. In some embodiments, at least one polynucleotide with at least one chemical modification has a 5'-end cap. In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, the composition comprises at least one polynucleotide, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame have a chemical modification, and optionally the composition is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine.

[0350] In some embodiments, the immunogenic fragment encoded by the additional polypeptide or polynucleotide (e.g., in an mRNA composition) is selected from gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, pp65, and IE1 antigens.

[0351] In some embodiments, a first composition and a second composition are administered to a mammal. In some embodiments, the first composition comprises a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV gB; and the second composition comprises a polynucleotide encoding HCMV pp65 or an antigenic fragment or epitope thereof. In some embodiments, the first composition comprises a polynucleotide encoding a polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV gB; and the second composition comprises a polynucleotide encoding at least one polynucleotide encoding an additional polypeptide selected from HCMV gH, gL, UL128, UL130, and UL131, or an antigenic fragment or epitope thereof.

[0352] In another aspect, the invention relates to a method of inducing an immune response in a mammal, comprising administering to the mammal an effective amount of a composition for inducing an immune response, the composition comprising a polynucleotide encoding a polypeptide comprising at least two introduced amino acid mutations compared to the amino acid sequence of wild-type HCMV gB.

[0353] In some embodiments, the immune response comprises a T cell response or a B cell response. In some embodiments, the immune response comprises a T cell response and a B cell response. In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method further comprises administering a booster dose of the composition to the subject. The compositions comprising the polynucleotides disclosed herein can be formulated in an effective amount to generate an antigen-specific immune response in a mammal.

[0354] The immunogenic composition may include an adjuvant. Exemplary adjuvants for enhancing the effectiveness of the composition include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations (with or without other specific adjuvants such as muramyl peptides (see below) or bacterial cell wall components), such as (a) MF59 containing 5% squalene, 0.5% TWEEN 80, and 0.5% Span 85 formulated into submicron particles using a microfluidizer (PCT Publication No. WO 90 / 14837); (b) submicron emulsions. (c) SAF containing 10% squalane, 0.4% Tween 80, 5% Pluronic block polymer L121, and thr-MDP, microfluidized into a squalene solution or vortexed to generate a larger particle size emulsion, and (d) RIBI™ Adjuvant System (RAS) (Ribi™) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™). Immunochem, Hamilton, Mont.; (3) saponin adjuvants that can be used, such as QS-21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), or particles produced therefrom, such as ISCOMs (immunostimulating complexes); (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as adjuvants to enhance the effectiveness of the composition. In a preferred embodiment, the adjuvant is a saponin adjuvant, i.e., QS-21. In some embodiments, the composition does not include an adjuvant. In some embodiments, the composition further comprises lipid nanoparticles.In some embodiments, the composition is formulated in nanoparticles. In some embodiments, the composition further comprises a cationic or polycationic compound, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL).

[0355] Each of the immunogenic compositions discussed herein can be used alone or in combination with one or more antigens, the latter from the same viral pathogen or from a different pathogenic source or sources. These compositions can be used for prophylactic (to prevent infection) or therapeutic (to treat disease after infection) purposes.

[0356] In one embodiment, the composition may contain a "pharmaceutically acceptable carrier," which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those skilled in the art. Additionally, these carriers may function as adjuvants. Furthermore, antigens can be conjugated to bacterial toxoids, such as toxoids derived from pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori (H. pylori).

[0357] In one embodiment, the composition comprises a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0358] The compositions described herein may optionally contain an immunologically effective amount of the polypeptide or polynucleotide, as well as any other components described above. By "immunologically effective amount," it is meant that administration of that amount to an individual, either in a single dose or as part of a series, is effective to elicit an immune response. The elicited immune response may be sufficient, for example, to treat and / or prevent and / or reduce the incidence of an illness, infection, or disease. This amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by routine testing.

[0359] The composition can be administered parenterally, for example, by injection, subcutaneously or intramuscularly. In some embodiments, the composition is administered to a mammal by intradermal or intramuscular injection. Additional formulations suitable for other administration modes include oral and pulmonary formulations, nasal formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. Dosage treatment may be a single-dose schedule or a multiple-dose schedule. The immunogenic composition can be administered together with other immunomodulators.

[0360] In another aspect, the present invention provides methods for eliciting an immune response against cytomegalovirus, comprising administering to a subject in need thereof an immunologically effective amount of a polypeptide and / or immunogenic composition described herein, comprising the protein, DNA molecule, RNA molecule (e.g., a self-replicating RNA molecule), or VRP. In certain embodiments, the immune response comprises the production of neutralizing antibodies against CMV.

[0361] The immune response may include a humoral immune response, a cellular immune response, or both. In some embodiments, the immune response is induced against each delivered CMV protein. The cellular immune response may include a helper T cell (Th) response, a CD8+ cytotoxic T cell (CTL) response, or both. In some embodiments, the immune response includes a humoral immune response, and the antibody is a neutralizing antibody.

[0362] Neutralizing antibodies block viral infection of cells. CMV infects epithelial cells and also infects fibroblasts. In some embodiments, the immune response reduces or prevents infection of both cell types. The neutralizing antibody response may be complement-dependent or complement-independent. In some embodiments, the neutralizing antibody response is complement-independent. In some embodiments, the neutralizing antibody response is cross-neutralizing; i.e., antibodies generated against the administered composition neutralize CMV virus strains other than the strain used in the composition.

[0363] The polypeptides and / or immunogenic compositions described herein may also elicit an effective immune response to reduce the likelihood of CMV infection in uninfected mammals or to reduce symptoms in infected mammals, e.g., to reduce the number of outbreaks, CMV shedding, and the risk of spreading the virus to other mammals.

[0364] In one aspect, the present invention relates to a method for reducing viral shedding of CMV in a mammal. In some embodiments, the present invention relates to a method for reducing viral shedding of CMV in urine in a mammal. In some embodiments, the present invention relates to a method for reducing viral shedding of CMV in saliva in a mammal. In another aspect, the present invention relates to a method for reducing viral titer of CMV in a mammal. In one aspect, the present invention relates to a method for reducing CMV nucleic acid in serum in a mammal. The term "viral shedding" is used herein in accordance with its plain and ordinary meaning in medicine and virology and refers to the production and release of virus from infected cells. In some embodiments, the virus is released from cells of the mammal. In some embodiments, the virus is released from an infected mammal into the environment. In some embodiments, the virus is released from cells within the mammal.

[0365] In one aspect, the present invention relates to a method for reducing CMV viral shedding in a mammal. The method comprises administering a modified CMV gB polypeptide and / or immunogenic composition described herein to a mammal infected with or at risk of infection with CMV. In one embodiment, the reduction in CMV viral shedding in the mammal is compared to viral shedding in a mammal that has not been administered the modified CMV gB. In another embodiment, the reduction in CMV viral shedding in the mammal is compared to viral shedding after administration of a CMV pentamer alone or after administration of a CMV pentamer in the absence of the polypeptide.

[0366] In some embodiments, the mammal is a human. In some embodiments, the human is a child, e.g., an infant. In some other embodiments, the human is a woman, including an adolescent female, a woman of childbearing age, a woman planning a pregnancy, a pregnant female, and a woman who has recently given birth. In some embodiments, the human is a transplant patient.

[0367] In one embodiment, the challenge cytomegalovirus strain is a human CMV strain. In one embodiment, the challenge cytomegalovirus strain is allogeneic to the CMV strain from which the polypeptide is derived. In another embodiment, the challenge cytomegalovirus strain is allogeneic to CMV strain VR1814. In another embodiment, the challenge cytomegalovirus strain is allogeneic to CMV strain Towne.

[0368] In one embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the CMV strain from which the modified CMV gB polypeptide is derived. In another embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the VR1814 CMV strain. In another embodiment, the challenge cytomegalovirus strain is the VR1814 CMV strain. In another embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the CMV strain Towne. In another embodiment, the challenge cytomegalovirus strain is the CMV strain Towne.

[0369] In another embodiment, the challenge cytomegalovirus strain is a rhesus CMV strain that is homologous to Macaque herpesvirus 3 isolate 21252 CMV strain. In another embodiment, the challenge cytomegalovirus strain is Macaque herpesvirus 3 isolate 21252 CMV strain.

[0370] A useful measure of antibody potency in the art is the "50% neutralization titer." Another useful measure of antibody potency is any one of the following: "60% neutralization titer," "70% neutralization titer," "80% neutralization titer," and "90% neutralization titer." For example, to determine the 50% neutralization titer, serum from an immunized animal is diluted to assess how diluted the serum retains its ability to block 50% of infectious viruses from entering cells. For example, a titer of 700 means that the serum retained its ability to neutralize 50% of infectious viruses after being diluted 700 times. Thus, a higher titer indicates a stronger neutralizing antibody response. In some embodiments, the titer is in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The 50%, 60%, 70%, 80%, or 90% neutralization titer ranges are about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 1100 The upper limit may be about 0, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000. For example, a 50% neutralization titer may be about 3,000 to about 6,500. "About" means plus or minus 10% of the stated value. Neutralization titers can be measured as described in the specific examples below.

[0371] An immune response can be stimulated by administering a protein, DNA molecule, RNA molecule (e.g., a self-replicating RNA molecule or a nucleoside-modified RNA molecule), or VRP to an individual, typically a mammal, including a human. In some embodiments, the induced immune response is a protective immune response, i.e., the response reduces the risk or severity or clinical outcome of CMV infection. Stimulation of a protective immune response is particularly desirable in certain populations at risk for CMV infection and disease. For example, at-risk populations include solid organ transplant (SOT) patients, bone marrow transplant patients, and hematopoietic stem cell transplant (HSCT) patients. VRP can be administered to transplant donors before transplantation or to transplant recipients before and / or after transplantation. Because vertical transmission from mother to child is a common source of infection in infants, administration of VRP to pregnant women or women who may become pregnant is particularly useful.

[0372] The administration of the compositions provided by the present disclosure, such as pharmaceutical compositions, can be carried out using standard administration routes. Any suitable administration route can be used. For example, the compositions can be administered intramuscularly, intraperitoneally, subcutaneously, or transdermally. Some embodiments will be administered via intramucosal routes, such as oral, nasal, vaginal, and rectal. The compositions can be administered according to any suitable schedule.

[0373] Also provided herein is a method of inhibiting cytomegalovirus entry into a cell, comprising contacting the cell with an immunogenic composition described herein.

[0374] In one aspect, the present invention relates to compositions comprising the above-described polypeptides. In another aspect, the present invention relates to compositions comprising nucleic acid molecules or vectors encoding such polypeptides. In a further aspect, the present invention relates to compositions comprising the above-described polypeptides and nucleic acid molecules or vectors encoding such polypeptides.

[0375] In some embodiments, the composition is an immunogenic composition that can induce an immune response against CMV in a subject. In some specific embodiments, the immunogenic composition is a pharmaceutical composition comprising a polypeptide provided by the present disclosure and a pharmaceutically acceptable carrier. In yet other embodiments, the pharmaceutical composition is a vaccine.

[0376] In some embodiments, a composition, such as an immunogenic composition or vaccine, comprises two or more different polypeptides described above. The two or more different polypeptides may contain the same introduced amino acid mutations, but may be derived from gBs derived from different HCMV strains or subtypes. In another embodiment, the two or more different polypeptides may contain different amino acid mutations compared to each other in native HCMV gB.

[0377] In preferred embodiments, the polypeptide is soluble in an aqueous solution, hi some embodiments, the polypeptide is soluble in a solution lacking detergent.

[0378] Antibodies and diagnostic uses The above polypeptides can be used to produce both polyclonal and monoclonal antibodies. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, guinea pig, horse, etc.) is immunized with an immunogenic polypeptide bearing a CMV epitope. Serum from the immunized animal is collected and processed according to known procedures. If the serum containing polyclonal antibodies against the CMV epitope contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

[0379] Those skilled in the art can easily produce monoclonal antibodies against CMV epitopes. General methods for producing monoclonal antibodies by hybridomas are known. Immortal antibody-producing cell lines can be created by cell fusion or by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against CMV epitopes can be screened for various properties, i.e., isotype, epitope affinity, etc.

[0380] Antibodies, both monoclonal and polyclonal, directed against CMV epitopes are particularly useful in diagnosis, and those that are neutralizing are useful in passive immunotherapy. In particular, monoclonal antibodies can be used to raise anti-idiotypic antibodies.

[0381] Both polypeptides immunologically reactive with serum containing CMV antibodies and antibodies raised against these polypeptides can be useful in immunoassays to detect the presence of CMV antibodies or the presence of the virus in biological samples, including, for example, blood or serum samples. The design of immunoassays is subject to numerous variations, a variety of which are known in the art. For example, an immunoassay may utilize a polypeptide having a sequence set forth in any one of SEQ ID NOS: 2-106.

[0382] Alternatively, immunoassays may use a combination of viral antigens derived from the polypeptides described herein. For example, they may use monoclonal antibodies against at least one polypeptide described herein, a combination of monoclonal antibodies against the polypeptides described herein, monoclonal antibodies against different viral antigens, polyclonal antibodies against the polypeptides described herein, or polyclonal antibodies against different viral antigens. Protocols may be, for example, based on competitive or direct reactions, or may be sandwich-type assays. Protocols may also use, for example, solid supports or may rely on immunoprecipitation. Many assays involve the use of labeled antibodies or polypeptides; labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signal from the probe are also known; examples include assays utilizing biotin and avidin, as well as enzyme-labeled and enzyme-mediated immunoassays, such as ELISA assays.

[0383] Kits suitable for immunodiagnosis and containing suitable labeled reagents are constructed by packaging the appropriate materials, including a polypeptide of the invention containing a CMV epitope or an antibody to the epitope, in a suitable container, along with the remaining reagents and materials required for carrying out the assay, and a suitable set of instructions for using the assay.

