WO2015181142A1 - Cytomegalovirus complexes and uses thereof - Google Patents

Abstract

This disclosure provides modified cytomegalovirus (CMV) gL proteins and complexes comprising gL proteins, in particular pentameric complexes comprising gH, gL, pUL128, pUL130, pUL131. The disclosure also provides methods of purifying pentameric complexes and reducing contaminating dimeric complexes consisting of gH and gL. Also provided are uses of pentameric complexes in immunogenic compositions and vaccines.

Description

CYTOMEGALOVIRUS COMPLEXES AND USES THEREOF

TECHNICAL FIELD

[1] This invention is in the field of cytomegalovirus (CMV) vaccines.

BACKGROUND

[2] Cytomegalovirus is a genus of virus that belongs to the viral family known as Herpesviridae or herpesviruses. The species that infects humans is commonly known as human cytomegalovirus (HCMV) or human herpesvirus-5 (HHV-5). Within Herpesviridae, HCMV belongs to the Betaherpesvirinae subfamily, which also includes cytomegaloviruses from other mammals.

[3] Although they may be found throughout the body, HCMV infections are frequently associated with the salivary glands. HCMV infects between 50% and 80% of adults in the United States (40%> worldwide), as indicated by the presence of antibodies in much of the general population. HCMV infection is typically unnoticed in healthy people, but can be life-threatening for the

immunocompromised, such as HIV-infected persons, organ transplant recipients, or new born infants. HCMV is the virus most frequently transmitted to a developing fetus. After infection, HCMV has an ability to remain latent within the body for the lifetime of the host, with occasional reactivations from latency. Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health top priority (Sung, H., et al, (2010) Expert review of vaccines 9, 1303-1314; Schleiss, Expert Opin Ther Pat. Apr 2010; 20(4): 597-602).

[4] The genomes of over 20 different HCMV strains have been sequenced, including those of both laboratory strains and clinical isolates. For example, the following strains of HCMV have been sequenced: Towne

(GL239909366), AD169 (GL219879600), Toledo (GL290564358) and Merlin (GI: 155573956). HCMV strains AD 169, Towne and Merlin can be obtained from the American Type Culture Collection (ATCC VR538, ATCC VR977 and ATCC

VR1590, respectively). [5] CMV contains an unknown number of membrane protein complexes. Of the approximately 30 known glycoproteins in the viral envelope, gH and gL have emerged as particularly interesting due to their presence in several different complexes: dimeric gH/gL, trimeric gH/gL/gO (also known as the gCIII complex), and the pentameric gH/gL/pUL128/pUL130/pUL131 (pUL131 is also referred to as "pUL131A", "pUL131a", or "UL131A"; pUL128, pUL130, and pUL131 subunits sometimes are also referred as UL128, UL130, UL131). CMV is thought to use the pentameric complexes to enter epithelial and endothelial cells by endocytosis and low-pH-dependent fusion but it is thought to enter fibroblasts by direct fusion at the plasma membrane in a process involving gH/gL or possibly gH/gL/gO. The gH/gL and/or gH/gL/gO complex(es) is/are sufficient for fibroblast infection, whereas the pentameric complex is required to infect endothelial and epithelial cells.

[6] The pentameric complex is considered as a major target for CMV vaccination. Viral genes UL128, UL130 and UL131 are needed for endothelial entry (Hahn, Journal of Virology 2004; 78: 10023-33). Fibroblast-adapted non-endothelial tropic strains contain mutations in at least one of these three genes. Towne strain, for example, contains a 2 base pair insertion causing a frame shift in UL130 gene, whereas AD 169 contains a 1 base pair insertion in UL131 gene. Both Towne and AD 169 could be adapted for growth in endothelial cells, and in both instances, the frame shift mutations in UL130 or UL131 genes were repaired.

[7] US7704510 discloses that pUL 131 A is required for epithelial cell tropism. US7704510 also discloses that pUL128 and pUL130 form a complex with gH/gL, which is incorporated into virions. This complex is required to infect endothelial and epithelial cells but not fibroblasts. Anti-CD46 antibodies were found to inhibit HCMV infection of epithelial cells.

[8] CMV vaccines tested in clinical trials include Towne vaccine,

Towne-Toledo chimeras, an alpha virus replicon with gB as the antigen, gB/MF59 vaccine, a gB vaccine produced by GlaxoSmithKline, and a DNA vaccine using gB and pp65. pp65 is viral protein that is a potent inducer of CD8+ responses directed against CMV. These vaccines are all poor inducers of antibodies that block viral entry into endothelial/epithelial cells (Adler, S. P. (2013), British Medical Bulletin, 107, 57-68. doi: 10.1093/bmb/ldt023).

[9] Preclinical animal studies in CMV vaccines include an inactivated

AD 169 which has been repaired in the UL131 gene, a DNA vaccine using a wild-type UL130 gene and peptide vaccines using peptides from pUL130 and 131 (Sauer, A, et al, Vaccine 2011 ;29:2705- 1. doi: 10.1016).

[10] CMV gB antigen is considered a poor inducer of antibodies that block entry into endothelial/epithelial cells. In a Phase II clinical trial, the gB/MF59 vaccine was only 50% effective at preventing primary infection among young women with a child at home (Pass, RF, et al, N Engl J Med 2009;360: 1191-9).

[11] It is generally believed that neutralizing antibodies against the pentameric complex (gH/gLpUL128/pUL130/pUL131) will be significantly more potent than neutralizing antibodies raised against CMV gB subunit, or gH/gL dimeric complex. Therefore, a purified pentameric complex would be useful as antigen for diagnostic applications, and as an immunogen for vaccines against CMV.

[12] One problem for obtaining purified CMV pentameric complex is the presence of contaminating gH/gL dimers. Because neutralizing antibodies raised against gH/gL dimer are far less potent as compared to that of pentamer, there is a need to develop techniques to obtain purified CMV pentamer having reduced amount of contaminating gH/gL dimers.

SUMMARY OF THE INVENTION

[13] The inventors have discovered that when pentameric complex

(gH/gL/pUL128/pUL130/pUL131) is recombinantly expressed and purified, substantial amount of contaminating gH/gL dimers is found. Contaminating gH/gL dimers reduce the efficacy of pentamer-based vaccines, because neutralizing antibodies against pentamer are estimated to be 100 times more potent than the one raised against gH/gL dimers. [14] To solve this problem, the invention provides compositions and methods for producing gH/gL/pUL128/pUL130/pUL131 pentameric complex with significantly reduced amount of contaminating gH/gL dimeric complexes.

[15] In one aspect, the invention provides a modified CMV gL protein that interferes with the formation of gH/gL dimers. Surprisingly, the modification reduces the formation of gH/gL dimer without significantly affecting the formation of gH/gL/pUL128/pUL130/pUL131 pentamer. The pentameric complexes obtained using the modified gL proteins have properties that are not substantially different from pentamer formed by wild type gL. In particular embodiments, the modified gL proteins have amino acid modifications at amino acid residues corresponding to Cys47 of SEQ ID NO : 1 , Cys54 of SEQ ID NO : 1 , or Cys 144 of SEQ ID NO : 1.

[16] In another aspect, the invention provides a method of purifying

CMV pentameric complex comprising gH, gL, pUL128, pUL130 and pUL131 (or a complex- forming fragment from each of the subunit) using ion exchange

chromatography.

[17] In a further aspect, the invention provides a method of purifying

CMV pentameric complex comprising gH, gL, pUL128, pUL130 and pUL131 (or a complex- forming fragment from each of the subunit) using affinity chromatography.

[18] The invention also provides CMV gL proteins and CMV protein complexes for use in therapy, such as for use in inducing an immune response in a subject. The invention further provides CMV gL proteins and CMV protein complexes for use in in the manufacture of a medicament for inducing an immune response in a subject.

BRIEF SUMMARY OF THE DRAWINGS

[19] FIGS. 1A-1D show that mutations at Cys47 and Cys54 were effective in removing gH/gL dimers from pentameric complex. FIG. 1 A shows that substantial amount of contaminating gH/gL dimers (monomeric form and dimeric form) were formed, as shown in non-reduced coomassie gel. FIG. IB shows that the contaminating gH/gL dimers (especially dimeric form) could not be removed by sizing column, as they co-eluted with the pentamer. FIG. 1C shows the results of mutagenesis studies. Cysteins at 47, 54, 144, 154, 159, and 233 in gL were mutated to Ser, respectively. Among these mutations, Cys47, Cys54, and Cysl44 mutants reduced gH/gL dimer contamination, allowing pentamer to be purified. When co- expressed with the gH and pUL 128-131 subunits, the proteins formed a stable pentameric complex. FIG. ID is a SEC chromatography graph showing that pentameric incorporating gL mutant (C54S) can bind to neutralizing antibodies as the wild type pentameric complex.

[20] FIG. 2 shows that pentameric complex incorporating gL mutant

(C54S, open and closed squares) elicited immune response in Balb C mice, similar to wild type pentamer (open and closed circles). In this experiment, 1 μg of soluble wild type or C54S mutant pentameric complex was formulated with MF59. Formulated proteins were used to immunize BalbC mice (5 animals per group) three times at day 0, day 21 , and day 42. Blood samples were collected by retroorbital phlebotomy (RO) at 3wpl, 3wp2, and 3wp3. Neutralization titers were determined using CMV strain VR1814 on ARPE19 cells, and pentamer-specific binding titers were determined by ELISA. 3wpl : 3 weeks after the 1st vaccination; 3wp2: 3 weeks after the 2nd vaccination; 3 wp3 : 3 weeks after the 3rd vaccination.

