BE1023213B1 - COMPLEXES FROM CYTOMEGALOVIRUS AND USES THEREOF - Google Patents

Abstract

This disclosure relates to modified cytomegalovirus (CMV) gL proteins and complexes comprising gL proteins, in particular pentameric complexes comprising gH, gL, pUL128, pUL130, pUL131. The disclosure also relates to methods for purifying pentameric complexes and for reducing contaminating dimeric complexes consisting of gH and gL. The uses of the pentameric complexes in immunogenic compositions and vaccines are further described.

Description

COMPLEXES FROM CYTOMEGALOVIRUS AND THEIR USES TECHNICAL FIELD

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

CONTEXT

Cytomegalovirus is a kind of virus that belongs to the viral family known as Herpesviridae or Herpesvirus. The species that infects humans is commonly known as human cytomegalovirus (HCMV) or human herpesvirus-5 (HHV-5). In the family Herpesviridae, HCMV belongs to the subfamily Beta-herpesviridae, which also includes cytomegaloviruses from other mammals.

Although they can be found throughout the body, HCMV infections are frequently associated with salivary glands. HCMV infects between 50 and 80% of adults in the United States (40% worldwide), as evidenced by the presence of antibodies in a large proportion of the population. HCMV infection typically goes unnoticed in healthy individuals, but can be fatal in immunocompromised individuals, such as HIV-infected individuals, organ transplant recipients, or neonates. HCMV is the virus most commonly transmitted to a developing fetus. After infection, HCMV can remain latent in the body throughout the life of the host, and out of latency during occasional reactivations. Given the severity and importance of this disease, obtaining an effective vaccine is considered an absolute public health priority (Sung, H., et al., (2010) Expert review of vaccines 9, 1303-1314; Schleiss, Expert Opin Ther Ther Apr. 2010; 20 (4): 597-602).

Genomes from more than 20 different strains of HCMV were sequenced, including laboratory strains and clinical isolates. For example, the following strains of HCMV were sequenced: Towne (GL239909366), AD169 (GI: 219879600), Toledo (GL290564358) and Merlin (GI: 155573956). The AD169, Towne and Merlin strains of HCMV can be obtained from the American Type Culture Collection (ATCC VR538, ATCC VR977 and ATCC VR1590, respectively).

CMV contains an unknown number of membrane protein complexes. Of the approximately 30 known glycoproteins in the viral envelope, gH and gL emerged particularly interestingly due to their presence in several different complexes: dimer gH / gL, trimer gH / gL / gO (also known as complex gCIII), and pentamer gH / gL / pUL128 / pUL130 / pUL131 (pUL131 is also referred to as "pUL131A", "pUL131a", or "UL131A", and the pUL128, pUL130, and pUL131 subunits are also sometimes referred to as UL128, UL130 , UL131). CMV is thought to use pentamer complexes to penetrate epithelial and endothelial cells by endocytosis and low-pH pH fusion, but is thought to enter the fibroblasts by direct fusion at the plasma membrane in a process involving gH / gL or optionally gH / gL / gO. The gH / gL and / or gH / gL / gO complexes are sufficient to infect the fibroblasts, whereas the pentamer complex is required to infect the endothelial and epithelial cells.

The pentamer complex is considered a major target for vaccination against CMV. The viral genes UL128, UL130 and UL131 are required for endothelial entry (Hahn, Journal of Virology 2004; 78: 10023-33). Non-endothelial tropics adapted to fibroblasts contain mutations in at least one of these three genes. The Towne strain, for example, contains a 2-base pair insert that shifts the reading frame in the UL130 gene, while AD169 contains a single-base insert in the UL131 gene. The Towne and AD169 strains could both be adapted for growth in endothelial cells, and in both cases the frameshift mutations in the UL130 or UL131 genes were repaired. US7704510 discloses that pUL131A is required for tropism of epithelial cells. 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 have been shown to inhibit HCMV infection of epithelial cells.

CMV vaccines tested in clinical trials include Towne vaccine, Towne-Toledo chimeras, an alphavirus replicon with gB as an antigen, the gB / MF5 9 vaccine, a gB vaccine produced by GlaxoSmithKline, and a DNA vaccine using gB and pp65, pp65 being a viral protein that is a potent inducer of CD8 + responses to CMV. These vaccines are poor antibody inducers that block viral entry into endothelial / epithelial cells (Adler, S. P. (2013),

British Medical Bulletin, 107, 57-68. doi: 10.1093 / bmb / ldt023).