[0384] Polynucleotide probes can also be packaged in diagnostic kits. Diagnostic kits include probe DNAs that can be labeled; alternatively, probe DNAs can be unlabeled, and labeling components can be included in the kit. Kits can also contain other suitable packaged reagents and materials required for specific hybridization protocols, such as standards, and instructions for carrying out tests.

[0385] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or immunogenic fragment thereof and at least one 5'-end cap. In some embodiments, the 5'-end cap is 7mG(5')ppp(5')NlmpNp.

[0386] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or immunogenic fragment thereof, the at least one ribonucleic acid (RNA) polynucleotide having at least one chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide further comprises a second chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide having at least one chemical modification has a 5'-terminal cap. In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0387] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0388] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame have a chemical modification, and optionally, the vaccine is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine.

[0389] Some embodiments of the present disclosure provide an HCMV vaccine formulated in cationic lipid nanoparticles, also referred to herein as ionizable cationic lipid nanoparticles, ionizable lipid nanoparticles, and lipid nanoparticles, which are used interchangeably. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyric acid (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid nanoparticles have a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. In some embodiments, the nanoparticles have a polydiversity value of less than 0.4. In some embodiments, the nanoparticles have a net neutral charge at neutral pH. In some embodiments, the nanoparticles have an average diameter of 50-200 nm.

[0390] In some embodiments, 80% of the uracils in the open reading frame have a chemical modification. In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine, N1-ethylpseudouridine. In some embodiments, the vaccine is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0391] Some embodiments of the present disclosure provide a method for inducing an antigen-specific immune response in a subject, the method comprising administering to the subject an HCMV RNA vaccine in an amount effective to generate an antigen-specific immune response. In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response. In some embodiments, the antigen-specific immune response comprises a T cell response and a B cell response. In some embodiments, the method for generating an antigen-specific immune response comprises a single administration of the vaccine. In some embodiments, the method further comprises administering a booster dose of the vaccine to the subject. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0392] Also provided herein is an HCMV RNA vaccine for use in a method for inducing an antigen-specific immune response in a subject, the method comprising administering to the subject an amount of the vaccine effective to generate an antigen-specific immune response.

[0393] Further provided herein is the use of an HCMV RNA vaccine in the manufacture of a medicament for use in a method for inducing an antigen-specific immune response in a subject, the method comprising administering to the subject an amount of the vaccine effective to generate an antigen-specific immune response.

[0394] Further provided herein is a method for preventing or treating HCMV infection, comprising administering to a subject a vaccine of the present disclosure. The HCMV vaccine disclosed herein can be formulated in an effective amount to generate an antigen-specific immune response in a subject.

[0395] The term "polypeptide variant" refers to a molecule that differs in its amino acid sequence from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least 50% identity to the native or reference sequence. In some embodiments, variants have at least 80% or at least 90% identity to the native or reference sequence.

[0396] In some embodiments, a "variant mimic" is provided. As used herein, the term "variant mimic" refers to a mimic containing at least one amino acid that mimics an activated sequence. For example, glutamate can act as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, a variant mimic can result in inactivation or in an inactivated product containing the mimic; for example, phenylalanine can act as an inactivating substitution for tyrosine; or alanine can act as an inactivating substitution for serine. "Ortholog" refers to genes in different species that have evolved from a common ancestral gene by speciation. Orthologs typically retain the same function during evolution. Identifying orthologs is important for reliable prediction of gene function in newly sequenced genomes. "Analog" is meant to include polypeptide variants that differ by one or more amino acid changes, such as substitution, addition, or deletion of amino acid residues, while still maintaining one or more properties of the parent or starting polypeptide.

[0397] The present disclosure provides several types of polynucleotide or polypeptide-based compositions, including variants and derivatives. These include, for example, substitution, insertion, deletion and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that is modified and / or changed in any way compared to a reference or starting molecule.

[0398] Therefore, peptides or polypeptides containing substitutions, insertions, and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly polynucleotides encoding the polypeptide sequences disclosed herein, are within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to a peptide sequence (e.g., at the N- or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase the solubility of the peptide or to enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of a peptide or protein amino acid sequence can be deleted, as needed, to provide a truncated sequence. Alternatively, certain amino acids (e.g., C- or N-terminal residues) can be deleted depending on the use of the sequence, for example, for expression of the sequence as part of a larger sequence that is soluble or linked to a solid support. A "substitutional variant" when referring to a polypeptide is one in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid has been inserted in its place. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0399] In some embodiments, the mutant CMV gB polypeptide is a truncated polypeptide that lacks one or more of the following domain sequences compared to SEQ ID NO: 1: (1) domain V (residues 124-344), (2) MPR domain (residues 705-750), (3) TM domain (residues 751-772), or (3) CT domain (residues 773-907). As used herein, the term "truncated" means that the sequence is missing some or all of the residues that comprise the domains described herein.

[0400] As described herein, a CMV gB polypeptide comprises the following domains and residues (SEQ ID NO: 1): (i) Domain I (residues 134-344), (ii) Domain II (residues 121-133 and 345-436), (iii) Domain III (residues 97-111, 475-539 and 640-648), (iv) Domain IV (residues 88-96, 540-639 and 551-641), (v) Domain V (residues 649-707), (vi) Membrane Proximal Region (MPR) (residues 705-750), (vii) Transmembrane Domain (TM) (residues 751-772), and (viii) Cytoplasmic Domain (CT) (residues 773-907). In another embodiment, the extracellular domain of CMV gB comprises residues 1-707 or 23-707 (excluding the signal sequence) of SEQ ID NO: 1. In another embodiment, the extracellular domain lacks domain V and includes residues 1-646 or 23-646 (not including the signal sequence). [Example]

[0401] The present invention will now be further described by the following illustrative examples, which do not limit the invention in any way and serve only to clarify the invention.

[0402] Example 1 Cross-linking with fusion inhibitors and isolation and purification of native HCMV gB (Towne strain) During HCMV sample preparation, a fusion inhibitor (compound 28 described in Bloom et al., Bioorganic & Medicinal Chemistry Letters, 14 (2004) 3401-3406; see also Figure 5D) was added at each step during virus concentration, processing, extraction, and purification to inhibit the conversion of gB to its post-fusion form.

[0403] Proteins on the virion surface are isolated using bis(sulfosuccinimidyl) glutarate (BS) 2G), gB was extracted from the virion using detergent, and then SM5-1 His / Strep-tagged Fab (Potzsch et al., PLoS pathogens, 7(8):e1002172, 2011) was added to aid in the purification and identification of gB by electron cryomicroscopy. The Fab-gB complex was purified by affinity column.

[0404] These extracted and purified proteins were then analyzed for the presence of prefusion gB by electron cryomicroscopy and used to solve the structure of the prefusion form.

[0405] Example 2 Electron microscope observation Electron microscopy grids supported on graphene oxide films were prepared. The gB sample solution was vitrified using a Vitrobot (ThermoFisher). The frozen grids were transferred to an FEI Titan Krios transmission electron microscope operated at 300 kV. The target position was set in the SerialEM program, and high-magnification (18,000x) images were automatically collected by the program using a K2 direct detection camera (Gatan) using super-resolution movie mode. The unbinned pixel size was 0.638 Å, and the beam intensity was approximately 8 e / unbin pixel / sec. The total electron dose to the sample for each movie was approximately 40 e / Å. 2 A total of 7,771 videos, each with 28 frames, were collected over three sessions.

[0406] Image Processing Drift correction was performed using the MotionCor2 program (Zheng S. et al., Nature Methods, 14, 331-332 (2017)). Final micrographs were binned at 2x and averaged across all frames. Contrast transfer function parameters were calculated using Gctf (Kai Zhang, Journal of Structural Biology, 193(1), 1-12 (2016)). For particle selection, a 30 Å density map was generated using pdb2mrc (EMAN) using the published structure of HCMV gB in the post-fusion conformation (PDB:5CXF) (Ludtke, S. et al., Journal of Structural Biology, 128(1), 82-97 (1999)). A projection from this density map was created in project3d (EMAN) (Figure 1) and used as a template for automated particle selection using the Gautomatch program (Urnavicius L et al., Science, 347(6229):1441-1446 (2015)). Relion v2.1-beta (Scheres, SH, Journal of Structural Biology, 180(3):519-530 (2012)) was used to extract the resulting ~1.9 million particles and perform all subsequent image processing steps, including 2D classification, 3D classification, automated refinement, and post-processing. The 2D classes were divided into three groups based on image features. The first group consisted of 2D classes that displayed features resembling the crystallographically determined post-fusion gB structure (>50%). The second group contained 2D classes with well-resolved protein features that did not resemble structural features from post-fusion gB (<10%). The third group contained 2D classes that did not contain well-defined proteins (approximately 40%) (Figure 1). The first and second groups were further processed using Relion for 3D classification, automatic refinement, and post-processing. After this processing, an electron density map at approximately 3.5 Å resolution, representing the post-fusion conformation, was reconstructed from the first group, and an electron density map at approximately 3.6 Å resolution, representing the pre-fusion conformation, was reconstructed from the second group.Based on these density maps and the known HCMV gB amino acid sequence (Towne strain P13201, SEQ ID NO: 1), atomic models were constructed using the Coot program for the pre- and post-fusion conformational structures (Emsley P. et al., Acta Crystallogr D Biol Crystallogr, 66(Pt4):486-501(2010)). The post-fusion gB crystal structure (PDB accession code 5CXF) and the crystal structure of the complex between SM5-1 Fab and gB domain II (PDB accession code 4OT1) were used as initial models for both structures. For the post-fusion structural model, fine-tuning was sufficient to obtain a good fit to the electron density. For the pre-fusion conformational model, domains I, II, III, and IV from the reference PDB model were docked as rigid bodies into the electron density map as a starting point. Individual residue adjustments were then performed to obtain an optimal fit. Models for domains V, MPR, and TM were constructed de novo. These models were iteratively refined with the Phenix.real_space_refine tool (Afonine PV et al. Acta Crystallogr D Struct Biol, 74(Pt6):531–544(2018)), followed by several rounds of local manual adjustments.

[0407] result Sample screening by cryo-EM The pre-fusion conformation of gB is unstable and tends to rearrange into the post-fusion state, including during sample handling. Therefore, the studied samples contained a mixture of gB conformers, complicating structure determination. Furthermore, there was no prior reliable information on the domain arrangement or unique structural features of pre-fusion gB. We screened different sample preparation conditions using direct visualization by electron microscopy and image processing. Image sorting by 2D and 3D classification allows for the determination of multiple structures from heterogeneous samples. However, this requires large datasets so that sufficient particles for each structure can be combined to produce class averages with good signal. This was particularly true in the case of gB samples, since pre-fusion gB was a minor component of the mixture. Therefore, we collected approximately 1,000 movies for each state to determine whether to continue image processing with more data from the same sample or switch to a different one at the 2D classification stage. The structure of gB in the post-fusion conformation bound to antibody Fab was easily obtained from many datasets. Projections from these Fab-bound post-fusion conformational structures were used as references to avoid selecting images from the pre-fusion reconstruction. We selected all good class averages with protein features that did not resemble any of the post-fusion gB projections for further image processing. Using this strategy, we screened dozens of conditions for sample preparation, ultimately finding a sample that yielded a number of alternative 2D classes as minor species in the particle population (Figure 1B, circled). A total of 7,771 movies were then collected from that sample and used to determine the pre-fusion gB structure.

[0408] A projection of the post-fusion gB structure bound to antibody Fab is shown in Figure 1A. 2D class averages from the collected dataset are shown in Figure 1B. Some classes that do not resemble any of the post-fusion gB reference 2D projections are circled.

[0409] Acquisition of pre-fusion conformational structure Approximately 1.9 million raw particle images were automatically selected from the dataset. After 2D classification, the images were grouped into post-fusion (55% of the particle population) and pre-fusion (10% of the particle population) classes. The two groups were further processed in 3D by applying C3 symmetry to obtain density maps of post-fusion gB bound to SM5-1 Fab at 3.5 Å resolution and pre-fusion gB bound to SM5-1 Fab at 3.6 Å resolution.

[0410] X-ray crystallography-based models of the SM5-1 Fab and the ectodomain of postfusion gB were fitted to the postfusion density map using rigid-body docking. Except for the constant domain of the Fab, which is likely too flexible to generate strong electron density, the density map and model of the postfusion gB-Fab complex were in good agreement with each other (Fig. 3A). The membrane-proximal, transmembrane, and cytoplasmic domains were not resolved in our final postfusion gB density map, suggesting that these regions of postfusion gB are flexible, either intrinsically or through detergent solubilization in sample preparations (Fig. 2, bottom row). The interaction of the Fab with DII in the electron cryomicroscopy-based model of postfusion gB is in good agreement with the previously determined crystal structure of the complex (PDB accession code 4OT1).

[0411] To construct a prefusion gB model guided by the known Fab binding locations, domains I, II, and III and a portion of domain IV from the postfusion gB crystal structure were individually docked into the density map of the prefusion gB-Fab complex, and individual residues were manually adjusted as needed for optimal fit of the electron density. The remainder of the prefusion gB structure was constructed de novo. The gB amino acids modeled in the prefusion structure are shown in the top row of Figure 2. The model of the prefusion gB-Fab complex fit most of the prefusion density map, and the presence of Fab density confirmed the identity of gB in the new structure (Figure 3B).

[0412] The coordinates and structure factors of the model of pre-fusion gB relevant to this example are provided in Table 1A.

[0413] Structure of gB in the prefusion conformation and comparison with postfusion gB The electron density for the complex of prefusion gB with SM5-1 Fab allowed the construction of a prefusion gB model that included the gB ectodomain, the membrane-proximal region (MPR—a helical region oriented parallel to the viral membrane), and the single-span transmembrane helix (TM) (Fig. 3B and Fig. 4B). The MPR and TM regions were not resolved in the structural data for postfusion gB and were not included in the postfusion gB model.