DETAILED DESCRIPTION

1. OVERVIEW

[21] As disclosed and exemplified herein, one problem for obtaining purified CMV pentameric complex is the presence of contaminating gH/gL dimers. The inventors discovered that during the expression and purification of the pentameric complex, there existed a significant amount of contaminating gH/gL dimer. In a typical purification process, the contaminating gH/gL dimer represented about 10-20% of the total amount of purified complexes. In one particular experimental setting, the amount of gH/gL dimer was up to 40%> to 50%>. The gH/gL dimers exist both as a "monomeric" dimer (a dimer consisting of one gH subunit and one gL subunit), or a "dimeric" dimer (two "monomeric" dimers associate with each other, resulting in a complex consisting of two copies of gH subunit and two copies of gL subunit).

[22] The presence of contaminating gH/gL dimers is undesirable. The inventors overcame this problem by three different methods. First, the inventors identified three mutations in the gL subunit, at Cys47, Cys54, or Cysl44, that can interfere with the formation of gH/gL dimer. Surprisingly, the mutant gL proteins do not substantially interfere with the gH/gL/pUL128/pUL130/pUL131 pentamer formation. In fact, the pentameric complexes obtained using the mutant gL proteins have properties that are not substantially different from pentamers formed by wild type gL. Using this method, large amounts of pentameric complex can be purified, with gH/gL contamination nearly undetectable.

[23] Second, the inventors used a cation exchange method to purify pentameric complex, and successfully removed the contaminating gH/gL dimers to undetectable level.

[24] Third, by attaching a Strep tag on the C terminal region of pUL130 subunit, and using affinity purification, the inventors obtained substantially homogeneous pentamer is a single step. Again, gH/gL contamination was nearly undetectable after purification.

[25] Accordingly, in one aspect, the invention provides modified gL protein from CMV that favors the formation of gH/gL/pUL128/pUL130/pUL131 pentamer, and/or disfavors the formation of gH/gL dimer. In some embodiment, in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), at least 90% of the gL protein molecules, or complex-forming fragment thereof, form a pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits). In some embodiments, in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131(or a complex-forming fragment from each of the subunits), no more than 10% of the gL protein molecules, or complex- forming fragment thereof, form a dimeric complex consisting of gH and gL (or a complex- forming fragment from each of the subunits). In some embodiments, the modification in gL reduces the amount of dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits) by at least 50%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. [26] In another aspect, the invention provides a method of purifying

CMV pentameric complex from a sample using ion exchange chromatography. The method comprises: (i) providing a sample comprising the mixture of: (a) pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), and (b) dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits); (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction that comprises said pentameric complex from the ion exchange column. The methods described herein can result in purified product in which: (i) no more than 10% of protein complexes obtained by affinity purification are dimeric complex consisting of gH and gL (or a complex- forming fragment from each of the subunits); or (ii) at least 90% of protein complexes obtained by affinity purification are pentameric complex comprising CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex-forming fragment from each of the subunits).

[27] In another aspect, the invention provides a method of purifying

CMV pentameric complex that comprises CMV gH, gL, pUL128, pUL130, pUL131 (or a complex-forming fragment from each of the subunit), comprising: (i) providing a sample comprising the mixture of: (a) pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), and (b) dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits), wherein an affinity-purification tag is attached to one of the following sites: (i) C-terminal region of pUL130, (ii) N-terminal region of pUL130, (iii) C-terminal region of pUL131, (iv) N-terminal region of pUL131, (v) C- terminal region of pUL128, (vi) N-terminal region of pUL128, or a combination thereof, and wherein said affinity-purification tag binds specifically to a binding partner; (ii) purifying said pentameric complex by an affinity chromatography matrix, wherein said affinity chromatography matrix comprises said binding partner attached to a solid support. The methods described herein can result in purified product in which: (i) no more than 10% of protein complexes obtained by affinity purification are dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits); or (ii) at least 90% of protein complexes obtained by affinity purification are pentameric complex comprising CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex-forming fragment from each of the subunits). [28] The three strategies (gL modification, ion-exchange, and affinity purification) can be used singularly, or in any combination, to obtain purified pentameric complex.

[29] The invention also provides purified complexes comprising CMV gH, gL, pUL128, pUL130, pUL131 (or a complex-forming fragment from each of the subunits), and methods of using the purified pentameric complexes.

2. MODIFIED CMV gL PROTEINS AND COMPLEXES

A. Modified gL Proteins

[30] In one aspect, the invention provides a modified CMV gL protein, or a complex- forming fragment thereof, that reduces formation of contaminating gH/gL dimer.

[31] The gL protein described herein, or a complex-forming fragment thereof, comprises an amino acid modification, such that in the presence of

substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or a complex- forming fragment from each of the subunits): (i) at least about 80% (preferably about 90%) of the gL molecules, or complex- forming fragment thereof, form a pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex- forming fragment from each of the subunits); (ii) no more than about 20% (preferably about 10%) of the gL molecules, or complex- forming fragment thereof, form a dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits); or (iii) said amino acid modification reduces the amount of dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits) by at least about 30%> (preferably 50%>), relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification.

[32] Human CMV glycoprotein L (gL) is encoded by the UL115 gene. gL is thought to be essential for viral replication and all known functional properties of gL are directly associated with its dimerization with gH. The gH/gL complex is required for the fusion of viral and plasma membranes leading to virus entry into the host cell. gL from HCMV strain Merlin (GL39842115, SEQ ID NO: 1) and HCMV strain Towne (GL239909463, SEQ ID NO: 2) have been reported to be 278 amino acids in length. gL from HCMV strain AD169 (GL2506510, SEQ ID NO: 3) has been reported to be 278 amino acids in length, include a signal sequence at its N- terminus (amino acid residues 1-35), have two N-glycosylation sites (at residues 74 and 114) and lack a TM domain (Rigoutsos, I, et al, Journal of Virology 77 (2003): 4326-44). The N-terminal signal sequence in SEQ ID NO: 1 is predicted to comprise amino acid residues 1-30. SEQ ID NO: 2 shares 98% amino acid identity with SEQ ID NO: 1. Sequencing of the full-length gL gene from 22 to 39 clinical isolates, as well as laboratory strains AD 169, Towne and Toledo revealed less than 2% variation in the amino acid sequences among the isolates (Rasmussen, L, et al, Journal of Virology 76 (2002): 10841-10888).

[33] Typically, the N-terminal signal sequence of gL proteins is cleaved by a host cell signal peptidase to produce mature gL proteins. The gL proteins in HCMV membrane complexes of the invention may lack an N-terminal signal sequences. An example of gL protein lacking N-terminal signal sequences is SEQ ID NO: 4, which lacks an N-terminal signal sequence and consists of amino acid residues 31-278 of SEQ ID NO: 1.

[34] While gL is thought to be essential for viral replication, all known functional properties of gL are directly associated with its dimerization with gH.

[35] gL proteins of the invention can be gL variants that have various degrees of identity to SEQ ID NO: 1 such as at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence recited in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. gL proteins of the invention can have various degrees of identity to SEQ ID NO: 4 such as at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence recited in SEQ ID NO: 4. In certain embodiments, the gL variant proteins: (i) do not form substantial amount of dimeric complex with gH; (ii) form part of the trimeric gH/gL/gO complex; (iii) form part of the pentameric

gH/gL/pUL128/pUL130/pUL131 complex; (iv) comprise at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3, or SEQ ID NO: 4; and/or (v) can elicit antibodies in vivo which immunologically cross react with a CMV virion. [36] Also encompassed in the invention are complex- forming fragments of gL proteins described herein. A complex- forming fragment of gL can be any part or portion of the gL protein that retain the ability to form a complex with another CMV protein. In certain embodiments, a complex-forming fragment of gL forms part of the pentameric gH/gL/pUL128/pUL130/pUL131 complex. A complex- forming fragment of gL can be obtained or determined by standard assays known in the art, such as co-immunoprecipitation assay, cross-linking, or co-localization by fluorescent staining, etc. In certain embodiments, the complex-forming fragment of gL also (i) comprises at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and/or (ii) can elicit antibodies in vivo which immunologically cross react with a CMV virion.

[37] In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunit), at least about 80% of the gL molecules, or complex-forming fragment thereof, form a pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits). For example, the modification can result in at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%>, at least about 96%>, at least about 97%>, at least about 98%>, or at least about 99%,of the gL molecules, or complex-forming fragment thereof, form a pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits).

[38] In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), no more than about 20%> of the gL molecules, or complex-forming fragment thereof, form a dimeric complex consisting of gH and gL (or a complex- forming fragment from each of the subunits). For example, the modification can result in no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% of the gL molecules, or complex- forming fragment thereof, form a dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits).