Preclinical animal studies on CMV vaccines include an inactivated strain AD169 that has been repaired in the UL131 gene, a wild-type UL130 gene-based DNA vaccine, and peptide vaccines using peptides from pUL130 and 131 (Sauer, A, et al., Vaccine 2011; 29: 2705-1. Doi: 10.1016). The 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 demonstrated an efficacy of only 50% in the prevention of primary infection in young women with a child at home (Pass, RF, et al., N Engl J Med 2009; 360: 1191-9).

In general, it is believed that the neutralizing antibodies to the pentamer complex (gH / gLpUL128 / pUL130 / pUL131) will be significantly more potent than the neutralizing antibodies to the CMV gB subunit, or the gH / gL dimer complex. Therefore, a purified pentamer complex would be useful as an antigen for diagnostic applications, and as an immunogen for CMV vaccines.

One of the problems in obtaining a pentameric complex of purified CMV is the presence of contaminating gH / gL dimers. As the neutralizing antibodies to the gH / gL dimers are much less potent compared to those directed against the pentamer, there is a need in terms of developing techniques to obtain a purified CMV pentamer containing a reduced amount of contaminating gH / gL dimers.

SUMMARY OF THE INVENTION

The inventors have found that when the pentamer complex (gH / gL / pUL128 / pUL130 / pUL131) is expressed by recombinant techniques and purified, a substantial amount of contaminating gH / gL dimer is formed. Contaminant gH / gL dimers reduce the efficacy of pentamer vaccines, since it is estimated that neutralizing antibodies to pentamers are 100 times more potent than those directed against gH / gL dimers.

To solve this problem, the invention provides compositions and methods for producing a pentamer complex gH / gL / pUL128 / pUL130 / pUL131 containing a significantly reduced amount of gH / gL contaminant dimer complexes.

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 the dimer gH / gL without significantly affecting the formation of the pentamer gH / gL / pUL128 / pUL130 / pUL131. The pentameric complexes obtained with the modified gL proteins have properties that are essentially not different from the wild-type gL pentamer. In particular embodiments, the modified gL proteins carry amino acid modifications on the amino acid residues corresponding to Cys47 of SEQ ID No: 1, Cys54 of SEQ ID No: 1, or Cys144 of SEQ ID No: 1.

In another aspect, the invention provides a method of purifying the CMV pentameric complex comprising gH, gL, pUL128, pUL130 and pUL131 (or a complex fragment of each of these subunits) by ion exchange chromatography.

In another aspect, the invention provides a method of purifying the CMV pentameric complex comprising gH, gL, pUL128, pUL130 and pUL131 (or a complex fragment of each of these subunits) by affinity chromatography. The invention also provides CMV gL proteins and CMV protein complexes that can be used to induce an immune response in a subject. The invention further provides CMV gL proteins and CMV protein complexes that can be used in the manufacture of a medicament for inducing an immune response in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1D show that mutations on Cys47 and Cys54 efficiently removed the gH / gL dimers of the pentamer complex. FIG. IA shows that a substantial amount of gH / gL dimer contaminants (monomeric forms and dimeric forms) had formed, as shown in the non-reduced Coomassie gel. FIG. IB shows that gH / gL dimer contaminants (especially dimeric forms) could not be removed by the size exclusion column, since they were co-eluted with the pentamer. FIG. IC shows the results of mutagenesis studies. Cysteines at position 47, 54, 144, 154, 159, and 233 in gL were mutated to Ser, respectively. Among these mutations, the mutants Cys47, Cys54, and Cys144 reduced the contamination with the dimer gH / gL, thus allowing the purification of the pentamer. When co-expressed with gH and pUL128-131 subunits, the proteins formed a stable pentamer complex. FIG. 1D is a SEC chromatographic graph showing that the mutant gL incorporating the pentamer complex (C54S) can bind to neutralizing antibodies such as the wild-type pentamer complex.

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

DETAILED DESCRIPTION

1. GENERAL

As described and illustrated herein, one of the problems in obtaining a purified CMV pentamer complex is the presence of contaminating gH / gL dimers. The inventors have discovered that, during expression and purification of the pentamer complex, a significant amount of gH / gL contaminants are formed. In a typical purification method, the contaminating gH / gL dimers accounted for about 10-20% of the total amount of the purified complexes. In a particular experimental setup, the amount of dimer gH / gL was up to 40-50%. The dimers gH / gL exist both in the form of "monomer" dimers (dimers constituted by a gH subunit and a gL subunit), or "dimeric" dimers (two "monomer" dimers are associated with to one another, to form a complex consisting of two copies of the gH subunit and two copies of the gL subunit).