[0414] The overall dimensions of pre-fusion and post-fusion gB are different (Figure 4A vs. Figure 4B). The post-fusion gB trimeric ectodomain has a rod shape, with an approximate height of 165 Å (the distance between the planes formed by proline 570 of each protomer at the membrane-distal end and tryptophan 240 of each protomer at the membrane-proximal end, Figure 4A). Its width is approximately 65 Å (the distance between alanine 315 on adjacent protomers). The structure described here was derived from gB of the HCMV Towne strain. Although some natural variation in the gB amino acid sequence exists, the overall post-fusion structure of Towne gB is nearly identical to the post-fusion structure of gB from the AD169 strain (PDB accession code 5CXF). Therefore, the description of the post-fusion gB structure applies to both strains using measurements from equivalent amino acids from sequence alignments.

[0415] The prefusion gB trimer has a chunkier shape than the postfusion gB trimer (Figure 4A vs. Figure 4B). The distance between the plane formed by W240 of each protomer and Q483, the most membrane-distal modeled residue in the prefusion structure, is approximately 115 Å. The width of the prefusion model is 95 Å (measured by the distance between any two A315 residues from different protomers).

[0416] The individual subunit structures of domains I, II, III, and IV are similar in the pre-fusion and post-fusion conformations. However, the overall arrangement of these domains is very different in the two conformations (Figures 4A-4B and 6A-6C). In the pre-fusion conformation, the fusion loop at the tip of the DI and the C-terminus of the central helix bundle in domain III all point in the same direction toward the virion envelope, as identified by the position of the TM region (Figures 4A and 6A). In contrast, in the post-fusion conformation, the fusion loop and the C-terminus of the central helix bundle point in opposite directions (Figures 4B and 6C).

[0417] In the prefusion structure, hydrophobic residues in the fusion loop (residues Y155, I156, H157, and W240, L241) are in close proximity to the MPR and are likely surrounded by surfactant (Figures 4A and 6A).

[0418] During the pre- to post-fusion transition, domain II shifts from a position up in the middle of the central coiled-coil of domain III to a position at the membrane-proximal end of the coiled-coil and the proximal end of domain I, opposite the fusion loop (Figure 4A and Figure 4B).

[0419] The structures of DIII (Figures 4A-4B and 6A-6C) are very similar in the pre- and post-fusion conformations. The central helix in both conformations shows minimal rearrangements during the pre- to post-fusion transition, spanning L479 to P525. However, the other domains change their position relative to the central helix of domain III, as noted above, such that the orientation of the DIII helix bundle (N- to C-terminus) points away from the fusion loop and toward the distal end of the trimer in the post-fusion conformation and toward the viral membrane, in the same direction as the fusion loop in the pre-fusion conformation.

[0420] In the pre-fusion structure, domain IV (Figures 4A and 6A) is buried at the interface between domain I on the exterior of the trimer and domains III and V in the center of the trimer. In contrast, in the post-fusion structure, domain IV forms a highly exposed "crown" at the membrane-distal tip of the trimer.

[0421] Domain V has a different structure in pre-fusion gB (Figures 4A and 6A) and post-fusion gB (Figures 4B and 6C). In pre-fusion gB, the N-terminal half of the domain (approximately residues 642-660) is sandwiched between domains I and IV of the adjacent protomer, isolating it from the solvent. The region from residues 683 to 704 of domain V forms a trimeric helix bundle with its counterpart in the other protomer. This helix bundle is mostly nested within the "crown" pocket formed by domain IV. An additional short helix (approximately residues 710-719) connects the helix bundle from domain V with the MPR region. In contrast, in the post-fusion conformation (Figures 4B and 6C), domain V is solvent-exposed, extending outside the domain III helix bundle and along the groove formed by the boundary between domain I and the adjacent protomer.

[0422] Comparison of the pre- and post-fusion gB structures suggests a progression of conformational changes familiar from other well-studied fusion proteins (Harrison, SC, Virology, 0:498-507 (2015)). This comparison provides confirmation that the structures described herein are indeed in the pre-fusion conformation. In the pre-fusion state (Figure 6A), the fusion loop of domain I is buried by interactions with the MPR and potentially the viral membrane. In the pre-fusion structure of the vesicular stomatitis virus G glycoprotein, a distant gB homolog, the fusion loop also points toward the viral membrane (also in the predicted location of the MPR region, which is not seen in this structure) (Roche et al., Science, 315:843-8 (2007)).

[0423] Based on similarities with other fusion proteins, it is likely that the extension of the central helix undergoes rearrangement as part of the transition between the pre- and post-fusion states to the proposed extended intermediate (Figure 6B). In the proposed extended intermediate state, the TM region is still tethered in the viral membrane, and the fusion loop, now extended away from the viral membrane at the tip of rotated and repositioned domain I, would interact with the cellular membrane. The transition from the proposed extended intermediate to the post-fusion conformation involves folding such that the transmembrane region and fusion loop are again in close proximity to each other at the same end of the molecule, this time both of which would interact with the fused viral and cellular membranes (Figure 6C).

[0424] We speculate that in pre-fusion gB there may be dynamic changes in the length of the central helix, and that the pre-fusion structure we determined represents a "snapshot" of a "breathing" molecule locked into the conformation seen in the electron density by fusion inhibitors and by the cross-linking agents used to prepare the samples studied by electron cryomicroscopy.

[0425] Stabilizing factors of the observed prefusion conformation After modeling the gB amino acids into the electron density map, an unfilled density region remained between the MPR and the tip of domain I, which contained domain V and the fusion loop (Figure 5A). The size and shape of the unfilled density fit the chemical structure of the HCMV fusion inhibitor, N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide (Figure 5D), which was present throughout the generation of samples studied by electron cryomicroscopy (Figure 5B). The compound adopted a pose with a twist between the trifluoromethylphenyl moiety and the rest of the compound. The thiazole forms contacts with hydrophobic residues L712, A738, and Y153 and Y155 from the adjacent promoter. The phenyl is surrounded by a hydrophobic environment formed by residues L715, the aliphatic hydrocarbon of D714 from domain V, G734 and I730 from the MPR, and F752 from the TM domain of the adjacent protomer. The trifluoromethylphenyl resides in the hydrophobic environment near the hinge between the MPR and TM helices from another protomer. This may act as a hook to prevent outward movement of the MPR and TM domains. In addition to the interactions coordinated by the inhibitor compound, W240 and Y242 from the other fusion loop form van der Waals interactions with the hydrophobic patch from the MPR region and L715 in domain V, respectively (Figure 5C). These specific interactions around the fusion inhibitor would be predicted to keep domain I, domain V, and MPR together and restrict movement between domains I, V, and MPR during the fusion process (Figure 6A-6C).

[0426] The effect of cross-linking on the stability of the prefusion conformation was also examined. 2 G cross-linking reagent was added or not. In the absence of cross-linking agent, the ratio of particles in the pre- to post-fusion conformation was 1:100, while in the presence of BS 2In the sample cross-linked with G reagent, the ratio was 1:4. The cross-linking agent was not identified in the electron density.

[0427] Color versions of the CMV gB pre-fusion structure and pre- and post-fusion structures depicted in the figures described herein can also be found in Liu et al., Science Advances, 7(10):eabf3178 (2021), which is hereby incorporated by reference in its entirety.

[0428] Example 3 Expression and purification of gB1666 To produce gB1666, the constructed PSB1666 was transiently transfected into Expi293F cells. Cell pellets were harvested 96 hours post-transfection. PSB1666 protein was purified through a series of steps: solubilization, affinity column chromatography, and size-exclusion chromatography in 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS, and 3 μg / ml WAY-174865 (inhibitor, see Figure 5D). Protein was analyzed on SDS-PAGE and by EM with negative staining to ensure that at least 50% of the protein exhibited the pre-fusion conformation. PSB1666 protein was efficiently expressed in transfected Expi293F cells, and 1 L of expression would yield approximately 0.1 mg of purified PSB1666 at high quality.

[0429] Polypeptide gB1666 (PSB1666) (SEQ ID NO: 57) contains mutations in domains I and IV. This polypeptide contains the following mutations compared to the corresponding wild-type gB (Towne) set forth in SEQ ID NO: 1: D217C and Y589C.

[0430] Example 4 DNA-expressed gB1666 is immunogenic in Balb / c mice. One of the proposed stabilized full-length prefusion gB constructs, gB1666 (SEQ ID NO: 57), was shown by EM to have an increased proportion of molecules in the prefusion conformation compared to wild-type gB in the Towne strain in the presence of a fusion inhibitor (WAY-174865, see Figure 5D) after purification from transfected mammalian cells. To evaluate whether this molecule could elicit an immune response in vivo, DNA sequences corresponding to gB1666 and wild-type gB were cloned into an in-house mammalian expression vector. Ten Balb / c mice were electroporated twice, 3 weeks apart (D0 and D21), with 100 μg of DNA encoding gB1666. An additional 10 mice were electroporated using the same protocol with DNA encoding wild-type gB, and a third group was electroporated with a placebo consisting of phosphate-buffered saline. Serum samples were collected on day 28. Anti-gB IgG responses were determined by ELISA against recombinant gB protein produced from mammalian cells based on the wild-type sequence of the Towne strain but with the transmembrane domain deleted (Sino Biologicals) according to standard protocols. Ten of ten animals from mice immunized with wild-type gB DNA and nine of ten mice immunized with gB1666 DNA developed detectable anti-gB IgG titers (Figure 11, showing mean ± SD, LLOQ = 25). This study demonstrates that gB1666 is immunogenic in Balb / c mice.

[0431] Example 5 Immunogenicity studies of stabilized pre-fusion gB1666 protein Immunogenicity study of gB1666 in mice. To evaluate antibody responses in mice, the following immunization scheme is followed: At week 8, mice are exsanguinated and neutralizing titers from the sera of immunized animals are determined and compared to those immunized with gB705 (post-fusion) and / or gB wild-type protein.

[0432] [Table 5]

[0433] Example 6 In Example 2, we disclose the electron cryomicroscopy (cryo-EM) structure of pre-fusion human cytomegalovirus (HCMV) Towne strain glycoprotein B (gB) complexed with an antibody fragment. The gB used for structure determination was prepared by adding the small molecule fusion inhibitor WAY-174865 to fermentations of authentic HCMV in mammalian cell culture, maintaining the presence of the inhibitor throughout gB production and analysis, purifying the virus, and cross-linking it with the chemical cross-linking agent, bis(sulfosuccinimidyl) glutarate (BS). 2The gB was obtained by treating gB with 1000-kDa antibody (G, 7.7 Å spacer arm), extracting gB from the virus using detergent, binding gB on the virion with an affinity-tagged antibody fragment, and purifying gB through affinity and sizing columns. We also disclosed the use of the cryo-EM structure of pre-fusion gB to engineer mutations that stabilize gB in the pre-fusion state. Specifically, we disclosed a recombinant gB protein, gB1666, in which two residues were mutated to cysteines (D217C, Y589C). The resulting engineered disulfide bond between C217 and C589 increases the conformational stability of recombinant gB in the pre-fusion state. gB1666 maintained pre-fusion structural characteristics when expressed in Expi293F cells and purified in the presence of the fusion inhibitor compound WAY-174865. In the absence of an inhibitor, gB1666 tends to undergo conformational changes and lose its pre-fusion structure. Loss of stability of the pre-fusion conformation in the absence of an inhibitor is not a desirable feature for using a recombinant glycoprotein as an antigen for immunization. Even when gB1666 is formulated with an inhibitor, there is a risk that in vivo dilution of the inhibitor will result in its dissociation from gB1666 and loss of the pre-fusion conformation upon injection into humans or animals. Therefore, it is desirable to sufficiently stabilize HCMV gB in the pre-fusion conformation so that it remains in the pre-fusion state in the absence of WAY-174865. It is also preferred that the pre-fusion gB immunogen contain a soluble ectodomain to improve manufacturability, solubility, and homogeneity, and to reduce or eliminate the need for formulation with detergents or other excipients to prevent aggregation or precipitation mediated by the gB transmembrane domain.

[0434] We now report, through a structure-based genetic engineering approach, new mutations in HCMV gB that confer these improved characteristics for use as an immunogen. First, we determined the structure by cryo-EM of gB1666, solubilized by tethering in Nanodiscs and stabilized in the prefusion conformation by the presence of WAY-174865 (Figure 12). While most of the new structures of the recombinant, D217C, and Y589C mutant gB resemble our previously determined structures of virion-derived, chemically cross-linked, and antibody-fragment-bound HCMV Towne prefusion gB, subtle differences exist between the two structures in specific local regions. The structural differences may reflect several differences in the preparations: first, the presence of an engineered disulfide bond in gB1666 that should restrict respiratory movements of the glycoprotein; second, the tethering of gB1666 in nanodiscs, which provides a more natural local lipid environment for the transmembrane domain than the detergent used to extract gB and keep it in solution for previous structure determination; third, the absence of chemical cross-linking of gB1666; and fourth, the higher resolution of the new structure at 3.3 Å compared to 3.6 Å of the previous structure, which allows for more accurate modeling of amino acid side chains.

[0435] Based on the new structural information, we designed additional stabilizing mutations on the full-length gB construct pSB1666 background (Tables 6 and 7). We hypothesized that adding these additional mutations to the pSB1666 background would further stabilize gB in the pre-fusion state (Figure 13). For example, cysteine ​​mutations at residues M371 and W506 may introduce a disulfide bond between domains II and III, cysteine ​​mutations at the pair (F541, E681) and (N524, M684) may introduce a disulfide bond between domains IV and V, and mutation of residues E686 and D679 to hydrophobic residues may remove a locally destabilizing patch of like charge repulsion, increasing protein stability. Recombinant glycoproteins carrying a selection of the new added mutations were expressed and purified in the absence of fusion inhibitors and without chemical cross-linking. Electrophoretic mobility of the expressed glycoproteins by SDS-PAGE showed the expected apparent molecular weight and heterogeneity consistent with glycosylation (Figure 14). Samples were stored at 4°C, and aliquots were taken for negative-stain electron microscopy analysis at days 1 and 7. Triangular-shaped features resembling the "top view" of the pre-fusion conformation of gB were evident in 2D class average images. The ratio of particles in the population belonging to the pre-fusion and post-fusion classes was 5:1 at day 1 and 3:1 at day 7 (Figure 15).