[39] In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), said amino acid modification reduces the amount of dimeric complex consisting of gH and gL (or a complex- forming fragment from each of the subunits) by at least about 50%, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification (such as a wild type gL or complex- forming fragment thereof). For example, the amino acid modification can reduce the amount of dimeric complex consisting of gH and gL (or a complex- forming fragment from each of the subunits) by at least about 30%>, at least about 40%>, at least about 50%>, at least about 60%>, at least about 70%>, at least about 75%, at least about 80%, at least about 85%), at least about 90%>, at least about 95%, at least about 96%>, at least about 97%), at least about 98%>, or at least about 99%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification (such as a wild type gL or a complex-forming fragment thereof). Alternatively, the amino acid

modification can reduce the amount of dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits) by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, or at least 50 fold, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification (such as a wild type gL or a complex-forming fragment thereof).

[40] In certain embodiments, the amino acid modification is at an amino acid residue corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: l, Cysl44 of SEQ ID NO: 1, or a combination thereof. Standard alignment methods can be used to identify residues that correspond to Cys47 of SEQ ID NO: 1 , Cys54 of SEQ ID NO: 1 , Cysl44 of SEQ ID NO: 1 , or a combination thereof. The modification can be addition, deletion, or substitution of an amino acid residue. The modification can also be the introduction of a non-naturally occurring amino acid or an amino acid analog into a polypeptide chain.

[41] It was discovered that mutations at the residue corresponding to

Cys47 of SEQ ID NO: 1 or Cys54 of SEQ ID NO: 1 resulted in a modified gL protein that does not form with gH either monomeric dimer (one copy of gH and gL) or dimeric dimer (two copies of gH and gL). Alternatively, gH/gL monomeric or dimeric dimers are present at a level that was not detectable when the inventors used standard detection methods.

[42] Interestingly, modification at the amino acid residue corresponding to Cysl44 of SEQ ID NO: l also allows efficient purification of pentamer because this modification substantially eliminated dimeric form of gH/gL dimer. It is possible that residual amount of monomeric form of gH/gL dimer may still exist with

modifications at a residue corresponding to Cysl44 of SEQ ID NO: l. However, the monomeric form of gL/gH dimer that can be separated from pentamer by a sizing column, whereas the dimeric form co-elutes with pentamer (see, FIG. IB). Basically, even if gL protein with a modification at the residue corresponding to Cysl44 still forms a monomeric dimer with gH, the peak of the gH/gL monomeric dimer is sufficiently separated from the peak of pentamer (compare FIG. IB), such that pentamer and monomeric dimer does not co-elute, and pentamer can be purified by a standard sizing column.

[43] Preferably, the amino acid modification disrupts the ability of a cysteine residue to form disulfide bond, such as deletion of the cysteine residue, mutation from cysteine to another amino acid (such as glycine, serine, threonine, alanine, valine, leucine, isoleucine, etc.).

[44] In certain embodiments, the amino acid modification is at the amino acid residue adjacent to the position corresponding to Cys47 of SEQ ID NO: 1 , Cys54 of SEQ ID NO: 1 , Cysl44 of SEQ ID NO: 1 , or a combination thereof. The amino acid residue can either be a residue immediately next (i.e., residues

corresponding to positions 46, 48, 53, 55, 143, or 145 of SEQ ID NO: 1), or in conformational proximity (e.g., within 30 angstroms, or within 25 angstroms, or within 20 angstroms, or within 15 angstroms, or within 10 angstroms, or within 7 angstroms, or within 5 angstroms, from one of the atoms from the residue

corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: 1, or Cysl44 of SEQ ID NO: 1). In certain embodiment, the amino acid residue adjacent to the position corresponding to Cys47, Cys54, or Cysl44 of SEQ ID NO:l comprises a bulky side chain. A bulky side chain can sterically hinder the formation of disulfide bond.

[45] The invention also provides modified gL (e.g., Cysl44

modification) that reduces the formation of dimeric gH/gL dimers (two copies of gH and two copies of gL). In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV protein gH, and said gL or a complex- forming fragment thereof, at least about 80% of the gL molecules, or complex- forming fragment thereof, form a monomeric dimer consisting of: one copy of gH, and one copy of said gL or complex- forming fragment thereof. For example, the modification can result in at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%>, at least about 97%, at least about 98%>, or at least about 99%,of the gL molecules, or complex-forming fragment thereof, form a monomeric dimer consisting of: one copy of gH, and one copy of said gL or complex- forming fragment thereof.

[46] In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV protein gH, and said gL or a complex- forming fragment thereof, no more than about 20% of the gL molecules, or complex- forming fragment thereof, form a dimeric dimer consisting of: two copies of gH, and two copies of said gL or complex- forming fragment thereof. For example, the modification can result in no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% of the gL molecules, or complex-forming fragment thereof, form a dimeric dimer consisting of: two copies of gH, and two copies of said gL or complex- forming fragment thereof.

[47] In certain embodiments, the gL protein described herein, or a complex- forming fragment thereof, comprises an amino acid modification, such that in the presence of substantially equal molar amounts of CMV protein gH, and said gL or a complex- forming fragment thereof, said amino acid modification reduces the amount of dimeric dimer, consisting of two copies of gH, and two copies of said gL or complex-forming fragment thereof, by at least about 50%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. For example, the amino acid modification can reduce the amount of dimeric dimer consisting of: two copies of gH, and two copies of said gL (or a complex-forming fragment from each of the subunits) by at least about 30%, at least about 40%, at least about 50%), at least about 60%>, at least about 70%>, at least about 75%, at least about 80%), at least about 85%, at least about 90%>, at least about 95%, at least about 96%>, at least about 97%, at least about 98%, or at least about 99%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification (such as a wild type gL or a complex- forming fragment thereof). Alternatively, the amino acid modification can reduce the amount of dimeric dimer consisting of: two copies of gH, and two copies of said gL (or a complex- forming fragment from each of the subunits) by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, or at least 50 fold, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification (such as a wild type gL or a complex- forming fragment thereof).

B. CMV Protein Complexes

[48] In another aspect, the invention provides a complex comprising the modified CMV gL protein, or a complex- forming fragment thereof, described herein. Such complexes include, e.g., isolated trimeric complex comprising the modified gL protein, or a complex- forming fragment thereof, described herein, and CMV proteins gH or a complex- forming fragment thereof, and gO or a complex- forming fragment thereof; isolated pentameric complex comprising the the modified gL protein, or a complex- forming fragment thereof, described herein, and CMV proteins pUL128 or a complex- forming fragment thereof, pUL130 or a complex- forming fragment thereof, pUL131 or a complex- forming fragment thereof, and gH or a complex- forming fragment thereof. Also included are any other complexes comprising gL (or a complex- forming fragment thereof) as a component.

[49] Although gH, gL, gO, gB and pUL130 may be referred to as glycoproteins, this nomenclature should not be taken to mean that these proteins must be glycosylated when used with the invention. On the contrary, in some embodiments of the invention, one or more of polypeptides are not glycosylated. Usually, however, one or more (or all) polypeptides in a complex of the invention are glycosylated. In some embodiments, one or more (or all) polypeptides in a complex of the invention are glycosylated by glycosylation mutants of cultured cells, such as mutated mammalian cells. Such glycosylation mutants produce a pattern of polypeptide glycosylation which differs from a wild-type pattern of glycosylation, i.e. the resulting polypeptide glyco forms differs from wild-type glycoforms.

[50] In certain embodiments, the glycosylation pattern of the gL (or a complex- forming fragment thereof), or a complex comprising gL (or a complex- forming fragment thereof) has a mammalian glycosylation pattern; and/or does not have an insect cell pattern of glycosylation. In some embodiments, one or more of the proteins of the complex contain complex N-linked side chains with penultimate galactose and terminal sialic acid.

C. Nucleic acid encoding modified gL proteins and complexes

[51] In another aspect, the invention provides a nucleic acid comprising a sequence that encodes the modified gL protein, or a complex- forming fragment thereof, described herein. The nucleic acid can be DNA or R A.

[52] In certain embodiments, the nucleic acid is DNA. DNA-based expression systems for expression and purification of recombinant proteins are well- known in the art. For example, the expression system may be a vector comprising a nucleotide sequence that encodes the modified gL or gL fragment described herein, which is operably linked to an expression control sequence that regulates the expression of the modified gL or gL fragment in a host cell, such as a mammalian host cell, a bacterial host cell, or an insect host cell. The expression control sequence may be a promoter, an enhancer, a ribosome entry site, or a polyadenylation sequence, for example. Other expression control sequences contemplated for use in the invention include introns and 3' UTR sequences.

[53] The recombinantly expressed modified gL protein of fragment thereof, or a complex comprising the modified gL protein or fragment thereof can be purified using methods described herein, such as purification methods disclosed in WO 2014/005959, or other methods known in the art.

[54] In certain embodiments, the nucleic acid molecule is a vector derived from an adenovirus, an adeno-associated virus, a lentivirus, or an alphavirus. In certain embodiments, the nucleic acid molecule is a replication-deficient viral vector.

[55] In certain embodiments, the nucleic acid is RNA. In certain embodiments, the nucleic acid is a self-replicating RNA molecule, such as an alphavirus-derived RNA replicon.