The presence of contaminating gH / gL dimers is undesirable. The inventors overcame this problem by three different strategies. First, the inventors have identified three mutations in the gL subunit, on Cys47, Cys54, or Cys144, that may interfere with the formation of the dimer gH / gL. Surprisingly, the mutant gL proteins practically do not interfere with the formation of the pentamer gH / gL / pUL128 / pUL130 / pUL131. Indeed, the pentamer complexes obtained with the mutant gL proteins have properties that are not substantially different from those of the pentamers formed by wild-type gL. Using this method, large amounts of pentamer complex can be purified at a substantially undetectable gH / gL contamination.

Second, the inventors used a cation exchange method to purify the pentamer complex, and successfully removed the contaminant gH / gL dimers to an undetectable level.

Third, by attaching a Strep tag to the C-terminal region of the pUL130 subunit, and using affinity purification, the inventors obtained a substantially homogeneous pentamer in a single step. Again, gH / gL contamination was virtually undetectable after purification.

Accordingly, in one aspect, the invention relates to a modified CMV-derived protein that promotes the formation of the pentamer gH / gL / pUL128 / pUL130 / pUL131, and / or disadvantages the formation of the gH / gL dimer. In some embodiments, in the presence of substantially equal molar amounts of the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits), at least 90% of the molecules of the gL protein, or complex fragment thereof, form a pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits). In some embodiments, in the presence of substantially equal molar amounts of the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits), no more than 10% of the molecules of the gL protein, or complex-forming fragment thereof, form a dimer complex consisting of gH and gL (or a complex fragment of each of these subunits). In some embodiments, the modification in gL reduces the amount of dimer complexes consisting of gH and gL (or a complex fragment of each of these subunits) by at least 50%, relative to a gL protein, or to a complex-forming fragment thereof without said amino acid modification.

In another aspect, the invention relates to a method for purifying the pentameric CMV complex from a sample by ion exchange chromatography. The method comprises: (i) using a sample comprising the following mixture: (a) pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits), and (b) a dimer complex consisting of gH and gL (or a complex fragment of each of these subunits); (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction that comprises said pentamer complex from the ion exchange column. The methods described herein may yield a purified product wherein: (i) not more than 10% of the affinity purification protein complexes are dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits) ); or (ii) at least 90% of the affinity purification protein complexes are pentamer complexes comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits).

In another aspect, the invention relates to a method of purifying the CMV pentameric complex which comprises the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits) comprising: (i) using a sample comprising the following mixture: (a) pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits), and (b) a dimer complex consisting of gH and gL (or a complex fragment of each of these 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, said affinity purification tag binding specifically to a portion thereof liaison officer; (ii) purifying said pentamer complex with an affinity chromatography matrix, said affinity chromatography matrix comprising said binding partner immobilized on a solid support. The methods described herein may provide a purified product wherein: (i) not more than 10% of the protein complexes obtained by affinity purification are dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits). units); or (ii) at least 90% of the protein complexes obtained by affinity purification are pentamer complexes comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits) .

The three strategies (Ig modification, ion exchange purification, and affinity) can be used individually, or in any combination, to obtain the purified pentamer complex. The invention also relates to purified complexes comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits), and methods of using the purified pentamer complexes. 2. Modified CMV Proteins and Complexes A. Modified G1 Proteins

In one aspect, the invention relates to a modified CMV gL protein, or complex fragment thereof, which reduces the formation of the contaminating gH / gL dimer.

The gL protein described herein, or a complex-forming fragment thereof, carries an amino acid modification such that in the presence of substantially equal molar amounts of CMV proteins gH, gL, pUL128, pUL130, and pUL131 (or of a complex-forming fragment of each of these subunits): (i) at least about 80% (preferably about 90%) of the gL molecules, or the complex-forming moiety thereof, form a pentamer complex comprising gH , gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits); (ii) not more than about 20% (preferably about 10%) of the gL molecules, or complex moiety thereof, form a dimer complex consisting of gH and gL (or a complex moiety of each of subunits); or (iii) said amino acid modification reduces the amount of dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits) by at least about 30% (preferably 50%), by relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification.