[0436] Based on the new structural information, we designed several soluble, detergent-free gB ectodomains with prefusion-stabilizing mutations, as illustrated in Figures 16A-16D (Table 8). The purified ectodomain of HCMV gB, residues 1-707, formed rosette-like aggregates in which the gB protein associated through its exposed fusion loop. To eliminate aggregation and increase protein secretion into the conditioned medium, we replaced four exposed hydrophobic residues within the fusion loop with corresponding more hydrophilic amino acids from herpes simplex virus-1 (HSV-1) gB, e.g., YIH(155-157) → GHR, W240 → A. We also mutated the exposed Cys246 to Ser (C246 → S) to prevent the formation of a spurious disulfide bond. To further stabilize the pre-fusion trimeric state of the antigen, we introduced either cysteine ​​residues capable of forming interproton disulfide bonds or an additional C-terminal trimerization motif, such as GCN4 or foldon from T4-bacteriophage fibritin. Disulfide mutations, such as D217C-Y589C, M317C-W506C, and N524C-M684C, were further introduced to lock the protein in the pre-fusion state. The recombinant glycoproteins were expressed, secreted into conditioned medium, and purified in the absence of fusion inhibitors and chemical detergents. Notably, the recombinant variants fused with the GCN4 trimerization motif exhibited optimal size-exclusion chromatography profiles (Figure 17). Negative-stain electron microscopy revealed the recombinant proteins gB2555 and gB2556 as monodisperse proteins in the absence of inhibitors and detergents. The expected gB protein features were observed in the 2D class average images (Figures 18 and 19). These results confirm that these engineered constructs are suitable for use as a framework to add, if desired, mutations that make the pre-fusion form of gB more stable in the absence of inhibitors and detergents.

[0437] The coordinates and structure factors of the model of pre-fusion gB relevant to this example are provided in Table 1B.

[0438] [Table 6]

[0439] [Table 7]

[0440] [Table 8]

[0441] The constructs listed in Table 8 were made to test the presence of pre-fusion gB in recombinant protein preparations purified under different conditions.

[0442] [Table 9]

[0443] Example 7 A global strategy for engineering stabilized prefusion gB Two strategies were used to engineer stabilized pre-fusion gB: i) The first strategy is to strengthen interactions in the pre-fusion structure, including engineering disulfide bonds between residues that are in close proximity in the pre-fusion structure and grafting stable structural motifs to the C-terminus without disrupting the structure from other parts of the molecule; ii) The second strategy is to remove energetically unfavorable local structures from the pre-fusion conformation. These include (a) removal of surface-exposed hydrophobic residues, (b) changing proline to non-proline and hydrophilic residues in the loop region, and (c) removing and re-engineering domain V, which undergoes a large conformational change during the fusion process. Using a combination of such designs, we obtained sufficient stabilization for pre-fusion gB. Proteins carrying such designed mutations were individually purified and screened by their characteristics in size-exclusion chromatography (SEC), thermal melting assays (TM), and electron microscopy (EM) images.

[0444] A panel of mutant cysteine ​​pair constructs was screened against the CMV gB full-length post-fusion construct pSB1764. In the first round, each new construct contained one mutant cysteine ​​pair. A complete list of the mutants screened is in Table 10.

[0445] [Table 10]

[0446] [Table 11-1]

[0447] [Table 11-2]

[0448] The screen determined that the cysteine ​​pair D217C-Y589C (pSB1666), in combination with the inhibitor WAY-174865, stabilized CMV gB in the prefusion state.

[0449] In the presence of fusion inhibitors, construct gB1666 (engineered disulfide by mutating D217C and Y589C) showed a right-shift in SEC retention volume and a distinct transition temperature at approximately 73 °C, indicating a conformation different from that of wild-type gB (Figures 22A and 22B). gB1666 was further characterized by EM imaging experiments. 2D classification results of the cryo-EM dataset showed the pre-fusion class as the dominant population, and 3D structural density maps confirmed the pre-fusion gB structure (data not shown). However, in the absence of inhibitors, the right-shifted peak from SEC and the transition temperature peak at approximately 73 °C were less distinct (Figure 23). This suggests that the single disulfide bond had limited effect on maintaining gB1666 in the pre-fusion state.

[0450] Therefore, a second round of mutations was screened in frame with gB1666 to create further stability of the pre-fusion conformation in the absence of inhibitors (Table 11). A new panel of mutants was cloned and screened that introduced a second pair of cysteines into construct pSB1666. The goal was to add another stabilizing disulfide bridge to further stabilize CMV gB in the pre-fusion state.

[0451] [Table 12]

[0452] [Table 13]

[0453] This screen determined that the cysteine ​​pair D217C-Y589C, combined with either the pair M371C-W506C (pSB2457) or the pair N524C-M684C (pSB2459), stabilized CMV gB in the prefusion state without the need for a stabilizing inhibitor. The gB2457 and gB2459 proteins exhibited profiles similar to those of the prefusion form (gB1666 + inhibitor) in SEC and TM analyses (Figures 24A-24B). gB2457 had a more pronounced transition at approximately 73°C in the thermal stability assay (Figure 24B). Furthermore, both gB2457 and gB2459 exhibited features of prefusion gB in negative-stain EM 2D class average images (data not shown). In the absence of fusion inhibitors, gB2457 and gB2459 represent more stable versions of pre-fusion gB than their parental design, gB1666. Such improvements also confirm that the correct combination of stabilizing mutation sites can contribute synergistically to the overall stability of the pre-fusion conformation.

[0454] Compared with the full-length and membrane-bound forms of pre-fusion gB, the soluble form of pre-fusion gB does not require detergents, offering advantages in scalability of protein production and ease of purification. Based on the known structure, the membrane-interacting domains from gB are the membrane proximal region (MPR), the transmembrane (TM) domain, and the fusion loop. The cytoplasmic terminal domain may also interact with membranes from the cytosol. Therefore, these membrane-interacting hydrophobic regions were either removed or converted to hydrophilic forms in the design of the soluble form of pre-fusion gB. Additionally, a structurally stable trimerization tag (e.g., GCN4, cysteine ​​ring, trimer foldon) was added to the carboxy terminus of the truncated protein before the MPR domain. After testing several truncation designs, the GCN4 tag (gB2267) was selected for use as the ectodomain construction frame. Stabilizing mutation sites identified from the full-length frame were generated and screened on this ectodomain frame (Table 12).

[0455] For the resulting proteins, gB2555 and gB2556, which were well-behaved after purification (Figure 25A), the lower phase transition peak at approximately 73°C was not evident (Figure 25B). However, 2D classification from negative-stain EM images indicated that both pre- and post-fusion forms were present (data not shown), indicating that further stabilization was still required for the ectodomain pre-fusion gB. A third pair of disulfide bonds was screened against the pSB2556 background (Table 12), but no significant improvement was observed.

[0456] [Table 14]

[0457] [Table 15]

[0458] The novel approach was to remove domain V from the ectodomain gB because domain V was predicted to undergo a large conformational change and provide the energy to drive membrane fusion between the virus and the host cell. SB2562 was selected as the starting construct (Tables 13 and 14).

[0459] [Table 16]

[0460] One of the designs removed the entire domain V, truncating the residue after D646. Because a disulfide pair could no longer form at N524C and M684C, N524C was reverted to its wild-type asparagine residue (N) to eliminate the free cysteine ​​(pSB2796) (Table 14). This construct was designated gB2796, and a well-behaved protein was demonstrated as a single peak in the SEC profile with a distinct phase transition temperature at approximately 70°C (Figures 26A and 26B). From the cryo-EM dataset, 2D class averages showed a clear, predicted protein density that corresponded to structural features from the model of the pre-ectodomain gB. The 3D reconstructed density envelope also corresponded to the predicted ectodomain structure (data not shown).

[0461] [Table 17]

[0462] Construct pSB2796 was redesigned into different N-terminally tagged versions for use in animal studies (Table 15). The Flag tag was exchanged for a 6xHis tag, which is more amenable to scale-up of purification. An untagged version was also made. The HA signal sequence was also replaced with an IgK signal sequence because it is more compatible with the His tag.

[0463] [Table 18]

[0464] These data demonstrated that pre-fusion gB is metastable and requires stabilizing mutations at multiple sites to achieve sufficient constraint to maintain its pre-fusion structure. Disulfide bond combinations and modifications of the unstable domain V provide examples of locking the pre-fusion conformation. Stabilized gB can be engineered in full-length (gB1666, gB2457, gB2459) and ectodomain protein formats (gB2796), making them suitable for use as pre-fusion gB antigens. Furthermore, SEC, thermal shift assays, and EM imaging techniques provided methods to assess the conformational state of gB samples without the need for specific antibodies for pre- or post-fusion.

[0465] method Design and cloning of cysteine ​​mutant constructs All constructs were cloned into the pcDNA3.1(+) vector backbone (ThermoFisher Scientific, Waltham, MA). Primers containing each cysteine ​​point mutation were ordered from IDT (Coralville, Iowa). Site-directed mutagenesis was performed using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilient, Santa Clara, CA). Mutagenized DNA was transformed into DH5 alpha cells. Colonies were selected and subjected to sequencing. Positive transformants were confirmed by DNA sequencing analysis. Plasmid DNA was amplified using a DNA preparation kit.

[0466] Mammalian cell expression 3 × 10 EXPI293 cells 6 The cells were grown to an OD of 100 cells / mL. Plasmid DNA was diluted in OptiMem medium and mixed 1:1 with PEI solution. The DNA-PEI mixture was then transfected into EXPI293 cells at a concentration of 1µg of DNA per mL of culture. Enhancer was added 24 hours after transfection. Cells were monitored and harvested 4-5 days after transfection by centrifugation.

[0467] Protein purification CMVgB protein was purified through a series of processes: solubilization, affinity column chromatography, and size exclusion chromatography (SEC). SEC was performed on a Superose 6 increase column in a buffer of 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, and 0.002% CHS. In experiments using inhibitors, 3 μg / ml of WAY-174865 was added.

[0468] Thermostability assay As a complementary biophysical study of gB mutants to aid in the identification of prefusion stable mutants, the thermal stability of purified gB protein was analyzed in a Tycho NT.6 from 35°C to 95°C using a heating rate of 20°C / min.

[0469] Preparation of negative stained grids Thin carbon-supported grids were glow-discharged for 30 seconds using an EZ-glow at -20 mA before use. A 4 μL aliquot of sample solution with a protein concentration of 0.02 mg / ml was applied to the carbon surface of the grid and allowed to stand for 45 seconds. The sample solution was then blotted with filter paper, and the same carbon surface was rinsed with filtered water. The excess water was then blotted with filter paper. The grids were stained with a 2% uranyl acetate solution, air-dried, and mounted in a TF20 electron microscope for imaging.

[0470] Cryogrid preparation Quantifoil grids were plasma-cleaned with argon / oxygen. A graphene oxide stock solution (Sigma) diluted to a concentration of 0.2 mg / ml was applied to the surface of the treated grid and allowed to stand for 2 minutes. Excess solution was blotted with filter paper and washed with a drop of water to remove excess graphene oxide. The grids were allowed to dry overnight before use. Aliquots of the sample solution were vitrified onto the graphene oxide film-supported grids using a Vitrobot (ThermoFisher). The grids were stored in liquid nitrogen until they were mounted on a microscope under cryogenic conditions for imaging.

[0471] Cryo-EM data collection and image processing Data collection was performed on a Thermo Fisher Titan Krios transmission electron microscope operating at 300 kV using the SerialEM program at a nominal magnification (165,000×) with a K2 direct detection camera (Gatan) in super-resolution movie mode. The unbinned pixel size was 0.434 Å, and the beam intensity was approximately 8 e / unbin pixel. The total electron dose to the sample for each movie was approximately 40 e / Ų. Both the Relion and cisTEM programs were used for data processing.

[0472] Example 8 Immunogenicity of gB2796 One of the engineered pre-fusion stabilized ectodomain gB proteins (gB2796), containing amino acids 23-646 from YIH with the GHR(155-157), D217C, W240A, C246S, M371C, W506C, and Y589C mutations, was purified from transfected mammalian cells via the FLAG tag in the absence of fusion inhibitors. Briefly, the protein was purified by adding 20 mM Tris pH 7.5, 200 mM NaCl to 3 L of cell culture medium overexpressing SB2796. After 4 hours of incubation with rotation at 4°C using a 10 ml M2 column, the supernatant was removed and the column was washed extensively with PBS. The protein was then eluted with PBS containing 150 μg / ml of FLAG peptide and subsequently concentrated. Size-exclusion chromatography was performed on Superose 6 increase 10 / 300 in PBS at 0.5 ml / min, and fractions were collected. The purified protein was analyzed on a 4-20% Mini-PROTEAN® TGX Stain-Free™ protein gel (Biorad) in Tris / glycine / SDS buffer (Figure 20A) and by electron microscopy using negative staining (Figure 20B). EM images showed the triangular shape of the protein particles, a typical prefusion conformation.

[0473] To evaluate whether gB2796 could induce a better immune response compared to postfusion gB, we generated a secreted postfusion gB based on the same strain of HCMV, but with the transmembrane domain removed. This protein was designated Sanofi gB because it is equivalent to the gB protein used in a previous clinical trial by Sanofi (Pass et al., 2009). Purification of this tag-free gB was achieved using affinity chromatography on a lentil lectin-Sepharose (GE Healthcare) column, followed by anion-exchange chromatography on a Toyopearl GigaCap Q-650M (Tosoh) column and size-exclusion chromatography on a Superdex 200 (GE Healthcare) column.

[0474] In vivo immunogenicity studies were performed in mice using the pre- and post-fusion gB described above, as shown in Table 16 below.

[0475] [Table 19]

[0476] At week 5 (2 weeks after the second dose), serum samples from mice were analyzed by ELISA to determine IgG titers to gB2796. Results showed a dose-dependent IgG response in both gB2796- and Sanofi gB-immunized mice (Figure 21).