[56] Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is typically a (+)-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA- dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells. Cells trans fected with self-replicating RNA briefly produce of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super- activate Dendritic Cells. Thus, the enhanced immunogenicity of self-replicating RNA may be a result of the production of pro -inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

[57] One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon. Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus, including, Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. As such, the self-replicating RNA of the invention may incorporate a RNA replicase derived from semliki forest virus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross-River virus (RRV), eastern equine encephalitis virus, or other viruses belonging to the alphavirus family.

[58] An alphavirus-based "replicon" expression vectors can be used in the invention. Replicon vectors may be utilized in several formats, including DNA, RNA, and recombinant replicon particles. Such replicon vectors have been derived from alphaviruses that include, for example, Sindbis virus (Xiong et al. (1989) Science 243: 1188-1191; Dubensky et al, (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol. 72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus (Liljestrom (1991) Bio/Technology 9: 1356-1361; Berglund et al. (1998) Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus (Pushko et al. (1997) Virology 239:389-401). Alphaviruses-derived replicons are generally quite similar in overall characteristics (e.g., structure, replication), individual alphaviruses may exhibit some particular property (e.g., receptor binding, interferon sensitivity, and disease profile) that is unique. Therefore, chimeric alphavirus replicons made from divergent virus families may also be useful.

[59] Use of Alphavirus-based RNA replicon is known in the art, see, e.g., WO 2013006837, paragraphs [0155] to [0179]. The RNA replicon can be administered without the need for purification of the protein encoded therein.

[60] In certain embodiments, the nucleic acid molecule is part of a vector derived from an adenovirus. The adenovirus genome is a linear double- stranded DNA molecule of approximately 36,000 base pairs with the 55-kDa terminal protein covalently bound to the 5' terminus of each strand. Adenoviral ("Ad") DNA contains identical Inverted Terminal Repeats ("ITRs") of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are located within the ITRs exactly at the genome ends. Adenoviral vectors for use with the present invention may be derived from any of the various adenoviral serotypes, including, without limitation, any of the over 40 serotype strains of adenovirus, such as serotypes 2, 5, 12, 40, and 41.

[61] In certain embodiments, the nucleic acid molecule is part of a vector derived from an Adeno Associated Virus (AAV). The AAV genome is a linear single- stranded DNA molecule containing approximately 4681 nucleotides. The AAV genome generally comprises an internal nonrepeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including serving as origins of DNA replication and as packaging signals for the viral genome. AAV is a helper- dependent virus; that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAV virions in the wild. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus rescues the integrated genome, allowing it to replicate and package its genome into infectious AAV virions. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells co-infected with a canine adenovirus.

[62] In certain embodiments, the nucleic acid molecule is part of a vector derived from a retroviruses. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described. See, e.g., U.S. Pat. No.

5,219,740; Miller and Rosman (1989) BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991) Virology 180:849-52; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop. 3 : 102-09. [63] The invention also provides host cells comprising the nucleic acid molecules disclosed herein. Host cells suitable for harboring the nucleic acid molecules and/or for expressing recombinant proteins, and methods of introducing a nucleic acid into a suitable host cell, are known in the art.

3. PURIFICATION OF CMV PENTAMERIC COMPLEXES BY ION

EXCHANGE

[64] In another aspect, the invention provides a method of purifying

CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV gH, gL, pUL128, pUL130, and pUL131 proteins, comprising: (i) providing a sample comprising (a) pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), and (b) dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits); (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction that comprises said pentameric complex from the ion exchange column.

[65] As disclosed and exemplified herein, it was discovered that ion exchange chromatography was surprisingly effective in removing contaminating gH/gL dimers from pentameric complex. In certain embodiments, no more than about 20% of protein complexes collected from the collected fraction are dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits). For example, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% of protein complexes collected from the collected fraction are dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits).

[66] In certain embodiments, at least about 80% of protein complexes collected from the collected fraction are pentameric complex comprising CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex-forming fragment from each of the subunits). For example, at least about 85%, at least about 90%>, at least about 91%), at least about 92%, at least about 93%>, at least about 94%>, at least about 95%, at least about 96%>, at least about 97%, at least about 98%>, or at least about 99%, of protein complexes collected from the collected fraction are pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits).

[67] Examples of materials useful in the ion exchange chromatography include DEAE-cellulose, QAE-cellulose, DEAE-cephalose, QAE-cephalose, DEAE- Toyopearl, QAE-Toyopearl, Mono Q, Mono S, Q sepharose, SP sepharose, etc. In one exemplary embodiment, the method uses Mono S column. In another exemplary embodiment, the method uses Mono Q column.

4. PURIFICATION OF CMV PENTAMERIC COMPLEXES BY AFFINITY TAGS

[68] In another aspect, the invention provides a method of purifying

CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV gH, gL, pUL128, pUL130, and pUL131 proteins, comprising: (i) providing a sample comprising: (a) pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex-forming fragment from each of the subunits), and (b) dimeric complex consisting of gH and gL (or a complex-forming fragment from each of the subunits), wherein an affinity-purification tag is attached to one of the following sites: (i) C-terminal region of pUL130, (ii) N-terminal region of pUL130, (iii) C-terminal region of pUL131, (iv) N-terminal region of pUL131, (v) C- terminal region of pUL128, (vi) N-terminal region of pUL128, or a combination thereof, and wherein said affinity-purification tag binds specifically to a binding partner; and (ii) purifying said pentameric complex by an affinity chromatography matrix, wherein said affinity chromatography matrix comprises said binding partner attached to a solid support.

[69] The structure of gH/gL/pUL128/pUL130/ pUL131 pentameric complex is unknown; therefore, if the affinity-purification tag is attached to a site that interferes with the formation of pentameric complex, or a site that is buried within the complex, affinity-purification would not be successful. As disclosed and exemplified herein, the following sites are believed to be suitable for attaching an affinity- purification tag, as the tag does not appear to interfere with formation of pentameric complex, and appears to be expose at the surface of an assembled pentamer: (i) C- terminal region of pUL130, (ii) N-terminal region of pUL130, (iii) C-terminal region of pUL131, (iv) N-terminal region of pUL131, (v) C-terminal region of pUL128, (vi) N-terminal region of pUL128, or a combination thereof.

[70] Examples of affinity-purification tags include, e.g., His tag (binds to metal ion), an antibody (binds to protein A or protein G), maltose-binding protein (MBP) (binds to amylose), glutathione- S -transferase (GST) (binds to glutathione), FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (binds to an anti-flag antibody), Strep tag (binds to streptavidin or a derivative thereof).

[71] One exemplary embodiment is Strep tag (or streptavidin affinity tag), a tag that binds to streptavidin or a derivative thereof, such as Strep-Tactin.

Strep tag comprises a peptide of nine amino acids: Ala-Trp-Arg-His-Pro-Gln-Phe- Gly-Gly, or eight amino acids (also called strep-tag II): Trp-Ser- His-Pro-Gln-Phe- Glu-Lys. Elution of a protein attached to a strep-tag from the column can be performed using biotin or a derivative or homologue thereof, such as desthio-biotin.

[72] The affinity-purification tag may be attached by any suitable means, and may be attached directly or indirectly. For example, the tag may be covalently attached at the N-terminus of the polypeptide sequence, or at the C-terminus of the polypeptide sequence. This can be achieved by recombinant expression of a fusion protein comprising the polypeptide and the tag, or by standard conjugation techniques that links the polypeptide to the tag. The tag may be attached to the side chain functional group of an amino acid residue of the polypeptide using standard

conjugation techniques.

[73] Alternatively, the tag may be attached non-covalently, for example, an antibody that binds to the C-terminal region of pUL130 may be used as a tag. The antibody is attached to the pentamer non-covalently. The pentamer-antibody complex can then be purified by a protein-A or protein-G column. Protein-A or protein-G is the binding partner for the antibody tag.

[74] In a certain embodiment, the tag is attached at the C-terminal residue of pUL130 (or a complex-forming fragment thereof). In a certain

embodiment, the tag is attached at a residue that is within 20 amino acids, within 15 amino acids, within 10 amino acids, within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids within 5 amino acids within 4 amino acids within 3 amino acids, or within 2 amino acids, from the C-terminal residue of pUL130 (or a complex-forming fragment thereof).

[75] In a certain embodiment, the tag is attached at the C-terminal residue of pUL131 (or a complex-forming fragment thereof). In a certain

embodiment, the tag is attached at a residue that is within 20 amino acids, within 15 amino acids, within 10 amino acids, within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids within 5 amino acids within 4 amino acids within 3 amino acids, or within 2 amino acids, from the C-terminal residue of pUL131 (or a complex-forming fragment thereof).

[76] Attachment of the tag may be direct, or indirect (through a linker).

Suitable linkers are known to those skilled in the art and include, e.g., straight or branched-chain carbon linkers, heterocyclic carbon linkers, carbohydrate linkers and polypeptide linkers.

[77] In a certain embodiment, cleavable linkers may be used to attach the molecule of interest to the tag. This allows for the tag to be separated from the purified complex, for example by the addition of an agent capable of cleaving the linker. A number of different cleavable linkers are known to those of skill in the art. Such linkers may be cleaved for example, by irradiation of a photolabile bond or acid- catalyzed hydrolysis. There are also polypeptide linkers which incorporate a protease recognition site and which can be cleaved by the addition of a suitable protease enzyme.

5. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

[78] The invention also provides pharmaceutical compositions comprising the CMV proteins, complexes, and nucleic acids described herein. The invention also provides pharmaceutical compositions comprising nucleic acid encoding CMV proteins, complexes, and nucleic acids described herein. [79] The CMV proteins, complexes, and nucleic acids described herein can be incorporated into an immunogenic composition, or a vaccine composition. Such compositions can be used to raise antibodies in a mammal (e.g. a human).

[80] The invention provides pharmaceutical compositions comprising the CMV proteins, complexes, and nucleic acids described herein, and processes for making a pharmaceutical composition involving combining the CMV proteins, complexes, and nucleic acids described herein with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention typically include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in Remington: The Science and Practice of Pharmacy.

[81] The pH of the composition is usually between about 4.5 to about

9.0, such as between about 5 to about 9, between about 5.5 to about 9, between about 6 to about 9, between about 5 to about 8.5, between about 5.5 to about 8.5, between about 6 to about 8.5, between about 5 to about, between about 5.5 to about 8, between about 6 to about 8, about 4.5, about 5, about 6.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, etc. Stable pH may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, phosphate buffer, or a histidine buffer. Thus a composition will generally include a buffer.

[82] A composition may be sterile and/or pyrogen free. Compositions may be isotonic with respect to humans.

[83] A composition comprises an immunologically effective amount of its antigen(s). An "immunologically effective amount" is an amount which, when administered to a subject, is effective for eliciting an antibody response against the antigen. This amount can vary depending upon the health and physical condition of the individual to be treated, their age, the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The antigen content of compositions of the invention will generally be expressed in terms of the mass of protein per dose. A dose of 10-50(^g (e.g. 50μg) per antigen can be useful. [84] Immunogenic compositions may include an immunological adjuvant. Exemplary adjuvants include the following: 1. mineral-containing compositions; 2. oil emulsions; 3. saponin formulations; 4. virosomes and virus-like particles; 5. bacterial or microbial derivatives; 6. bioadhesives and mucoadhesives; 7. liposomes; 8. polyoxyethylene ether and polyoxy ethylene ester formulations; 9.

polyphosphazene (pcpp); 10. muramyl peptides; 11. imidazoquinolone compounds; 12. thiosemicarbazone compounds; 13. tryptanthrin compounds; 14. human

immunomodulators; 15. lipopeptides; 16. benzonaphthyridines; 17. microparticles; 18. immunostimulatory polynucleotide (such as rna or dna; e.g., cpg-containing

oligonucleotides) .

[85] For example, the composition may include an aluminium salt adjuvant or an oil in water emulsion (e.g. an oil-in-water emulsion comprising squalene, such as MF59 or AS03). Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of Vaccine Design... (1995) eds. Powell & Newman. ISBN:

030644867X. Plenum), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being an example. The concentration of A1+++ in a composition for administration to a patient may be less than 5mg/ml e.g. <4 mg/ml, <3 mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and lmg/ml. A maximum of 0.85mg/dose is preferred. Aluminum hydroxide and aluminium phosphate adjuvants are suitable for use with the invention.

[86] One suitable immunological adjuvant comprises a compound of

Formula (I) as defined in WO2011/027222, or a pharmaceutically acceptable salt thereof, adsorbed to an aluminum salt. Many further adjuvants can be used, including any of those disclosed in Powell & Newman (1995).

[87] Compositions may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thiomersal and 2

phenoxyethanol are commonly found in vaccines, but sometimes it may be desirable to use either a mercury- free preservative or no preservative at all. [88] Compositions may comprise detergent e.g. a polysorbate, such as polysorbate 80. Detergents are generally present at low levels e.g. <0.01%.

[89] Compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml.

[90] Compositions of the invention will generally be administered directly to a subject. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by any other suitable route. For example, intramuscular administration may be used e.g. to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dosage volume is 0.5 ml.

[91] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[92] The subject may be a human, and may also be, e.g., a cow, a pig, a chicken, a cat or a dog, as the pathogens covered herein may be problematic across a wide range of species. Where the vaccine is for prophylactic use, the human is preferably a child (e.g., a toddler or infant), a teenager, or an adult; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc..

[93] Vaccines of the invention may be prophylactic (i.e. to prevent disease) or therapeutic (i.e. to reduce or eliminate the symptoms of a disease).

[94] Nucleic acid molecules disclosed herein may be formulated and administered as a nucleic acid-based vaccine according to art-known standard. [95] Isolated and/or purified CMV proteins, complexes, and nucleic acids described herein can be administered alone or as either prime or boost in mixed- modality regimes, such as a RNA prime followed by a protein boost. Benefits of the RNA prime protein boost strategy, as compared to a protein prime protein boost strategy, include, for example, increased antibody titers, a more balanced IgGl :IgG2a subtype profile, induction of THl-type CD4+ T cell-mediated immune response that was similar to that of viral particles, and reduced production of non-neutralizing antibodies. The RNA prime can increase the immunogenicity of compositions regardless of whether they contain or do not contain an adjuvant.

[96] In the RNA prime-protein boost strategy, the RNA and the protein are directed to the same target antigen. Examples of suitable modes of delivering RNAs include virus-like replicon particles (VRPs), alphavirus RNA, replicons encapsulated in lipid nanoparticles (LNPs) or formulated RNAs, such as replicons formulated with cationic nanoemulsions (CNEs). Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012/006380 e.g. comprising an oil core (e.g. comprising squalene) and a cationic lipid (e.g. DOTAP, DMTAP, DSTAP, DC- cholesterol, etc.).

[97] WO2012/051211 discloses that antibodies to the pentameric complex are produced in mice that have been immunized with VRPs and formulated RNAs (CNEs and LNPs) that encode the protein constituents of the pentameric complex. These antibodies have been found to be capable of neutralizing CMV infection in epithelial cells. The RNA prime-protein boost regimen may involve first (e.g. at weeks 0-8) performing one or more priming immunization(s) with RNA (which could be delivered as VRPs, LNPs, CNEs, etc.) that encodes one or more of the protein components of a CMV protein complex of the invention and then perform one or more boosting immunization(s) later (e.g. at weeks 24-58) with: an isolated CMV protein complex of the invention, optionally formulated with an adjuvant or a purified CMV protein complex of the invention, optionally formulated with an adjuvant.

[98] In some embodiments, the RNA molecule is encapsulated in, bound to or adsorbed on a cationic lipid, a liposome, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, a cationic nanoemulsion, or combinations thereof.

[99] Also provided herein are kits for administration of nucleic acid

(e.g., R A), purified proteins, and purified complexes described herein, and instructions for use. The invention also provides a delivery device pre- filled with a composition or a vaccine disclosed herein.

[100] The pharmaceutical compositions described herein can be administered in combination with one or more additional therapeutic agents. The additional therapeutic agents may include, but are not limited to antibiotics or antibacterial agents, antiemetic agents, antifungal agents, anti-inflammatory agents, antiviral agents, immunomodulatory agents, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumour antibiotics, antimitotic agents, topoisomerase inhibitors, cytostatic agents, anti-invasion agents, antiangiogenic agents, inhibitors of growth factor function inhibitors of viral replication, viral enzyme inhibitors, anticancer agents, a-interferons, β-interferons, ribavirin, hormones, and other toll-like receptor modulators, immunoglobulins (Igs), and antibodies modulating Ig function (such as anti-IgE (omalizumab)).

[101] In certain embodiments, the compositions disclosed herein may be used as a medicament, e.g., for use in inducing or enhancing an immune response in a subject in need thereof, such as a mammal.

[102] In certain embodiments, the compositions disclosed herein may be used in the manufacture of a medicament for inducing or enhancing an immune response in a subject in need thereof, such as a mammal.

[103] One way of checking efficacy of therapeutic treatment involves monitoring pathogen infection after administration of the compositions or vaccines disclosed herein. One way of checking efficacy of prophylactic treatment involves monitoring immune responses, systemically (such as monitoring the level of IgGl and IgG2a production) and/or mucosally (such as monitoring the level of IgA production), against the antigen. Typically, antigen-specific serum antibody responses are determined post-immunization but pre-challenge whereas antigen-specific mucosal antibody responses are determined post-immunization and post-challenge.

6. DEFINITIONS

[104] The term "complex-forming fragment" of a CMV protein (such as gL) refers to any part or portion of the protein that retain the ability to form a complex with another CMV protein. Such complexes include, e.g., gH/gL dimeric complex, gH/gL/gO trimeric complex, or gH/gL/pUL128/pUL130/pUL131 pentameric complex.

[105] As used herein, "gH/gL dimeric complex," or "gH/gL dimer," refer to a protein complex formed by gH and gL. CMV gH and gL subunits can form either a "monomeric" dimer (a dimer consisting of one gH subunit and one gL subunit) or a "dimeric" dimer (two "monomeric" dimers associate with each other, resulting in a complex consisting of two copies of gH subunit and two copies of gL subunit).

Both forms of dimers that generally referred to as gH/gL dimeric complex, or gH/gL dimer. Although generally referred to as gH/gL dimer in the specification, the gH and gL subunit do not need to be full-length; the term also encompasses monomeric dimers and dimeric dimers formed by complex- forming fragments of gH and gL.