The glycoprotein L (gL) of human CMV is encoded by the UL115 gene. It is believed that gL is essential for viral replication and that all known functional properties of gL are directly associated with its dimerization with gH. The gH / gL complex is required for fusion of the viral and plasma membranes allowing entry of the virus into the host cell. GL from the HCMV Merlin strain (GI: 39842115, SEQ ID No: 1) and the HCMV Towne strain (GI: 239909463, SEQ ID No: 2) has been reported to have a

278 amino acids long. GL from HCMV strain AD169 (GI: 2506510, SEQ ID NO: 3) has been reported to be 278 amino acids in length, includes a signal sequence at its N-terminus (amino acid residues 1). -35), has two N-glycosylation sites (at residues 74 and 114) and lacks the TM domain (Rigoutsos, I, et al., Journal of Virology 77 (2003): 4326-44). The signal sequence at the N-terminus of SEQ ID No: 1 should include amino acid residues 1-30. SEQ ID NO: 2 shares a 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 AD169, Towne and Toledo revealed less than 2% variation in amino acid sequences among isolates (Rasmussen, L, et al., Journal of Virology 76 (2002): 10841-10888).

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

Although it is believed that gL is essential for viral replication, all known functional properties of gL are directly associated with its dimerization with gH.

The gL proteins according to the invention can be variants of gL having various degrees of identity with SEQ ID No: 1 such as for example an identity of at least 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the sequence indicated in SEQ ID No: 1, SEQ ID No: 2, or SEQ ID No: 3. The proteins gL according to the invention can have various degrees of identity. with SEQ ID No: 4 such as for example an identity of at least 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the sequence indicated in SEQ ID No: 4. In some embodiments, variants of the gL protein: (i) do not form significant amounts of dimeric complexes with gH; (ii) are part of the trimer complex gH / gL / gO; (iii) are part of the pentamer complex gH / gL / pUL128 / pUL130 / pUL131; (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 initiate the production of antibodies in vivo that have cross-immunological reactions with a CMV virion.

The complex-forming fragments of the gL proteins described herein are also part of the invention. A gL complex fragment may be any portion or portion of the gL protein that retains the ability to complex with other CMV proteins. In some embodiments, a gL complex fragment is part of the pentamer complex gH / gL / pUL128 / pUL130 / pUL131. A gL complex fragment can be obtained or determined by standard assays known in the state of the art, such as coimmunoprecipitation assay, cross-linking, or fluorescent colocalization, etc. In some embodiments, the gL (i) complex fragment also comprises at least one epitope derived from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and / or (ii) can also trigger the production of antibodies in vivo that have cross-immunological reactions with a CMV virion.

In some embodiments, the gL protein described herein, or a complex-forming fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of gH, gL, pUL128, pUL130 proteins, and CMV pUL131 (or a complex fragment of each of these subunits), at least about 80% of the gL molecules, or complex fragment thereof, form a pentamer complex comprising gH, gL, pUL128 , pUL130, and pUL131 (or a complex fragment of each of these 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 the complex moiety thereof, form a pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits).

In some embodiments, the gL protein described herein, or a complex-forming fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of gH, gL, pUL128, pUL130 proteins, and CMV pUL131 (or a complex-forming fragment of each of these subunits), no more than about 20% of the gL molecules, or the complex-forming moiety thereof, form a dimer complex consisting of gH and gL (or a complex fragment of each of these subunits). For example, the change may result in not more than about 20%, not more than about 15%, not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, or not more than about 1% of the gL molecules, or the complex moiety thereof, form a dimer complex consisting of gH and gL (or a complex fragment of each of these subunits).

In some embodiments, the gL protein described herein, or a complex-forming fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of gH, gL, pUL128, pUL130 proteins, and CMV pUL131 (or a complex fragment of each of these subunits), said amino acid modification reduces the amount of dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits). units) of at least about 50%, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification (such as wild-type gL or a complex fragment of that -this) . For example, the amino acid modification may reduce the amount of dimeric complexes consisting of gH and gL (or a complex fragment of each of these 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 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%, to a gL protein, or a complex-forming fragment thereof, without said amino acid modification (such as wild-type gL or a complex-forming fragment thereof). Alternatively, the amino acid modification can reduce the amount of the dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits) by at least 2-fold, at least 3-fold, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, 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, in relation to a gL protein, or a complex-forming fragment thereof, without said amino acid modification (such as wild-type gL or a complex-forming fragment thereof).