[0477] List of raw arrays

[0478] [Table 20-1]

[0479] [Table 20-2]

[0480] [Table 20-3]

[0481] [Table 20-4]

[0482] [Table 20-5]

[0483] [Table 20-6]

[0484] [Table 20-7]

[0485] [Table 20-8]

[0486] [Table 20-9]

[0487] [Table 20-10]

[0488] [Table 20-11]

[0489] [Table 20-12]

[0490] [Table 21]

[0491] Embodiments of the present invention are described in the following numbered paragraphs:

[0492] C1. A polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), wherein the polypeptide comprises a conformation that is not the HCMV gB post-fusion conformation.

[0493] C2. A polypeptide that binds to an HCMV gB prefusion-specific antibody.

[0494] C3. A polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), wherein the polypeptide is capable of binding to an HCMV gB prefusion-specific antibody.

[0495] C4. A polypeptide comprising at least one introduced amino acid mutation compared to the amino acid sequence of wild-type HCMV glycoprotein B (gB), wherein the polypeptide is capable of binding to a bis(aryl)thiourea compound.

[0496] C5. The polypeptide according to item C3, wherein the compound is N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide.

[0497] C6. The polypeptide of item C1, characterized by structural coordinates that include root mean square deviations (RMSDs) of backbone atoms of conserved residues when the polypeptide is superimposed onto the backbone atoms described by the structural coordinates of Table 1A.

[0498] C7. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a cysteine ​​substitution.

[0499] C8. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a mutation that allows for disulfide bond formation.

[0500] C9. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises an electrostatic mutation.

[0501] C10. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a phenylalanine substitution.

[0502] C11. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a leucine substitution.

[0503] C12. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type HCMV gB polypeptide, wherein the mutation stabilizes the prefusion conformation of the polypeptide, and wherein the polypeptide specifically binds to an HCMV gB prefusion-specific antibody.

[0504] C13. A polypeptide comprising a cysteine ​​at any one of the amino acid positions listed in column (ii) of Table 2, relative to SEQ ID NO:1.

[0505] C14. A polypeptide comprising an amino acid substitution at any one of the amino acid positions listed in column (ii) of Table 2, relative to SEQ ID NO:1.

[0506] C15. A polypeptide comprising the mutations Q98C and I653C according to the numbering of SEQ ID NO: 1.

[0507] C16. A polypeptide comprising the mutations T100C and S269C according to the numbering of SEQ ID NO: 1.

[0508] C17. A polypeptide comprising the mutations D217C and F584C according to the numbering of SEQ ID NO: 1.

[0509] C18. A polypeptide comprising the mutations Y242C and K710C according to the numbering of SEQ ID NO: 1.

[0510] C19. A polypeptide comprising the mutations Y242C and D714C according to the numbering of SEQ ID NO: 1.

[0511] C20. A polypeptide comprising the mutations S367C and L499C according to the numbering of SEQ ID NO: 1.

[0512] C21. A polypeptide comprising the mutations T372C and W506C according to the numbering of SEQ ID NO: 1.

[0513] C22. A polypeptide comprising the mutations S550C and D652C according to the numbering of SEQ ID NO: 1.

[0514] C23. A polypeptide comprising the mutations T608C and D679C according to the numbering of SEQ ID NO: 1.

[0515] C24. A polypeptide comprising the mutations K695C and K724C according to the numbering of SEQ ID NO: 1.

[0516] C25. A polypeptide comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-43, wherein the polypeptide comprises a mutation compared to SEQ ID NO: 1.

[0517] C26. The polypeptide of any one of items C1-C25, which does not comprise a mutation at any one of the following positions: R562, P577, S587, Y588, G592, G595, L601 / H605, C610, L612, P613, Y625, Y627, F632, and K633.

[0518] C27. The polypeptide according to any one of items C1 to C26, which does not contain the cytoplasmic tail of HCMV gB.

[0519] C28. The polypeptide according to any one of items C1 to C26, which does not contain a transmembrane region.

[0520] C29. A polypeptide according to any one of items C1 to C26, comprising the cytoplasmic tail of HCMV gB and not the transmembrane region.

[0521] C30. The polypeptide according to any one of paragraphs C1 to C29, which does not contain an insect cell glycosylation pattern.

[0522] C31. The polypeptide of any one of paragraphs C1-C30, which exhibits improved solubility or stability compared to native gB in the post-fusion conformation.

[0523] C32. The polypeptide according to any one of paragraphs C1 to C31, which is immunogenic.

[0524] C33. A nucleic acid encoding a polypeptide according to any one of items C1 to C32.

[0525] C34. The nucleic acid of item C33, comprising a self-replicating RNA molecule.

[0526] C35. The nucleic acid of item C33, comprising a modified RNA molecule.

[0527] C36. A composition comprising a nucleic acid according to any one of items C33 to C35.

[0528] C37. A composition comprising a polypeptide according to any one of paragraphs C1 to C32, and further comprising a CMV antigen.

[0529] C38. The composition of any one of items C36-C37, further comprising any one of the following polypeptides: gO, gH, gL, pUL128, pUL130, pUL131, and any combination thereof.

[0530] C39. A composition comprising a polypeptide according to any one of items C1 to C32, and a diluent.

[0531] C40. A composition comprising a polypeptide according to any one of items C1 to C32, and an adjuvant.

[0532] C41. The composition of any one of paragraphs C36-C40, which is immunogenic.

[0533] C42. The composition of any one of paragraphs C36-C41 for use in eliciting an immune response against cytomegalovirus.

[0534] C43. A method for eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a polypeptide according to any one of paragraphs C1 to C32.

[0535] C44. A method for reducing viral shedding of cytomegalovirus in a mammal, comprising administering to the mammal an effective amount of a polypeptide according to any one of paragraphs C1 to C32.

[0536] C45. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-43.

[0537] C46. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-106.

[0538] C47. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 47-106.

[0539] C48. A polypeptide comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO:57.

[0540] C49. A composition comprising at least one polynucleotide encoding an HCMV polypeptide selected from any one of gH, gL, UL128, UL130, and UL131; a polynucleotide encoding HCMV gB or a fragment thereof; a polynucleotide encoding pp65 or a fragment thereof; and a pharmaceutically acceptable carrier or diluent.

[0541] C50. A composition comprising: at least one polynucleotide comprising a sequence having at least 95% identity to a sequence selected from any one of SEQ ID NOs: 141-210; a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 95% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0542] C51. A composition comprising at least one polynucleotide comprising a sequence having at least 95% identity to a sequence selected from any one of SEQ ID NOs: 224-254; a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0543] C52. A composition comprising at least one polypeptide comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from any one of SEQ ID NOs: 211-223; a polypeptide comprising an amino acid sequence having at least 95% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0544] C53. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, the polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43, and comprising a mutation compared to SEQ ID NO: 1.

[0545] C54. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, the polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106, and comprising a mutation compared to SEQ ID NO: 1.

[0546] C55. A polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of wild-type HCMV gB, the polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 47-106, and comprising a mutation compared to SEQ ID NO: 1.

[0547] C56. A polypeptide comprising the sequence set forth in SEQ ID NO: 57.

[0548] A composition comprising at least one polynucleotide encoding an HCMV polypeptide selected from any one of C57.gH, gL, UL128, UL130, and UL131; a polynucleotide encoding HCMV gB or a fragment thereof; a polynucleotide encoding pp65 or a fragment thereof; and a pharmaceutically acceptable carrier or diluent.

[0549] C58. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NOs: 141-210; a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0550] C59. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NOs: 224-254; a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0551] C60. A composition comprising at least one polypeptide comprising a sequence selected from any one of SEQ ID NOs: 211-223; a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or diluent.

[0552] C61. The composition of any one of items C49-C51 and C57-C59, wherein the polynucleotide is DNA.

[0553] C62. The composition of any one of items C49-C51 and C57-C59, wherein the polynucleotide is RNA.

[0554] C63. The composition of any one of items C49-C51 and C57-C59, wherein at least one polynucleotide comprises at least one chemical modification.

[0555] C64. The composition of item C61, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0556] C65. The composition of any one of items C49-C51 and C57-C59, formulated in cationic lipid nanoparticles.

[0557] C66. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:210, SEQ ID NO:152, and SEQ ID NO:158; a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:1-106; and a pharmaceutically acceptable carrier or diluent.

[0558] C67. A composition comprising at least one polypeptide comprising a sequence selected from any one of SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217; a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:1-106; and a pharmaceutically acceptable carrier or diluent.

[0559] C68. A polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 106.

[0560] C69. The polypeptide according to item C68, wherein the sequence comprises SEQ ID NO: 56.

[0561] C70. The polypeptide according to item C68, wherein the sequence comprises SEQ ID NO: 57.

[0562] C71. The polypeptide according to item C68, wherein the sequence comprises SEQ ID NO: 58.

[0563] C72. The polypeptide according to item C68, wherein the sequence comprises SEQ ID NO: 75.

[0564] C73. A polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 106.

[0565] C74. A composition comprising a polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 1-106; and a diluent.

[0566] C75. The composition of item C74, wherein the sequence does not include any one of SEQ ID NO:59, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:71, SEQ ID NO:52, SEQ ID NO:96, and SEQ ID NO:50.

[0567] C76. The composition according to item C74, further comprising a polypeptide comprising any one of sequences selected from SEQ ID NOs: 211-224.

[0568] C77. A composition comprising a polynucleotide encoding a polypeptide comprising a sequence selected from any one of SEQ ID NOs: 1-106; and a diluent.

[0569] C78. The composition according to item C77, further comprising a polynucleotide comprising a sequence selected from any one of SEQ ID NOs: 141-210.

[0570] C79. The composition of item C77, further comprising a polynucleotide comprising a sequence selected from any one of SEQ ID NOs: 224-254.

[0571] C80. A method for eliciting an immune response in a mammal, comprising administering an effective amount of a composition comprising a polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 1-106; and a diluent.

[0572] C81. A method for eliciting an immune response in a mammal, comprising administering an effective amount of a composition comprising a polynucleotide encoding a polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 1-106; and a diluent.