[106] As used herein, "pentameric complex" or "pentamer" refers to CMV complex that comprises five different subunits: gH, gL, pUL128, pUL130, and pUL131. Although generally referred to as gH/gL/pUL128/pUL130/pUL131 pentamer (or pentameric complex comprising gH, gL, pUL128, pUL130, and pUL131) in the specification, each of the five subunits does not need to be full-length; the term also encompasses pentamers formed by complex- forming fragments of gH, gL, pUL128, pUL130, and pUL131.

[107] The term "substantially equal" herein refers to any numerical value with a variance of +/- 20% from the base numerical value.

[108] The term "about", as used here, refers to +/- 5% of a base value.

[109] The term "amino acid modification" refers to addition, deletion, or substitution of an amino acid residue. The term also includes modifications that introduce a non-naturally occurring amino acid or an amino acid analog into a polypeptide chain.

[110] An amino acid residue of a query sequence "corresponds to" a designated position of a reference sequence (e.g., Cys47 or Cys54 of SEQ ID NO: 1) when, by aligning the query amino acid sequence with the reference sequence, the position of the residue matches the designated position. Such alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, or "BLAST 2 Sequences" using default parameters.

[Ill] An amino acid residue comprises a "bulky side chain" when the side chain comprises a branched or cyclic substituent. Examples of amino acid residues with a bulky side chain include tryptophan, tyrosine, phenylalanine, homophenylalanine, leucine, iso leucine, histidine, 1-methyltryptophan, a- methyltyrosine, a-methylphenylalanine, a-methylleucine, a-methyliso leucine, a- methylhistidine, cyclopentylalanine, cyclohexylalanine, naphthylalanine, etc.

EXAMPLES

[1] The inventors have discovered that when pentameric complex

(gH/gL/pUL128/pUL130/pUL131) is recombinantly expressed and purified, substantial amount contaminating gH/gL dimers are found. In a typical experiment, a consistent presence of contaminating gH/gL dimers, representing about 10-20% of the entire amount, was observed. CMV gH/gL subunits can form either a monomeric complex (dimer of gH and gL) or a dimeric complex (dimer of heterodimers). Both complexes co-exist with the pentameric complex and need to be removed.

[2] The investors have identified several strategies to limit or eliminate the described excess gH/gL dimers during pentamer production for vaccine development.

2. EXAMPLE 1: MODIFIED gL

[3] The inventors identified 3 point-mutants on gL subunit that interfere with the gH/gL complex formation (both for monomeric and dimeric forms of gH/gL dimers). These mutants do not substantially interfere with formation of pentamers, and can be used to purify large amounts of pentamers. The purified pentameric complex does not comprise the gH/gL contamination (at least, not detectable by standard methods).

[4] The gL wild type sequence used for Pentameric production is the following (signal peptide in bold; Cys47, Cys54, and Cysl44 underlined):

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWL

RPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRS I FTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGI TLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

[5] The gL mutants used to avoid the gH/gL contamination are the following (signal peptide in bold; Cys47-Ser, Cys54-Ser, and Cysl44-Ser underlined): gL Cys47-Ser

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAESPELTRRCLLGEVFEGDKYESWL

RPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRS I FTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGI TLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

gL Cys54-Ser

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRSLLGEVFEGDKYESWL

RPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRS I FTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGI TLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

gL Cysl44-Ser

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWL

RPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSESGDGSPAVYTCVDDLCRGYDLTRLSYGRS I FTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGI TLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

[6] 293EBNA cells were transfected with plasmids encoding

individual subunits carrying the point mutations in gL. Proteins were purified from mammalian cells supernatants by affinity-chromatography on Strep-Tactin Superflow Plus resin (Qiagen, Valencia, CA, USA) using a Strep-tag II tag on pUL130 subunits. The complex was eluted from the resin by competition with elution buffer (25 mM Hepes pH 7.5, 300 mM NaCl) containing 5 mM Desthiobiotin. The complexes were then subjected to size exclusion chromatography (SEC) on a Superose 6 PC 3.2/30 column (GE Healthcare, Uppsala, Sweden) equilibrated in elution buffer. Strep tagged proteins were overexpressed and purified using a similar strategy and eluted in 25 mM Hepes pH 7.5, 150 mM NaCl. HCMV complexes were incubated for 2 hours on ice and purified by SEC.

[7] EM grids were prepared by depositing a thin layer of continuous carbon over a holey carbon layer on a 400-mesh copper grid (Electron Microscopy Sciences). Five microliters of purified sample (approximately 30ng) were placed onto a fresh glow discharged grid. After 30 second of incubation, the grid was laid on top of 75-μ1 drops of a freshly prepared 2% (w/v) uranyl formate solution and stirred gently for five subsequent 10-s staining steps. Samples were imaged using a Tecnai Spirit T12 transmission electron microscope operating at 120 keV at a nominal magnification of 49,000x (1.57 A/pixel at the detector level) using a defocus range of -0.8 to -1.2 μιη. Images were recorded under low-dose conditions on a Gatan 4096 x 4096 pixel CCD camera. For the random conical tilt (RCT) dataset, images were collected at -56° and 0°. Particle picking was done semi-automatically using e2boxer Tang, G., Peng, L., Baldwin, P.R., Mann, D.S., Jiang, W., Rees, I., and Ludtke, S.J. (2007). EMAN2: an extensible image processing suite for electron microscopy.

Journal of structural biology 157, 38-46) A 224 x 224-pixel particle window was used for all datasets. All datasets were band-pass filtered with a 200-A high-pass cutoff and either a 20-A low-pass cutoff. Iterative multivariate statistical analysis (MSA) and multi-reference alignment (MRA) in Imagic ( van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R., and Schatz, M. (1996). A new generation of the IMAGIC image processing system. Journal of structural biology 1 16, 17-24) of the extracted particles provided representative 2D views of the HCMV complexes. On average, about 20 particles were included per class average

[8] The pentameric complex obtained using the described procedure had substantially identical properties as compared to the wild type. It was able to bind neutralizing Fabs, it ran in SDS-PAGE and SEC with the similar apparent molecular weight, and looked indistinguishable by Electron Microscopy. See, FIGS 1A-1D.

[9] Significantly, these mutants also raised a similar immune-response in Balb C mice as compared to the wild type. See, FIG. 2. 3. EXAMPLE 2: AFFINITY-PURIFICATION

[10] In this Example, we show that, by attaching a Strep tag on the C- terminal region of pUL130, and subsequent affinity purification, gH/gL

contamination was substantially eliminated.

[11] 293EBNA were transfected with plasmids encoding individual subunits. Proteins were purified from mammalian cells supernatants by affinity- chromatography on Strep-Tactin Superflow Plus resin (Qiagen, Valencia, CA, USA) using a Strep-tag II tag on pUL130 subunits. The complex was eluted from the resin by competition with elution buffer (25 mM Hepes pH 7.5, 300 mM NaCl) containing 5 mM Desthiobiotin. The complexes were then subjected to size exclusion chromatography (SEC) on a Superose 6 PC 3.2/30 column (GE Healthcare, Uppsala, Sweden) equilibrated in elution buffer.

[12] The purification can occur in a single step and the resulting pentameric complex was substantially homogeneous, and substantially free of gH/gL dimers (data not shown).

4. EXAMPLE 3: ION EXCHANGE

[13] In this Example, we show that, by ion exchange chromatography, gH/gL contamination was substantially eliminated.

[14] Specifically, a cation exchange column was used. Pentameric complex containing gH/gL contamination was initially dialyzed in 20mM MES pH 6.0, 50mM NaCl buffer. This sample was then loaded on a MonoS PC 1.6/5 column. Pentameric complex was retained on the column while excess of gH/gL was present in the unbound fraction. Pentameric complex was purified without gH/gL

contamination from the MonoS column with 20mM MES pH 6.0, 250mM NaCl buffer (data not shown).

[15] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[16] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by claimed embodiments.

[17] Particular embodiments of the invention include:

1. An isolated cytomegalovirus (CMV) gL protein, or a complex- forming

fragment thereof, comprising an amino acid modification, such that in the presence of substantially equal molar amounts of CMV proteins gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof:

(i) at least 90% of the gL molecules, or complex-forming fragment

thereof, form a pentameric complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex- forming fragment thereof;

(ii) no more than 10% of the gL molecules, or complex- forming fragment thereof, form a dimeric complex consisting of: gH, and said gL or complex- forming fragment thereof; or

(iii) said amino acid modification reduces the amount of dimeric complex, consisting of gH, and said gL or complex- forming fragment thereof, by at least 50%, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification.