In some embodiments, the amino acid modification is performed on an amino acid residue corresponding to Cys47 of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID No: 1, or a combination of these. Standard alignment methods can be used to identify residues that correspond to Cys47 of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID No: 1, or a combination thereof. The modification may be an addition, a deletion, or a substitution of an amino acid residue. The modification may also be the introduction of an unnatural amino acid or an amino acid analogue into a polypeptide chain.

The mutations on the Cys47 residue of SEQ ID No: 1 or Cys54 of SEQ ID No: 1 were found to yield a modified gL protein that does not form with gH nor a "monomer" dimer (a copy of gH and a copy of gL), nor dimer "dimer" (two copies of gH and two copies of gL). Alternatively, the "monomer" or "dimer" gH / gL dimers are present at a level that was not detectable when the inventors used standard detection methods.

Interestingly, the modification to the amino acid residue corresponding to Cys144 of SEQ ID NO: 1 also allows effective purification of the pentamer since this modification has substantially eliminated the "dimer" form of the dimer gH / gL. It is possible that a residual amount of the "monomer" form of the dimer gH / gL still remains with modifications to a residue corresponding to Cys144 of SEQ ID NO: 1. However, the "monomeric" form of the dimer gL / gH can to be separated from the pentamer by a size exclusion column, while the "dimer" form is co-eluted with the pentamer (see, Fig. 1B). In fact, even if the gL protein having a modification on the residue corresponding to Cys144 still forms a "monomer" dimer with gH, the monomeric dimer peak gH / gL is sufficiently separated from the pentamer peak (compare FIG. that the pentamer and the "monomer" dimer are not co-eluted, and that the pentamer can be purified by an exclusion column of standard sizes.

Preferably, the amino acid modification alters the ability of a cysteine residue to form a disulfide bridge, such as, for example, a deletion modification of the cysteine residue, a cysteine to another amino acid mutation (such as glycine, serine, threonine). , alanine, valine, leucine, isoleucine, etc.).

In some embodiments, the amino acid change occurs on the amino acid residue adjacent to the Cys47 position of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID NO: 1, or a combination thereof. The amino acid residue may be either an immediately proximal residue (i.e., residues corresponding to positions 46, 48, 53, 55, 143, or 145 in SEQ ID NO: 1), or a conformational proximity residue (e.g. eg, within 30 angstroms, or 25 angstroms, or 20 angstroms, or 15 angstroms, or 10 angstroms, or 7 angstroms, or 5 angstroms, relative to one of the atoms of the residue corresponding to Cys47 of SEQ ID No: 1, to Cys54 of SEQ ID No: 1, to Cys144 of SEQ ID No: 1). In some embodiments, the amino acid residue adjacent to the position corresponding to Cys47, Cys54, or Cys144 of SEQ ID No: 1 comprises a cumbersome side chain. A bulky side chain can, by serum congestion, prevent the formation of a disulfide bridge. The invention also relates to a modified gL (e.g., Cys144 modification) which reduces the formation of dimeric gH / gL dimers (two copies of gH and two copies of gL). In some embodiments, the gL protein described herein, or a complex fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of the CMV gH protein, and said or a complex-forming fragment thereof, at least about 80% of the gL molecules, or the complex-forming moiety thereof, form a "monomer" dimer consisting of: a copy of gH, and a copy of said gL or complex 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 the complex moiety thereof, form a "monomer" dimer consisting of: a copy of gH, and a copy of said gL or complex fragment thereof.

In some embodiments, the gL protein described herein, or a complex fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of the CMV gH protein, and said or a complex-forming fragment thereof, no more than about 20% of the gL molecules, or a complex-forming moiety thereof, form a "dimer" dimer consisting of: two copies of gH, and two copies of said gL or complex fragment thereof. For example, the change may result in not more than about 20%, not more than about 15%, not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, or not more than about 1% of the gL molecules, or the complex moiety thereof, form a "dimer" dimer consisting of: two copies of gH, and two copies of said gL or complex fragment thereof.