[0573] TABLE 1A REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : PHENIX (dev_3092: phenix.real_space_refine) REMARK 3 AUTHORS : Adams,Afonine,Bunkoczi,Burnley,Chen,Dar,Davis, REMARK 3 : Draizen,Echols,Gildea,Gros,Grosse-Kunstleve,Headd, REMARK 3 : Hintze,Hung,Ioerger,Liebschner,McCoy,McKee,Moriarty, REMARK 3 : Oeffner,Poon,Read,Richardson,Richardson,Sacchettini, REMARK 3 : Sauter,Sobolev,Storoni,Terwilliger,Williams,Zwart REMARK 3 REMARK 3 REMARK 3 SCATTERING TABLE: N_GAUSSIAN REMARK 3 REMARK 3 REFINEMENT TARGET: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS) REMARK 3 REMARK 3 MODEL TO MAP FIT. REMARK 3 CC_mask : 0.7786 REMARK 3 CC_volume: 0.7566 REMARK 3 CC_peaks : 0.5466 REMARK 3 GEOMETRY RESTRAINTS LIBRARY: CDL v1.2 DEVIATIONS FROM IDEAL VALUES. BOND : 0.004 0.033 17103 ANGLE : 0.769 10.302 23835 CHIRALITY : 0.056 0.568 2595 PLANARITY : 0.004 0.043 2907 DIHEDRAL : 11.547 179.989 9717 MIN NONBONDED DISTANCE : 1.763 MOLPROBITY STATISTICS. ALL-ATOM CLASHSCORE : 7.96 RAMACHANDRAN PLOT: OUTLIERS : 0.00 % ALLOWED : 15.80 % FAVORED : 84.20 % ROTAMER OUTLIERS : 0.36 % CBETA DEVIATIONS : 0 PEPTIDE PLANE: CIS-PROLINE : 10.5263157895 CIS-GENERAL : 0.162337662338 TWISTED PROLINE : 0.0 TWISTED GENERAL : 0.162337662338 REMARK 3 HELIX 1 1 TRP A 174 ILE A 179 1 6 HELIX 2 2 ASP A 390 SER A 407 1 18 HELIX 3 3 LEU A 484 LYS A 522 1 39 HELIX 4 4 SER A 526 ILE A 532 1 7 HELIX 5 5 PHE A 661 TYR A 667 1 7 HELIX 6 6 ILE A 683 PRO A 704 1 22 HELIX 7 7 LYS A 710 LEU A 719 1 10 HELIX 8 8 VAL A 726 LYS A 749 1 24 HELIX 9 9 PHE A 752 TYR A 769 1 18 HELIX 10 1 TRP B 174 ILE B 179 1 6 HELIX 11 2 ASP B 390 SER B 407 1 18 HELIX 12 3 LEU B 484 LYS B 522 1 39 HELIX 13 4 SER B 526 ILE B 532 1 7 HELIX 14 5 PHE B 661 TYR B 667 1 7 HELIX 15 6 ILE B 683 PRO B 704 1 22 HELIX 16 7 LYS B 710 LEU B 719 1 10 HELIX 17 8 VAL B 726 LYS B 749 1 24 HELIX 18 9 PHE B 752 TYR B 769 1 18 HELIX 19 1 TRP C 174 ILE C 179 1 6 HELIX 20 2 ASP C 390 SER C 407 1 18 HELIX 21 3 LEU C 484 LYS C 522 1 39 HELIX 22 4 SER C 526 ILE C 532 1 7 HELIX 23 5 PHE C 661 TYR C 667 1 7 HELIX 24 6 ILE C 683 PRO C 704 1 22 HELIX 25 7 LYS C 710 LEU C 719 1 10 HELIX 26 8 VAL C 726 LYS C 749 1 24 HELIX 27 9 PHE C 752 TYR C 769 1 18 SHEET 1 1 1 CHAPTER 102 TO 103 0 SHEET 2 2 1 LEU A 121 LYS A 130 0 SHEET 3 3 1 HIS A 136 ALA A 154 0 SHEET 4 4 1 TYR A 160 VAL A 169 0 SHEET 5 5 1 TYR A 186 VAL A 192 0 SHEET 6 6 1 VAL A 197 TYR A 201 0 SHEET 7 7 1 LYS A 209 THR A 210 0 SHEET 8 8 1 ARG TO 225 VALUES TO 227 0 SHEET 9 9 1 LEU A 241 SER A 259 0 SHEET 10 10 1 PHE A 266 THR A 268 0 SHEET 11 11 1 ASP A 272 TO 274 0 SHEET 12 12 1 PHE A 297 SER A 307 0 SHEET 13 13 1 PAGE A 315 ARG A 327 0 SHEET 14 14 1 PAGE A 330 ASP A 335 0 SHEET 15 15 1 SECTION A 346 GLU A 359 0 SHEET 16 of 16 1 PART A 364 SER A 368 0 SHEET 17 of 17 1 PAGE A 373 SER A 377 0 SHEET 18 18 1 Glue A 413 TYR A 415 0 SHEET 19 of 19 1 PAGE A 419 GLU A 422 0 SHEET 20 20 1 VALUE A 428 LYS A 435 0 SHEET 21 21 1 PAGE A 539 MET A 542 0 SHEET 22 22 1 VAL A 545 CHAPTER A 548 0 SHEET 23 23 1 VAL A 559 CHAPTER A 562 0 SHEET 24 24 1 VAL A 579 ASN A 583 0 SHEET 25 25 1 VAL A 590 GLY A 596 0 SHEET 26 of 26 1 GLU A 600 LEGE A 602 0 SHEET 27 of 27 1 SECTION A 616 TO 620 0 SHEET 28 of 28 1 PAGE A 625 TO 629 0 SHEET 29 of 29 1 PAGE A 632 TO 637 0 SHEET 30 1 1 LION B 102 HOUSE B 103 0 SHEET 31 2 1 LION B 121 LYS B 130 0 SHEET 32 3 1 HIS B 136 ALA B 154 0 SHEET 33 4 1 TYR B 160 VAL B 169 0 SHEET 34 5 1 TYR B 186 VAL B 192 0 SHEET 35 6 1 VAL B 197 TYR B 201 0 SHEET 36 7 1 LYS B 209 THR B 210 0 SHEET 37 8 1 ARG B 225 VAL B 227 0 SHEET 38 9 1 LEU B 241 SER B 259 0 SHEET 39 10 1 PHE B 266 THR B 268 0 SHEET 40 11 1 ASP B 272 VAL B 274 0 SHEET 41 12 1 PHE B 297 SER B 307 0 SHEET 42 13 1 ALA B 315 ARG B 327 0 SHEET 43 14 1 SER B 330 ASP B 335 0 SHEET 44 15 1 LEU B 346 GLU B 359 0 SHEET 45 16 1 TYR B 364 SER B 368 0 SHEET 46 17 1 ALA B 373 SER B 377 0 SHEET 47 18 1 GLU B 413 TYR B 415 0 PAGE 48 of 19 1 PAGE B 419 GLU B 422 0 SHEET 49 20 1 VAL B 428 LYS B 435 0 SHEET 50 of 21 1 PAGE B 539 MET B 542 0 SHEET 51 22 1 VAL B 545 LEO B 548 0 SHEET 52 23 1 VAL B 559 LEO B 562 0 SHEET 53 24 1 VAL B 579 ASN B 583 0 SHEET 54 25 1 VAL B 590 GLY B 596 0 SHEET 55 of 26 1 GLU B 600 LEO B 602 0 SHEET 56 of 27 1 SHEEP B 616 HOUSE B 620 0 SHEET 57 of 28 1 PAGE B 625 VAL B 629 0 SHEET 58 of 29 1 LEO B 632 HOUSE B 637 0 SHEET 59 1 1 LEGEND C 102 HOUSE C 103 0 SHEET 60 2 1 LEGEND C 121 LYS C 130 0 SHEET 61 3 1 HIS C 136 ALA C 154 0 PAGE 62 4 1 PART C 160 VAL C 169 0 PAGE 63 5 1 PART C 186 VAL C 192 0 SHEET 64 6 1 VAL C 197 TYR C 201 0 PAGE 65 7 1 ROW C 209 THR C 210 0 PAGE 66 8 1 ARG C 225 VAL C 227 0 SHEET 67 9 1 LEGEND C 241 SER C 259 0 SHEET 68 10 1 PHE C 266 THR C 268 0 SHEET 69 11 1 ASP C 272 VAL C 274 0 SHEET 70 12 1 PHE C 297 SER C 307 0 SHEET 71 13 1 PAGE C 315 ARG C 327 0 PAGE 72 of 14 1 PAGE C 330 ASP C 335 0 SHEET 73 15 1 LEGEND C 346 GLU C 359 0 PAGE 74 of 16 1 SECTION C 364 SER C 368 0 SHEET 75 17 1 PAGE C 373 SER C 377 0 SHEET 76 18 1 GLU C 413 TYR C 415 0 PAGE 77 of 19 1 SERVICE C 419 GLU C 422 0 SHEET 78 20 1 VAL C 428 LYS C 435 0 SHEET 79 of 21 1 PAGE C 539 MET C 542 0 SHEET 80 22 1 VAL C 545 LEO C 548 0 SHEET 81 23 1 VAL C 559 LEO C 562 0 SHEET 82 24 1 VAL C 579 ASN C 583 0 SHEET 83 25 1 VAL C 590 GLY C 596 0 SHEET 84 26 1 GLU C 600 LEO C 602 0 SHEET 85 of 27 1 LEGEND C 616 HOUSE C 620 0 SHEET 86 of 28 1 PAGE C 625 VAL C 629 0 SHEET 87 of 29 1 LEGEND C 632 HOUSE C 637 0 LINK C1 NAG A1003 O3 NAG A1002 LINK C1 NAG A1008 O3 NAG A1007 LINK C1 NAG B1003 O3 NAG B1002 LINK C1 NAG B1008 O3 NAG B1007 LINK C1 NAG C1003 O3 NAG C1002 LINK C1 NAG C1008 O3 NAG C1007 LINK C1 NAG A1002 O4 NAG A1001 LINK C1 NAG B1002 O4 NAG B1001 LINK C1 NAG C1002 O4 NAG C1001 LINK C1 NAG A1001 ND2 ASN A 208 LINK C1 NAG A1004 ND2 ASN A 281 LINK C1 NAG A1005 ND2 ASN A 286 LINK C1 NAG A1007 ND2 ASN A 383 LINK C1 NAG A1009 ND2 ASN A 417 LINK C1 NAG A1010 ND2 ASN A 555 LINK C1 NAG A1011 ND2 ASN A 586 LINK C1 NAG A1012 ND2 ASN A 341 LINK C1 NAG A1013 ND2 ASN A 405 LINK C1 NAG A1014 ND2 ASN A 118 LINK C1 NAG A1015 ND2 ASN A 338 LINK C1 NAG B1001 ND2 ASN B 208 LINK C1 NAG B1004 ND2 ASN B 281 LINK C1 NAG B1005 ND2 ASN B 286 LINK C1 NAG B1007 ND2 ASN B 383 LINK C1 NAG B1009 ND2 ASN B 417 LINK C1 NAG B1010 ND2 ASN B 555 LINK C1 NAG B1011 ND2 ASN B 586 LINK C1 NAG B1012 ND2 ASN B 341 LINK C1 NAG B1013 ND2 ASN B 405 LINK C1 NAG B1014 ND2 ASN B 118 LINK C1 NAG B1015 ND2 ASN B 338 LINK C1 NAG C1001 ND2 ASN C 208 LINK C1 NAG C1004 ND2 ASN C 281 LINK C1 NAG C1005 ND2 ASN C 286 LINK C1 NAG C1007 ND2 ASN C 383 LINK C1 NAG C1009 ND2 ASN C 417 LINK C1 NAG C1010 ND2 ASN C 555 LINK C1 NAG C1011 ND2 ASN C 586 LINK C1 NAG C1012 ND2 ASN C 341 LINK C1 NAG C1013 ND2 ASN C 405 LINK C1 NAG C1014 ND2 ASN C 118 LINK C1 NAG C1015 ND2 ASN C 338 SSBOND 1 CYS A 94 CYS A 551 SSBOND 2 CYS A 111 CYS A 507 SSBOND 3 CYS A 185 CYS A 250 SSBOND 4 CYS A 344 CYS A 391 SSBOND 5 CYS A 574 CYS A 611 SSBOND 6 CYS B 94 CYS B 551 SSBOND 7 CYS B 111 CYS B 507 SSBOND 8 CYS B 185 CYS B 250 SSBOND 9 CYS B 344 CYS B 391 SSBOND 10 CYS B 574 CYS B 611 SSBOND 11 CYS C 94 CYS C 551 SSBOND 12 CYS C 111 CYS C 507 SSBOND 13 CYS C 185 CYS C 250 SSBOND 14 CYS C 344 CYS C 391 SSBOND 15 CYS C 574 CYS C 611 CRYST1 385.280 385.280 385.280 90.00 90.00 90.00 P 1 SCALE1 0.002596 0.000000 0.000000 0.00000 SCALE2 0.000000 0.002596 0.000000 0.00000 SCALE3 0.000000 0.000000 0.002596 0.00000 ATOM 1 N THR A 87 183.022 162.064 195.194 1.00 73.34 N ATOM 2 CA THR A 87 183.305 163.436 195.596 1.00 73.34 C ATOM 3 C THR A 87 184.805 163.704 195.604 1.00 73.34 C ATOM 4 O THR A 87 185.591 162.880 196.069 1.00 73.34 O ATOM 5 CB THR A 87 182.731 163.746 196.991 1.00 73.34 C ATOM 6 OG1 THR A 87 181.312 163.555 196.981 1.00 73.34 O ATOM 7 CG2 THR A 87 183.040 165.181 197.387 1.00 73.34 C ATOM 8 N LYS A 88 185.196 164.865 195.080 1.00 65.97 N ATOM 9 CA LYS A 88 186.590 165.285 195.056 1.00 65.97 C ATOM 10 C LYS A 88 186.914 166.277 196.167 1.00 65.97 C ATOM 11 O LYS A 88 187.902 167.011 196.066 1.00 65.97 O ATOM 12 CB LYS A 88 186.937 165.888 193.694 1.00 65.97 C ATOM 13 CG LYS A 88 186.203 167.181 193.383 1.00 65.97 C ATOM 14 CD LYS A 88 186.561 167.697 191.999 1.00 65.97 C ATOM 15 CE LYS A 88 185.846 169.001 191.690 1.00 65.97 C ATOM 16 NZ LYS A 88 186.189 169.509 190.334 1.00 65.97 N ATOM 17 N TYR A 89 186.103 166.313 197.222 1.00 56.58 N ATOM 18 CA TYR A 89 186.300 167.222 198.350 1.00 56.58 C ATOM 19 C TYR A 89 186.288 166.411 199.638 1.00 56.58 C ATOM 20 O TYR A 89 185.298 166.416 200.380 1.00 56.58 O ATOM 21 CB TYR A 89 185.229 168.311 198.376 1.00 56.58 C ATOM 22 CG TYR A 89 185.302 169.265 197.210 1.00 56.58 C ATOM 23 CD1 TYR A 89 186.228 170.297 197.192 1.00 56.58 C ATOM 24 CD2 TYR A 89 184.443 169.138 196.129 1.00 56.58 C ATOM 25 CE1 TYR A 89 186.300 171.173 196.130 1.00 56.58 C ATOM 26 CE2 TYR A 89 184.507 170.009 195.061 1.00 56.58 C ATOM 27 CZ TYR A 89 185.437 171.025 195.068 1.00 56.58 C ATOM 28 OH TYR A 89 185.505 171.897 194.007 1.00 56.58 O ATOM 29 N PRO A 90 187.370 165.699 199.932 1.00 48.30 N ATOM 30 CA PRO A 90 187.438 164.942 201.183 1.00 48.30 C ATOM 31 C PRO A 90 187.843 165.830 202.348 1.00 48.30 C ATOM 32 O PRO A 90 188.481 166.871 202.182 1.00 48.30 O ATOM 33 CB PRO A 90 188.516 163.893 200.897 1.00 48.30 C ATOM 34 CG PRO A 90 189.408 164.563 199.915 1.00 48.30 C ATOM 35 CD PRO A 90 188.521 165.424 199.057 1.00 48.30 C ATOM 36 N TYR A 91 187.448 165.397 203.542 1.00 40.22 N ATOM 37 CA TYR A 91 187.784 166.124 204.760 1.00 40.22 C ATOM 38 C TYR A 91 189.296 166.225 204.890 1.00 40.22 C ATOM 39 O TYR A 91 190.000 165.215 204.812 1.00 40.22 O ATOM 40 CB TYR A 91 187.197 165.415 205.981 1.00 40.22 C ATOM 41 CG TYR A 91 185.687 165.327 206.010 1.00 40.22 C ATOM 42 CD1 TYR A 91 184.905 166.099 205.169 1.00 40.22 C ATOM 43 CD2 TYR A 91 185.044 164.460 206.880 1.00 40.22 C ATOM 44 CE1 TYR A 91 183.531 166.008 205.189 1.00 40.22 C ATOM 45 CE2 TYR A 91 183.669 164.367 206.908 1.00 40.22 C ATOM 46 CZ TYR A 91 182.919 165.147 206.060 1.00 40.22 C ATOM 47 OH TYR A 91 181.548 165.068 206.074 1.00 40.22 O ATOM 48 N ARG A 92 189.803 167.438 205.087 1.00 37.78 N ATOM 49 CA ARG A 92 191.241 167.647 205.120 1.00 37.78 C ATOM 50 C ARG A 92 191.578 168.770 206.091 1.00 37.78 C ATOM 51 O ARG A 92 190.698 169.460 206.610 1.00 37.78 O ATOM 52 CB ARG A 92 191.777 167.926 203.717 1.00 37.78 C ATOM 53 CG ARG A 92 191.252 169.189 203.094 1.00 37.78 C ATOM 54 CD ARG A 92 191.640 169.237 201.636 1.00 37.78 C ATOM 55 NE ARG A 92 191.302 170.514 201.022 1.00 37.78 N ATOM 56 CZ ARG A 92 190.112 170.802 200.509 1.00 37.78 C ATOM 57 NH1 ARG A 92 189.140 169.904 200.544 1.00 37.78 N ATOM 58 NH2 ARG A 92 189.895 171.990 199.966 1.00 37.78 N ATOM 59 N VAL A 93 192.875 168.939 206.340 1.00 32.43 N ATOM 60 CA VAL A 93 193.369 169.752 207.446 1.00 32.43 C ATOM 61 C VAL A 93 194.285 170.854 206.920 1.00 32.43 C ATOM 62 O VAL A 93 195.354 171.111 207.483 1.00 32.43 O ATOM 63 CB VAL A 93 194.072 168.881 208.503 1.00 32.43 C ATOM 64 CG1 VAL A 93 194.368 169.686 209.763 1.00 32.43 C ATOM 65 CG2 VAL A 93 193.224 167.678 208.833 1.00 32.43 C ATOM 66 N CYS A 94 193.898 171.496 205.817 1.00 41.32 N ATOM 67 CA CYS A 94 194.795 172.415 205.122 1.00 41.32 C ATOM 68 C CYS A 94 195.091 173.643 205.972 1.00 41.32 C ATOM 69 O CYS A 94 194.589 174.736 205.692 1.00 41.32 O ATOM 70 CB CYS A 94 194.175 172.879 203.803 1.00 41.32 C ATOM 71 SG CYS A 94 193.803 171.599 202.601 1.00 41.32 S ATOM 72 N SER A 95 195.897 173.474 207.008 1.00 43.21 N ATOM 73 CA SER A 95 196.237 174.561 207.918 1.00 43.21 C ATOM 74 C SER A 95 197.730 174.703 208,162 1.00 43.21 C ATOM 75 O SER A 95,198,213,175,824 208,331 1.00 43.21 O ATOM 76 CB SER A 95 195.519 174.361 209.260 1.00 43.21 C ATOM 77 AND SER A 95 195.881 175.371 210.184 1.00 43.21 O ATOM 78 N MET A 96 198.470 173.601 208.195 1.00 44.84 N ATOM 79 CA MET A 96,199,854 173,603 208,644 1.00 44.84 C ATOM 80 C MET A 96 200.801 173.435 207.465 1.00 44.84 C ATOM 81 O MET A 96,200,516,172,689 206,525 1.00 44.84 O ATOM 82 CB MET A 96 200.080 172.489 209.665 1.00 44.84 C ATOM 83 CG MET A 96 199.896 171.095 209.098 1.00 44.84 C ATOM 84 SD MET A 96 199.923 169.814 210.365 1.00 44.84 S ATOM 85 CE MET A 96 198.306 170.020 211.106 1.00 44.84 C ATOM 86 N ALA A 97 201.933 174.132 207.525 1.00 42.33 N ATOM 87 CA ALA A 97 202.926 174.065 206.465 1.00 42.33 C ATOM 88 C ALA A 97 204.274 174.491 207.024 1.00 42.33 C ATOM 89 O ALA A 97 204.362 175.120 208.080 1.00 42.33 O ATOM 90 CB ALA A 97 202.540 174.941 205.271 1.00 42.33 C ATOM 91 N GLN A 98 205.319 174.148 206.282 1.00 45.39 N ATOM 92 CA GLN A 98 206.710 174.310 206.688 1.00 45.39 C ATOM 93 C GLN A 98 207.544 174.356 205.412 1.00 45.39 C ATOM 94 O GLN A 98 207.021 174.655 204.333 1.00 45.39 O ATOM 95 CB GLN A 98 207.136 173.195 207.659 1.00 45.39 C ATOM 96 CG GLN A 98 206.530 173.315 209.043 1.00 45.39 C ATOM 97 CD GLN A 98 206.918 172.172 209.950 1.00 45.39 C ATOM 98 OE1 GLN A 98 207.539 171.205 209.514 1.00 45.39 O ATOM 99 NE2 GLN A 98 206.566 172.283 211.225 1.00 45.39 N ATOM 100 N GLY A 99 208.837 174.081 205.530 1.00 46.28 N ATOM 101 CA GLY A 99 209.693 174.138 204.365 1.00 46.28 C ATOM 102 C GLY A 99 209.459 172.972 203.425 1.00 46.28 C ATOM 103 O GLY A 99 208.311 172.594 203.176 1.00 46.28 O ATOM 104 N THR A 100 210.537 172.404 202.891 1.00 42.54 N ATOM 105 CA THR A 100 210.597 171.424 201.813 1.00 42.54 C ATOM 106 C THR A 100 210.409 172.083 200.449 1.00 42.54 C ATOM 107 O THR A 100 210.626 171.423 199.433 1.00 42.54 O ATOM 108 CB THR A 100 209.558 170.292 201.943 1.00 42.54 C ATOM 109 OG1 THR A 100 208.248 170.824 201.720 1.00 