2. The gL protein of embodiment 1, wherein at least 90% of the gL molecules, or complex- forming fragment thereof, form a pentameric complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof. The gL protein of embodiment 1 or 2, wherein at least 95% of the gL molecules, or complex-forming fragment thereof, form a pentameric complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex- forming fragment thereof. The gL protein of embodiment 1, wherein no more than 10% of the gL molecules, or complex-forming fragment thereof, form a dimeric complex consisting of gH, and gL or a complex- forming fragment thereof. The gL protein of embodiment 1 or 4, wherein no more than 5% of the gL molecules, or complex-forming fragment thereof, form a dimeric complex consisting of gH, and gL or a complex- forming fragment thereof. The gL protein of embodiment 1, wherein said amino acid modification reduces the amount of dimeric complex consisting of gH, and gL or a complex-forming fragment thereof, by at least 50%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. The gL protein of embodiment 1 or 6, wherein said amino acid modification reduces the amount of dimeric complex consisting of gH, and gL or a complex-forming fragment thereof, by at least 75%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. The gL protein of any one of embodiments 1 and 6-7, wherein said amino acid modification reduces the amount of dimeric complex consisting of gH, and gL or a complex- forming fragment thereof, by at least 90%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification The gL protein of any one of embodiments 1-8, wherein said amino acid modification is at an amino acid residue corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: l, or Cysl44 of SEQ ID NO: l . The gL protein of embodiment 9, wherein the amino acid residue

corresponding to Cys47 of SEQ ID NO: 1 is absent, or is an amino acid residue other than Cysteine. The gL protein of embodiment 10, wherein the amino acid residue

corresponding to Cys47 of SEQ ID NO: 1 is Glycine, Serine or Alanine. The gL protein of any one of embodiments 9-11, wherein the amino acid residue corresponding to Cys54 of SEQ ID NO: l is absent, or is an amino acid residue other than Cysteine. The gL protein of embodiment 12, wherein the amino acid residue

corresponding to Cys54 of SEQ ID NO: 1 is Glycine, Serine or Alanine. The gL protein of any one of embodiments 9-13, wherein the amino acid residue corresponding to Cysl44 of SEQ ID NO: l is absent, or is an amino acid residue other than Cysteine. The gL protein of embodiment 14, wherein the amino acid residue

corresponding to Cysl44 of SEQ ID NO: l is Glycine, Serine or Alanine. The gL protein of any one of embodiments 1-15, wherein said amino acid modification is at an amino acid residue adjacent to the position corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: 1, or Cysl44 of SEQ ID NO: l . The gL protein of embodiment 16, wherein the amino acid residue adjacent to the position corresponding to Cys47 of SEQ ID NO: 1 comprises a bulky side chain. The gL protein of embodiment 16 or 17, wherein the amino acid residue adjacent to the position corresponding to Cys54 of SEQ ID NO: 1 comprises a bulky side chain. The gL protein of any one of embodiments 16-17, wherein the amino acid residue adjacent to the position corresponding to Cysl44 of SEQ ID NO: l comprises a bulky side chain. An isolated trimeric complex comprising the gL protein of any one of embodiments 1-19, and further comprises CMV proteins: gH or a complex- forming fragment of gH, and gO or a complex-forming fragment of gO. An isolated pentameric complex comprising the gL protein of any one of embodiments 1-19, and further comprises CMV proteins: pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, pUL131 or a complex-forming fragment of pUL131, and gH or a complex-forming fragment of gH. A nucleic acid comprising a sequence that encodes the gL protein of any one of embodiments 1-19. The nucleic acid of embodiment 22, further comprising a sequence that encodes a CMV protein selected from the group consisting of: pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, pUL131 or a complex-forming fragment of pUL131, and gH or a complex- forming fragment of gH, and a combination thereof. The nucleic acid of embodiment 22 or 23, wherein said nucleic acid is a DNA molecule. The nucleic acid of embodiment 22 or 23, wherein said nucleic acid is an RNA molecule. The nucleic acid of embodiment 25, wherein said RNA molecule is a self- replicating RNA molecule. A host cell comprising the nucleic acid of any one of embodiments 22-26. A composition comprising (i) the gL protein of any one of embodiments 1-19, the complex of embodiment 20 or 21, or the nucleic acid of any one of claims 22-26, and (ii) a pharmaceutically acceptable carrier. The composition of embodiment 28, further comprising an adjuvant. The gL protein of any one of embodiments 1-19, the complex of embodiment 20 or 21, the nucleic acid of any one of embodiments 22-26, or the

composition of embodiment 28 or 29, for use in inducing an immune response in a subject. The use of embodiment 30, wherein the immune response comprises the production of neutralizing antibodies. The use of embodiment 31 , wherein said neutralizing antibodies are complement-independent. A method of inducing an immune response against CMV in a subject in need thereof, comprising administering to said subject an immunologically effective amount of (i) the gL protein of any one of embodiments 1-19, (ii) the complex of embodiment 20 or 21, (iii) the nucleic acid of any one of embodiments 22- 26, or (iv) the composition of embodiment 28 or 29. The method of embodiment 33, wherein the immune response comprises the production of neutralizing antibodies. The method of embodiment 34, wherein said neutralizing antibodies are complement-independent. A method of purifying CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV proteins: gH or a complex-forming fragment of gH, gL or a complex- forming fragment of gL, pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, and pUL131 or a complex- forming fragment of pUL131, comprising:

(i) providing a sample comprising (a) said pentameric complex, and (b) dimeric complexes consisting of: gH or a complex-forming fragment of gH, and gL or a complex-forming fragment of gL;

(ii) passing said sample through an ion exchange chromatography column; and

(iii) collecting the fraction that comprises said pentameric complex from the ion exchange column; wherein (i) no more than 10% of protein complexes collected from said fraction are said dimeric complexes; or (ii) at least 90% of protein complexes collected from said fraction are said pentameric complexes. The method of embodiment 36, wherein no more than 10% of protein complexes collected from said fraction are dimeric complexes. The method of embodiment 36 or 37, wherein no more than 5% of protein complexes collected from said fraction are dimeric complexes. The method of any one of embodiments 36-38, at least 90% of protein complexes collected from said fraction are pentameric complexes. The method of any one of embodiments 36-39, at least 95% of protein complexes collected from said fraction are pentameric complexes. The method of any one of embodiments 36-40, wherein said ion exchange column is a MonoS column. A method of purifying CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV proteins: gH or a complex-forming fragment of gH, gL or a complex- forming fragment of gL, pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex- forming fragment of pUL130, and pUL131 or a complex- forming fragment of pUL131, comprising:

(i) providing a sample comprising: (a) said pentameric complex, and (b) dimeric complexes consisting of gH or a complex-forming fragment of gH, and gL or a complex- forming fragment of gL, wherein an affinity- purification tag is attached to one of the following sites: C-terminal region of pUL130, N-terminal region of pUL130, C-terminal region of pUL131, N-terminal region of pUL131, C-terminal region of pUL128, N-terminal region of pUL128, or a combination thereof, and wherein said affinity-purification tag binds specifically to a binding partner; (ii) purifying said pentameric complex by an affinity chromatography matrix, wherein said affinity chromatography matrix comprises said binding partner attached to a solid support; wherein (i) no more than 10% of protein complexes obtained by affinity purification are said dimeric complexes; or (ii) at least 90% of protein complexes obtained by affinity purification are said pentameric complexes. The method of embodiment 42, wherein no more than 10% of protein complexes obtained by affinity purification are said dimeric complexes. The method of embodiment 42 or 43, wherein no more than 5% of protein complexes obtained by affinity purification are said dimeric complexes. The method of any one of embodiments 42-44, at least 90% of protein complexes obtained by affinity purification are said pentameric complexes. The method of any one of embodiments 42-45, at least 95% of protein complexes obtained by affinity purification are said pentameric complexes. The method of any one of embodiments 42-46, wherein said affinity- purification tag is attached to one of the following sites: C-terminal region of pUL130, N-terminal region of pUL130, C-terminal region of pUL131, N- terminal region of pUL131, C-terminal region of pUL128, N-terminal region of pUL128, or a combination thereof, by a covalent bond. The method of any one of embodiments 42-47, wherein said affinity- purification tag is a strep tag. The method of any one of embodiments 42-48, further comprising removing said affinity-purification tag from the purified pentameric complex. A recombinant cytomegalovirus (CMV) pUL130 protein, or a complex- forming fragment thereof, comprising an affinity-purification tag that is attached to the N-terminus or C-terminus of said pUL130 protein. A recombinant cytomegalovirus (CMV) pUL128 protein, or a complex- forming fragment thereof, comprising an affinity-purification tag that is attached to the N-terminus or C-terminus of said pUL128 protein. A recombinant cytomegalovirus (CMV) pUL131 protein, or a complex- forming fragment thereof, comprising an affinity-purification tag that is attached to the N-terminus or C-terminus of said pUL131 protein. The recombinant protein of any one of embodiments 50-52, wherein said affinity-purification tag is a Strep tag. The recombinant protein of any one of embodiments 50-53, wherein said affinity-purification tag is covalently attached to the N-terminus or C-terminus of said protein. An isolated pentameric complex comprising the pUL130 protein, or a complex- forming fragment thereof, of any one of embodiments 50 and 53-54, and further comprises CMV proteins: gH or a complex- forming fragment of gH, gL or a complex- forming fragment of gL, pUL128 or a complex- forming fragment of pUL128, and pUL131 or a complex-forming fragment of pUL131. An isolated pentameric complex comprising the pUL128 protein, or a complex- forming fragment thereof, of any one of embodiments 51 and 53-54, and further comprises CMV proteins: gH or a complex- forming fragment of gH, gL or a complex-forming fragment of gL, pUL130 or a complex-forming fragment of pUL130, and pUL131 or a complex-forming fragment of pUL131. An isolated pentameric complex comprising the pUL131 protein, or a complex- forming fragment thereof, of any one of embodiments 52-54, and further comprises CMV proteins: gH or a complex- forming fragment of gH, gL or a complex-forming fragment of gL, pUL130 or a complex- forming fragment of pUL130, and pUL128 or a complex-forming fragment of pUL128. An isolated cytomegalovirus (CMV) gL protein, or a complex-forming fragment thereof, comprising an amino acid modification, such that in the presence of substantially equal molar amounts of CMV protein gH, and said gL or a complex- forming fragment thereof:

(i) at least 90% of the gL molecules, or complex-forming fragment

thereof, form a monomeric dimer consisting of: one copy of gH, and one copy of said gL or complex- forming fragment thereof;

(ϋ) no more than 10% of the gL molecules, or complex- forming fragment thereof, form a dimeric dimer consisting of: two copies of gH, and two copies of said gL or complex- forming fragment thereof; or

(iii) said amino acid modification reduces the amount of dimeric dimer, consisting of two copies of gH, and two copies of said gL or complex- forming fragment thereof, by at least 50%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid

modification. The gL protein of embodiment 58, wherein at least 90%> of the gL molecules, or complex- forming fragment thereof, form a monomeric dimer consisting of: one copy of gH, and one copy of said gL or complex- forming fragment thereof. The gL protein of embodiment 58 or 59, wherein at least 95% of the gL molecules, or complex-forming fragment thereof, form a monomeric dimer consisting of: one copy of gH, and one copy of said gL or complex- forming fragment thereof. The gL protein of embodiment 58, wherein no more than 10% of the gL molecules, or complex-forming fragment thereof, form a dimeric dimer consisting of: two copies of gH, and two copies of said gL or complex- forming fragment thereof. The gL protein of embodiment 58 or 61, wherein no more than 5% of the gL molecules, or complex-forming fragment thereof, form a dimeric dimer consisting of: two copies of gH, and two copies of said gL or complex- forming fragment thereof. The gL protein of embodiment 58, wherein said amino acid modification reduces the amount of dimeric dimer, consisting of two copies of gH, and two copies of said gL or complex-forming fragment thereof, by at least 50%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. The gL protein of embodiment 58 or 63, wherein said amino acid modification reduces the amount of dimeric dimer, consisting of two copies of gH, and two copies of said gL or complex-forming fragment thereof, by at least 75%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification. The gL protein of any one of embodiment 58 and 63-64, wherein said amino acid modification reduces the amount of dimeric dimer, consisting of two copies of gH, and two copies of said gL or complex- forming fragment thereof, by at least 90%, relative to a gL protein, or a complex- forming fragment thereof, without said amino acid modification The gL protein of any one of embodiments 58-65, wherein said amino acid modification is at an amino acid residue corresponding to Cysl44 of SEQ ID NO: l . The gL protein of embodiment 66, wherein the amino acid residue

corresponding to Cysl44 of SEQ ID NO:l is absent, or is an amino acid residue other than Cysteine. The gL protein of embodiment 67, wherein the amino acid residue

corresponding to Cysl44 of SEQ ID NO: l is Glycine, Serine or Alanine. The gL protein of any one of embodiments 58-65, wherein said amino acid modification is at an amino acid residue adjacent to the position corresponding to Cysl44 of SEQ ID NO: l . The gL protein of any one of embodiments 69, wherein the amino acid residue adjacent to the position corresponding to Cysl44 of SEQ ID NO: 1 comprises a bulky side chain. An isolated monomelic dimer consisting of one copy of the gL protein of any one of embodiments 58-70, and one copy of gH or a complex- forming fragment of gH.

Sequences

SEQ ID NO: 1 (gL from HCMV strain Merlin = GI : 39842115)

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWL RPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

SEQ ID NO: 2 (gL from HCMV strain To ne = GI : 239909463)

MCRRPDCGFSFSPGPVALLWCCLLLPIVSSATVSVAPTVAEKVPAECPELTRRCLLGEVFQGDKYESWL RPLVNVTRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

SEQ ID NO: 3 (gL from HCMV strain AD169 = GI: 2506510)

MCRRPDCGFSFSPGPWLLWCCLLLPIVSSVAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWL RPLVNVTRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVM RGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVR LPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVD AR

SEQ ID NO: 4 (gL mature protein consisting of amino acid residues 31- 278 of SEQ ID NO: 1)

AAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSV LLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLS YGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQ LDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR

Claims

An isolated cytomegalovirus (CMV) gL protein, or a complex-forming fragment thereof, comprising an amino acid modification, such that in the presence of substantially equal molar amounts of CMV proteins gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof:

(i) at least 90% of the gL molecules, or complex-forming fragment

thereof, form a pentameric complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex- forming fragment thereof;

(ii) no more than 10% of the gL molecules, or complex- forming fragment thereof, form a dimeric complex consisting of: gH and said gL or complex- forming fragment thereof; or

(iii) said amino acid modification reduces the amount of dimeric complex, consisting of gH and said gL or complex- forming fragment thereof, by at least 50%, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification.

The gL protein of claim 1, wherein at least 95% of the gL molecules, or complex- forming fragment thereof, form a pentameric complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof.

The gL protein of claim 1 or 2, wherein no more than 5% of the gL molecules, or complex- forming fragment thereof, form a dimeric complex consisting of gH, and gL or a complex forming fragment thereof.

The gL protein of any one of claims 1-3, wherein said amino acid modification is at an amino acid residue corresponding to Cys47 of SEQ ID NO: 1, Cys54 of SEQ ID NO: l, Cys 144 of SEQ ID NO: 1, or a combination thereof.

The gL protein of claim 4, wherein the amino acid residue corresponding to Cys47 of SEQ ID NO : 1 , Cys54 of SEQ ID NO : 1 , Cys 144 of SEQ ID NO : 1 , or a combination thereof, is absent, or is an amino acid residue other than Cysteine.

6. The gL protein of any one of claims 1-3, wherein said amino acid modification is at an amino acid residue adjacent to the position corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: l, Cysl44 of SEQ ID NO: 1, or a combination thereof.

7. The gL protein of claim 6, wherein the amino acid residue adjacent to the position corresponding to Cys47 of SEQ ID NO: l, Cys54 of SEQ ID NO: l, Cysl44 of SEQ ID NO: l, or a combination thereof, comprises a bulky side chain.

8. An isolated trimeric complex comprising the gL protein of any one of claims 1-7, and further comprises CMV proteins: gH or a complex- forming fragment of gH, and gO or a complex-forming fragment of gO.

9. An isolated pentameric complex comprising the gL protein of any one of claims 1-7, and further comprises CMV proteins: pUL128 or a complex- forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, pUL131 or a complex-forming fragment of pUL131, and gH or a complex- forming fragment of gH.

10. A nucleic acid comprising a sequence that encodes the gL protein of any one of claims 1-7.

11. A host cell comprising the nucleic acid of claim 10.

12. A composition comprising (i) the gL protein of any one of claims 1-7, the complex of claim 8 or 9, or the nucleic acid of claim 10, and (ii) a

pharmaceutically acceptable carrier.

13. The gL protein of any one of claims 1-7, the complex of claim 8 or 9, the nucleic acid of claim 10, or the composition of claim 12, for use in inducing an immune response in a subject.

14. A method of purifying CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV proteins: gH or a complex-forming fragment of gH, gL or a complex- forming fragment of gL, pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, and pUL131 or a complex- forming fragment of pUL131, comprising:

(i) providing a sample comprising (a) said pentameric complex, and (b) dimeric complexes consisting of: gH or a complex-forming fragment of gH, and gL or a complex-forming fragment of gL;

(ii) passing said sample through an ion exchange chromatography column; and

(iii) collecting the fraction that comprises said pentameric complex from the ion exchange column; wherein (i) no more than 10% of protein complexes collected from said fraction are said dimeric complexes; or (ii) at least 90% of protein complexes collected from said fraction are said pentameric complexes.

A method of purifying CMV pentameric complex from a sample, wherein said pentameric complex comprises CMV proteins: gH or a complex-forming fragment of gH, gL or a complex- forming fragment of gL, pUL128 or a complex-forming fragment of pUL128, pUL130 or a complex-forming fragment of pUL130, and pUL131 or a complex- forming fragment of pUL131, comprising:

(i) providing a sample comprising: (a) said pentameric complex, and (b) dimeric complexes consisting of gH or a complex-forming fragment of gH, and gL or a complex- forming fragment of gL, wherein an affinity- purification tag is attached to one of the following sites: C-terminal region of pUL130, N-terminal region of pUL130, C-terminal region of pUL131, N-terminal region of pUL131, C-terminal region of pUL128, N-terminal region of pUL128, or a combination thereof, and wherein said affinity-purification tag binds specifically to a binding partner;

(ii) purifying said pentameric complex by an affinity chromatography

matrix, wherein said affinity chromatography matrix comprises said binding partner attached to a solid support; wherein (i) no more than 10% of protein complexes obtained by affinity purification are said dimeric complexes; or (ii) at least 90% of protein complexes obtained by affinity purification are said pentameric complexes.