In some embodiments, the gL protein described herein, or a complex fragment thereof, carries an amino acid modification such that, in the presence of substantially equal molar amounts of the CMV gH protein, and said or a complex-forming moiety thereof, said amino acid modification reduces the amount of "dimer" dimers, consisting of two copies of gH, and two copies of said gL or complex fragment thereof. at least about 50%, based on a gL protein, or a complex-forming fragment thereof, without said amino acid modification. For example, the amino acid modification may reduce the amount of "dimer" dimers consisting of: two copies of gH, and two copies of said gL (or a complex fragment of each of these subunits) of 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 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 fragment of it). Alternatively, the amino acid modification can reduce the amount of dimeric complexes consisting of: two copies of gH, and two copies of said gL (or a complex fragment of each of these subunits) of at least 2 times , at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times, with respect 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

In another aspect, the invention relates to a complex comprising the CMV-modified gL protein, or a complex-forming fragment thereof, as described herein. These complexes include, e.g. eg, an isolated trimer complex comprising the modified CMV gL protein, or a complex fragment thereof, as described herein, and CMV gH proteins or a complex fragment thereof, and gO or a fragment thereof forming complex thereof; an isolated pentamer complex comprising the modified gL protein, or a complex fragment thereof, as described herein, and CMV pUL128 proteins 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 encompasses any other complex comprising gL (or a complex fragment thereof) as a component.

Although gH, gL, gO, gB and pUL130 may be considered as glycoproteins, this nomenclature should not be interpreted to mean that these proteins must be glycosylated when used in the context of the present invention. In contrast, in some embodiments of the invention, one or more of the polypeptides are not glycosylated. Generally, however, one or more (or all) polypeptides in a complex according to the invention are glycosylated. In some embodiments, one or more (or all) polypeptides in a complex according to the invention are glycosylated by cultured cell glycosylation mutants, such as mutated mammalian cells. These glycosylation mutants generate a polypeptide glycosylation profile which differs from the wild-type glycosylation pattern, i.e., the resulting polypeptide glycoforms differ from wild-type glycoforms.

In some embodiments, the glycosylation profile of the gL (or a complex fragment thereof), or a complex comprising gL (or a complex fragment thereof) has a profile of glycosylation of mammals; and / or has no glycosylation pattern of insect cells. In some embodiments, one or more of the complex proteins contains N-linked side chains complex with penultimate galactose and terminal sialic acid. C. Nucleic acid encoding modified gL proteins and complexes

In another aspect, the invention relates to a nucleic acid comprising a sequence that encodes the modified gL protein, or a complex fragment thereof, as described herein. The nucleic acid may be DNA or RNA.

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

The modified gL protein expressed by recombinant techniques or a fragment thereof, or a complex comprising the modified gL protein or a fragment thereof can be purified using the methods described herein, such as purification methods described in WO 2014/005959, or other methods known in the state of the art.

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

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

Self-replicating RNA molecules are well known in the state of the art and can be produced using derived replication elements, e.g. alphaviruses, which substitute the structural viral proteins with a nucleotide sequence coding for a protein of interest. A self-replicating RNA molecule is typically a (+) stranded molecule that can be directly translated after introduction into a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense transcripts. and sense from the introduced RNA. Therefore, the introduced RNA generates the production of multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can themselves be translated to provide in situ expression of an encoded antigen, or they can be transcribed to provide additional transcripts in the same sense as RNA. introduced that are translated to provide in situ expression of the antigen. The overall result of this transcription sequence is a gigantic amplification of the number of introduced replicon RNAs, so that the coded antigen becomes a major polypeptide product of the cells. Cells transfected with self-replicating RNA briefly produce the antigen before undergoing apoptotic death. This death is a likely result of the required double-stranded RNA (db) intermediates, which have been shown to over-activate dendritic cells. Therefore, the enhanced immunogenicity of self-replicating RNA may be a result of pro-inflammatory dsRNA production, which mimic an infection of the host cells by an RNA virus.

A suitable system for obtaining such self-replication is to use a replicon derived from an alphavirus. Alphaviruses are a group of arthropod-borne, generically, structurally, and serologically related viruses of the family Togaviridae. Twenty-six known viruses and virus subtypes have been classified as alphaviruses, including Sindbis virus, Semliki forest virus, Ross river virus, and Venezuelan equine encephalitis virus. As such, the self-replicating RNA according to the invention can incorporate a replicon RNA 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.