42.54 O ATOM 110 CG2 THR A 100 209.595 169.680 203.327 1.00 42.54 C ATOM 111 N ASP A 101 210.026 173.360 200.387 1.00 41.94 N ATOM 112 CA ASP A 101 209.945 174.113 199.141 1.00 41.94 C ATOM 113 C ASP A 101 209.545 175.557 199.420 1.00 41.94 C ATOM 114 O ASP A 101 208.847 175.831 200.400 1.00 41.94 O ATOM 115 CB ASP A 101 208.967 173.449 198.173 1.00 41.94 C ATOM 116 CG ASP A 101 207,615 173,210 198,794 1.00 41.94 C ATOM 117 OD1 ASP A 101 207.451 173.516 199.993 1.00 41.94 O ATOM 118 OD2 ASP A 101 206.721 172.701 198.089 1.00 41.94 O ATOM 119 N LEU A 102 209,966 176,483 198,564 1.00 40.67 N ATOM 120 CA LEU A 102 209,739 177,899 198,808 1.00 40.67 C ATOM 121 C LEU A 102 209,415 178,587 197,491 1.00 40.67 C ATOM 122 LEU A 102 209,878 178,165 196,429 1.00 40.67 O ATOM 123 CB LEU A 102 210,962 178,563 199,451 1.00 40.67 C ATOM 124 CG LEU A 102 211,281 178,345 200,932 1.00 40.67 C ATOM 125 CD1 READ A 102 211.852 176.967 201.172 1.00 40.67 C ATOM 126 CD2 READ A 102 212.256 179.403 201.412 1.00 40.67 C ATOM 127 N Ile A 103 208.615 179.652 197.566 1.00 42.52 N ATOM 128 CA IL A 103 208.299 180.454 196.387 1.00 42.52 C ATOM 129 C ile A 103 208.127 181.920 196.765 1.00 42.52 C ATOM 130 O IL A 103 207.306 182.266 197.620 1.00 42.52 O ATOM 131 CB ILE A 103 207.042 179.933 195.662 1.00 42.52 C ATOM 132 CG1 ILE A 103 205.828 179.942 196.587 1.00 42.52 C ATOM 133 CG2 ILE A 103 207.269 178.546 195.083 1.00 42.52 C ATOM 134 CD1 ILE A 103 204.525 179.696 195.863 1.00 42.52 C ATOM 135 N A R G A 104 208.907 182.788 196.129 1.00 55.38 N ATOM 136 CA ARG A 104 208.772 184.231 196.260 1.00 55.38 C ATOM 137 C ARG A 104 207.847 184.754 195.162 1.00 55.38 C ATOM 138 O ARG A 104 207.128 183.990 194.513 1.00 55.38 O ATOM 139 CB ARG A 104 210.141 184.897 196.208 1.00 55.38 C ATOM 140 CG ARG A 104 210.837 184.689 194.888 1.00 55.38 C ATOM 141 CD ARG A 104 212.175 185.384 194.839 1.00 55.38 C ATOM 142 NE ARG A 104 212.838 185.138 193.566 1.00 55.38 N ATOM 143 CZ ARG A 104 213.989 185.690 193.207 1.00 55.38 C ATOM 144 NH1 ARG A 104 214.599 186.536 194.022 1.00 55.38 N ATOM 145 NH2 ARG A 104 214.523 185.407 192.029 1.00 55.38 N ATOM 146 N PHE A 105 207.845 186.073 194.944 1.00 61.78 N ATOM 147 CA PHE A 105 206.981 186.681 193.941 1.00 61.78 C ATOM 148 C PHE A 105 207.699 187.528 192.895 1.00 61.78 C ATOM 149 O PHE A 105 207.109 187.780 191.839 1.00 61.78 O ATOM 150 CB PHE A 105 205.899 187.538 194.613 1.00 61.78 C ATOM 151 CG PHE A 105 204.849 186.735 195.324 1.00 61.78 C ATOM 152 CD1 PHE A 105 203.789 186.185 194.624 1.00 61.78 C ATOM 153 CD2 PHE A 105 204.925 186.524 196.689 1.00 61.78 C ATOM 154 CE1 PHE A 105 202.822 185.447 195.273 1.00 61.78 C ATOM 155 CE2 PHE A 105 203.960 185.786 197.344 1.00 61.78 C ATOM 156 CZ PHE A 105 202.907 185.246 196.634 1.00 61.78 C ATOM 157 N GLU A 106 208.936 187.968 193.144 1.00 76.47 N ATOM 158 CA GLU A 106 209.783 188.575 192.111 1.00 76.47 C ATOM 159 C GLU A 106 209.134 189.829 191.514 1.00 76.47 C ATOM 160 O GLU A 106 208.664 189.835 190.375 1.00 76.47 O ATOM 161 CB GLU A 106 210.113 187.554 191.017 1.00 76.47 C ATOM 162 CG GLU A 106 211.032 188.084 189.933 1.00 76.47 C ATOM 163 CD GLU A 106 212.395 188.462 190.469 1.00 76.47 C ATOM 164 OE1 GLU A 106 212.797 187.909 191.513 1.00 76.47 O ATOM 165 OE2 GLU A 106 213.064 189.313 189.847 1.00 76.47 O ATOM 166 N ARG A 107 209.097 190.877 192.347 1.00 87.65 N ATOM 167 CA ARG A 107 208.391 192.127 192.063 1.00 87.65 C ATOM 168 C ARG A 107 208.548 192.637 190.629 1.00 87.65 C ATOM 169 O ARG A 107 207.563 192.759 189.893 1.00 87.65 O ATOM 170 CB ARG A 107 208.867 193.220 193.030 1.00 87.65 C ATOM 171 CG ARG A 107 208.352 193.122 194.458 1.00 87.65 C ATOM 172 CD ARG A 107 209.321 192.376 195.358 1.00 87.65 C ATOM 173 NE ARG A 107 208.972 192.524 196.769 1.00 87.65 N ATOM 174 CZ ARG A 107 208.284 191.631 197.473 1.00 87.65 C ATOM 175 NH1 ARG A 107 208.007 191.857 198.749 1.00 87.65 N ATOM 176 NH2 ARG A 107 207.874 190.507 196.898 1.00 87.65 N ATOM 177 N ASN A 108 209.777 192.931 190.209 1.00104.17 N ATOM 178 CA ASN A 108 209.992 193.635 188.948 1.00104.17 C ATOM 179 C ASN A 108 209.913 192.681 187.757 1.00104.17 C ATOM 180 O ASN A 108 210.726 191.761 187.622 1.00104.17 O ATOM 181 CB ASN A 108 211.326 194.383 188.967 1.00104.17 C ATOM 182 CG ASN A 108 212.500 193.493 189.322 1.00104.17 C ATOM 183 OD1 ASN A 108 212.340 192.304 189.593 1.00104.17 O ATOM 184 ND2 ASN A 108 213.697 194.066 189.299 1.00104.17 N ATOM 185 IN THIS PLACE 109 208.925 192.912 186.893 1.00111.55 N ATOM 186 CA ILE A 109 208,701 192,129 185,682 1.00111.55 C ATOM 187 C ILE A 109 208.735 193.086 184.495 1.00111.55 C ATOM 188 THIS WEEK 109 207,919 192,972 183,573 1.00111.55 O ATOM 189 CB IL A 109 207.374 191.350 185.747 1.00111.55 C ATOM 190 CG1 ILE A 109 207.296 190.556 187.047 1.00111.55 C ATOM 191 CG2 ILE A 109 207.263 190.358 184.593 1.00111.55 C ATOM 192 CD1 ILE A 109 208.398 189.536 187.200 1.00111.55 C ATOM 193 N VAL A 110 209.638 194.070 184.552 1.00124.50 N ATOM 194 CA VAL A 110 209.718 195.191 183.613 1.00124.50 C ATOM 195 C VAL A 110 209.615 194.742 182.160 1.00124.50 C ATOM 196 O VAL A 110 210.365 193.871 181.704 1.00124.50 O ATOM 197 CB VAL A 110 211.015 195.993 183.832 1.00124.50 C ATOM 198 CG1 VAL A 110 211.030 196.604 185.223 1.00124.50 C ATOM 199 CG2 VAL A 110 212.237 195.108 183.622 1.00124.50 C ATOM 200 N CYS A 111 208.675 195.337 181.431 1.00128.31 N ATOM 201 CA CYS A 111 208.356 194.943 180.070 1.00128.31 C ATOM 202 C CYS A 111 209.149 195.782 179.073 1.00128.31 C ATOM 203 O CYS A 111 209.786 196.779 179.422 1.00128.31 O ATOM 204 CB CYS A 111 206.855 195.088 179.819 1.00128.31 C ATOM 205 SG CYS A 111 205.824 194.095 180.924 1.00128.31 S ATOM 206 N THR A 112 209.101 195.369 177.810 1.00137.95 N ATOM 207 CA THR A 112 209.823 196.046 176.742 1.00137.95 C ATOM 208 C THR A 112 208.880 196.310 175.579 1.00137.95 C ATOM 209 O THR A 112 208.140 195.416 175.156 1.00137.95 O ATOM 210 CB THR A 112 211,032 195,223 176,274 1.00137.95 C ATOM 211 OG1 THR A 112 211.767 195.964 175.291 1.00137.95 O ATOM 212 CG2 THR A 112 210.591 193.889 175.685 1.00137.95 C ATOM 213 N SER A 113 208,898 197,540 175,079 1.00136.24 N ATOM 214 CA SER A 113 208,144 197,898 173,890 1.00136.24 C ATOM 215 C SER A 113 209,009 197,629 172,660 1.00136.24 C ATOM 216 SER A 113 210.067 197.000 172.743 1.00136.24 O ATOM 217 CB SER A 113 207,681 199,350 173,974 1.00136.24 C ATOM 218 OG SER A 113 206,772 199,532 175,045 1.00136.24 O ATOM 219 N MET A 114 208,570 198,104 171,502 1.00129.10 N ATOM 220 CA MET A 114 209,253 197,854 170,242 1.00129.10 C ATOM 221 C MET A 114 209.385 199.167 169.470 1.00129.10 C ATOM 222 O MET A 114 208.786 200.186 169.824 1.00129.10 O ATOM 223 CB MET A 114 208.494 196.796 169.433 1.00129.10 C ATOM 224 CG MET A 114 209.213 196.273 168.200 1.00129.10 C ATOM 225 SD MET A 114 208.210 195.111 167.262 1.00129.10 S ATOM 226 CE MET A 114 207.022 196.231 166.527 1.00129.10 C ATOM 227 N LYS A 115 210.178 199.135 168.403 1.00131.31 N ATOM 228 CA LYS A 115 210.332 200.281 167.520 1.00131.31 C ATOM 229 C LYS A 115 209.617 199.992 166.202 1.00131.31 C ATOM 230 O LYS A 115 210.234 199.525 165.240 1.00131.31 O ATOM 231 CB LYS A 115 211.824 200.613 167.342 1.00131.31 C ATOM 232 CG LYS A 115 212.731 199.508 166.771 1.00131.31 C ATOM 233 CD LYS A 115 213.073 199.726 165.296 1.00131.31 C ATOM 234 CE LYS A 115 214.037 198.681 164.759 1.00131.31 C ATOM 235 NZ LYS A 115 214.317 198.890 163.308 1.00131.31 N ATOM 236 N PRO A 116 208.302 200.212 166.123 1.00130.15 N ATOM 237 CA PRO A 116 207.623 199.981 164.844 1.00130.15 C ATOM 238 C PRO A 116 207.996 201.074 163.864 1.00130.15 C ATOM 239 O PRO A 116 207.422 202.169 163.881 1.00130.15 THEIR ATOM 240 CB PRO A 116 206.138 200.029 165.219 1.00130.15 C ATOM 241 CG PRO A 116 206.098 200.918 166.416 1.00130.15 C ATOM 242 CD PRO A 116 207.371 200.660 167.174 1.00130.15 C ATOM 243 IN THIS PLACE 117 208.964 200.776 163.003 1.00134.54 N ATOM 244 CA ILE A 117 209,476 201,758 162,059 1.00134.54 C ATOM 245 C ILE A 117 210.110 201.052 160.863 1.00134.54 C ATOM 246 O ILE A 117 211.080 200.300 161.024 1.00134.54 O ATOM 247 CB ILE A 117 210,466 202,697 162,780 1.00134.54 C ATOM 248 CG1 VIEW 117 210.900 203.857 161.887 1.00134.54 C ATOM 249 CG2 VIEW 117 211.661 201.933 163.340 1.00134.54 C ATOM 250 CD1 ILE A 117 211.652 204.938 162.631 1.00134.54 C ATOM 251 N ASN A 118 209.569 201.285 159.659 1.00142.40 N ATOM 252 CA ASN A 118 210.235 200.929 158.398 1.00142.40 C ATOM 253 C ASN A 118 210.638 199.452 158.348 1.00142.40 C ATOM 254 O ASN A 118 211.665 199.085 157.773 1.00142.40 O ATOM 255 CB ASN A 118 211.446 201.842 158.192 1.00142.40 C ATOM 256 CG ASN A 118 211.048 203.296 157.999 1.00142.40 C ATOM 257 OD1 ASN A 118 210.835 204.006 158.979 1.00142.40 O ATOM 258 ND2 ASN A 118 211.000 203.773 156.755 1.00142.40 N ATOM 259 N GLU A 119 209.824 198.596 158.969 1.00123.74 N ATOM 260 CA GLU A 119 210.201 197.205 159.184 1.00123.74 C ATOM 261 C GLU A 119 209.066 196.275 158.777 1.00123.74 C ATOM 262 O GLU A 119 207.896 196.664 158.733 1.00123.74 O ATOM 263 CB GLU A 119 210,572 196,936 160,650 1.00123.74 C ATOM 264 CG GLU A 119 209,390 197,005 161,612 1.00123.74 C ATOM 265 CD GLU A 119 209,790 196,789 163,062 1.00123.74 C ATOM 266 OE1 GLU A 119 210.998 196.621 163.332 1.00123.74 O ATOM 267 OE2 GLU A 119 208,894 196,787 163,933 1.00123.74 O ATOM 268 N ASP A 120 209,438 195,028 158,491 1.00108.02 N ATOM 269 CA ASP A 120 208,514 193,936 158,218 1.00108.02 C ATOM 270 C ASP A 120 208,755 192,822 159,234 1.00108.02 C ATOM 271 ASP A 120 209.507 192.988 160.199 1.00108.02 O ATOM 272 CB ASP A 120 208,654 193,432 156,774 1.00108.02 C ATOM 273 CG ASP A 120 210,060 192,967 156,446 1.00108.02 C ATOM 274 OD1 ASP A 120 210.960 193.105 157.301 1.00108.02 O ATOM 275 OD2 ASP A 120 210.265 192.459 155.324 1.00108.02 O ATOM 276 N LEU A 121 208,115 191,674 159,020 1.00 94.90 N ATOM 277 CA LEU A 121 208,103 190,635 160,042 1.00 94.90 C ATOM 278 C LEU A 121 207,711 189,300 159,421 1.00 94.90 C ATOM 279 THE LEU A 121 207,075 189,246 158,365 1.00 94.90 O ATOM 280 CB LEU A 121 207,158 191,013 161,189 1.00 94.90 C ATOM 281 CG LEU A 121 205.649 191.025 160.928 1.00 94.90 C ATOM 282 CD1 READ A 121 204.890 191.120 162.237 1.00 94.90 C ATOM 283 CD2 READ A 121 205.249 192.169 160.009 1.00 94.90 C ATOM 284 N ASP A 122 208,104 188,221 160,099 1.00 95.78 N ATOM 285 CA ASP A 122 207,905 186,847 159,660 1.00 95.78 C ATOM 286 C ASP A 122 206,901 186,140 160,570 1.00 95.78 C ATOM 287 ASP A 122 206.271 186.755 161.437 1.00 95.78 O ATOM 288 CB ASP A 122 209.244 186.105 159.625 1.00 95.78 C ATOM 289 CG ASP A 122 210.161 186.605 158.528 1.00 95.78 C ATOM 290 OD1 ASP A 122 209.649 187.091 157.499 1.00 95.78 O ATOM 291 OD2 ASP A 122 211.395 186.505 158.692 1.00 95.78 O ATOM 292 N GLU A 123 206,749 184,832 160,366 1.00102.67 N ATOM 293 CA GLU A 123 205,846 184,029 161,182 1.00102.67 C ATOM 294 C GLU A 123 206.245 182.566 161.073 1.00102.67 C ATOM 295 O GLU A 123 206.483 182.069 159.968 1.00102.67 O ATOM 296 CB GLU A 123 204.391 184.224 160.745 1.00102.67 C ATOM 297 CG GLU A 123 204.092 183.742 159.339 1.00102.67 C ATOM 298 CD GLU A 123 202.659 184.010 158.930 1.00102.67 C ATOM 299 OE1 GLU A 123 201.905 184.580 159.747 1.00102.67 O ATOM 300 OE2 GLU A 123 202.284 183.654 157.793 1.00102.67 O ATOM 301 N GLY A 124 206.323 181.887 162.211 1.00103.93 N ATOM 302 CA GLY A 124 206.702 180.484 162.235 1.00103.93 C ATOM 303 C GLY A 124 206.171 179.800 163.473 1.00103.93 C ATOM 304 O GLY A 124 206.056 180.411 164.540 1.00103.93 O ATOM 305 NILE A 125 205.852 178.515 163.330 1.00111.88N ATOM 306 CA IL A 125 205.282 177.731 164.421 1.00111.88 C ATOM 307 C ile A 125 206.400 177.219 165.319 1.00111.88 C ATOM 308 O IL A 125 207.432 176.737 164.837 1.00111.88 O ATOM 309 CB IL A 125 204.427 176.578 163.859 1.00111.88 C ATOM 310 CG1 ILE A 125 205.251 175.673 162.940 1.00111.88 C ATOM 311 CG2 ILE A 125 203.236 177.124 163.093 1.00111.88 C ATOM 312 CD1 ILE A 125 205.906 174.503 163.636 1.00111.88 C ATOM 313 N MET A 126 206.197 177.311 166.634 1.00115.13 N ATOM 314 CA MET A 126 207.241 176.972 167.591 1.00115.13 C ATOM 315 C MET A 126 206.629 176.412 168.865 1.00115.13 C ATOM 316 O MET A 126 205.559 176.852 169.296 1.00115.13 O ATOM 317 CB MET A 126 208.113 178.193 167.915 1.00115.13 C ATOM 318 CG MET A 126 207.383 179.365 168.567 1.00115.13 C ATOM 319 SD MET A 126 207.276 179.304 170.366 1.00115.13 S ATOM 320 CE MET A 126 205.805 180.287 170.645 1.00115.13 C ATOM 321 N VAL A 127 207.322 175.445 169.464 1.00113.61 N ATOM 322 CA VAL A 127 206.956 174.869 170.751 1.00113.61 C ATOM 323 C VAL A 127 208.072 175.169 171.741 1.00113.61 C ATOM 324 O VAL A 127 209.253 175.155 171.380 1.00113.61 O ATOM 325 CB VAL A 127 206.710 173.351 170.649 1.00113.61 C ATOM 326 CG1 VAL A 127 205.547 173.068 169.717 1.00113.61 C ATOM 327 CG2 VAL A 127 207.966 172.645 170.169 1.00113.61 C ATOM 328 N VAL A 128 207.695 175.438 172.991 1.00114.43 N ATOM 329 CA VAL A 128 208.655 175.849 174.010 1.00114.43 C ATOM 330 C VAL A 128 208.740 174.790 175.100 1.00114.43 C ATOM 331 O VAL A 128 207.988 173.809 175.090 1.00114.43 O ATOM 332 CB VAL A 128 208.273 177.213 174.608 1.00114.43 C ATOM 333 CG1 VAL A 128 208.236 178.272 173.522 1.00114.43 C ATOM 334 CG2 VAL A 128 206.933 177.121 175.316 1.00114.43 C ATOM 335 N TYR A 129 209.651 174.984 176.049 1.00113.69 N ATOM 336 CA TYR A 129 209.794 174.079 177.182 1.00113.69 C ATOM 337 C TYR A 129 210.769 174.704 178.172 1.00113.69 C ATOM 338 O TYR A 129 211.405 175.723 177.887 1.00113.69 O ATOM 339 CB TYR A 129 210.244 172.687 176.732 1.00113.69 C ATOM 340 CG TYR A 1...

Claims

1. 1. A human cytomegalovirus (HCMV) immunogenic composition comprising RNA formulated in lipid nanoparticles, wherein the RNA encodes the amino acid sequence of a mutant of wild-type HCMV glycoprotein B (gB) protein as set forth in SEQ ID NO: 1, wherein the mutant comprises additional disulfide mutations compared to the amino acid sequence of SEQ ID NO: 1, the disulfide mutations comprising D217C and Y589C as numbered in SEQ ID NO: 1, the amino acid sequence of the mutant having at least 90% identity to residues 23-646, 23-702, or 23-907 as numbered in SEQ ID NO: 1, and wherein the mutant elicits an immune response against HCMV.

2. 2. The HCMV immunogenic composition of claim 1, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

3. 3. The HCMV immunogenic composition of claim 2, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol.

4. 3. The HCMV immunogenic composition of claim 2, wherein the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

5. 3. The HCMV immunogenic composition of claim 2, wherein the lipid nanoparticles have a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid.

6. 10. The HCMV immunogenic composition of claim 1, further comprising a polynucleotide encoding an additional HCMV antigenic polypeptide.

7. 7. The HCMV immunogenic composition of claim 6, wherein the additional HCMV antigenic polypeptide is selected from the group consisting of gH, gL, gO, gM, gN, UL128, UL130, UL131A, and pp65, and fragments thereof.

8. 8. The HCMV immunogenic composition of claim 7, wherein the additional HCMV antigenic polypeptide forms a trimer or a pentamer.

9. The HCMV immunogenic composition described in claim 1, wherein the disulfide mutations further include M371C and W506C or N524C and M684C according to the numbering of SEQ ID NO:

1.

10. The mutant (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; and (4) Substitution of I at position 675 with S 10. The HCMV immunogenic composition of claim 1 or 9, further comprising at least one additional mutation selected from the group consisting of:

11. 11. The HCMV immunogenic composition of claim 10, wherein the amino acid mutations are combinations of the following mutations: D217C and Y589C, M371C and W506C, Y155G, I156H, H157R, W240A, and C246S.

12. 2. The HCMV immunogenic composition of claim 1, wherein the RNA comprises the sequence set forth in SEQ ID NO:266.

Images