Alphavirus-derived "replicon" expression vectors can be used in the invention. The replicon vectors can be used in a variety of formats, including recombinant DNA, RNA, and replicon particles. These replicon vectors have been derived from alphaviruses which 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), the 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). In general, alphavirus derived replicons have very similar overall characteristics (eg, structure, replication), but individual alphaviruses may exhibit a particular property (eg, receptor binding, interferon sensitivity, and pathological profile) that is unique. Therefore, chimeric alphavirus replicons made from divergent virus families may also be useful. The use of an alphavirus-derived RNA replicon is known from the state of the art, see, p. eg, WO 2013006837, paragraphs [0155] to [0179]. The RNA replicon can be administered without the need to purify the protein for which it codes.

In some 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 about 36,000 base pairs, a 55 kDa terminal protein being covalently linked to the 5'-terminus of each strand. Adenoviral DNA ("Ad") contains identical Inverse Terminating Repeats ("ITRs") of about 100 base pairs, the exact length of which depends on the serotype. The origins of viral replication lie within the ITRs exactly at the ends of the genome. The adenoviral vectors which can be used in the present invention can be derived from any of the various adenoviral serotypes, including, inter alia, any of the more than 40 serotypes of the adenovirus, such as serotypes 2, 5, 12, 40, and 41.

In some embodiments, the nucleic acid molecule is part of a vector derived from an adeno-associated virus (AAV). The genome of AAV is a linear single-stranded DNA molecule containing about 4681 nucleotides. In general, the genome of AAV comprises a genome without internal repetitions flanked at each end by inverted terminal repeats (ITR). The ITRs are approximately 145 base pairs (bp) in length. ITRs perform multiple functions, including serving as origins of DNA replication and as encapsulation signals for the viral genome. AAV is a virus dependent on an auxiliary virus; that is, it requires co-infection with a helper virus (eg, adenovirus, herpesvirus, or vaccinia virus) to form AAV virions in the wild-type. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into the chromosome of a host cell, but no infectious virion is produced. Subsequent infection with a helper virus comes to the rescue of the integrated genome, allowing it to replicate and encapsulate in infectious AAV virions. Although AAV can infect cells from different species, the helper virus must belong to the same species as the host cell. For example, human AAV will replicate in canine cells co-infected with canine adenovirus.

In some embodiments, the nucleic acid molecule is part of a retrovirus derived vector. A selected gene may be inserted into a vector and encapsulated in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and administered to the subject's cells either in vivo or ex vivo. A number of retroviral systems have been described. See, p. eg, U.S. Patent 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. Broom. Develop. 3: 102-09. The invention also relates to host cells comprising the nucleic acid molecules described herein. Suitable host cells for harboring nucleic acid molecules and / or for expressing recombinant proteins, and methods for introducing a nucleic acid into a suitable host, are known in the state of the art. 3. Purification of CMV pentamer complexes by ion exchange

In another aspect, the invention relates to a method for purifying the pentameric CMV complex from a sample, said pentamer complex comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins, and the method comprising: i) using a sample comprising (a) the pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits), and (b) the dimer complex consisting of by gH and gL (or a complex fragment of each of these subunits); (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction that comprises said pentamer complex from the ion exchange column.

As described and illustrated herein, it has been surprisingly found that ion exchange chromatography effectively removes the contaminant dimers gH / gL from the pentamer complex. In some embodiments, no more than about 20% of the protein complexes collected in the fraction collected are dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits). For example, not more than about 20%, not more than about 15%, not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7% , not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, or not more than Protein complexes collected in the fraction collected are dimeric complexes consisting of gH and gL (or a complex fragment of each of these subunits).

In some embodiments, at least about 80% of the protein complexes collected in the fraction collected are pentamer complexes comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins (or a complex fragment of each of these subunits). units). 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 the protein complexes collected in the fraction collected are pentamer complexes comprising gH, gL, pUL128, pUL130, and pUL131 (or a each of these subunits). As examples of materials useful in ion exchange chromatography, there are DEAE-cellulose, QAE-cellulose, DEAE-cephalose, QAE-cephalose, DEAE-Toyopearl, QAE-Toyopearl, Mono Q, Mono S, Q Sepharose, SP Sepharose, etc. In an exemplary embodiment, the method uses a Mono S column. In another example embodiment, the method uses a Mono Q. column. 4. Purification of CMV Protein Complexes by Labels

AFFINITY

In another aspect, the invention relates to a method for purifying the pentameric CMV complex from a sample, said pentamer complex comprising the CMV gH, gL, pUL128, pUL130, and pUL131 proteins, and the method comprising: i) using a sample comprising: (a) the pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131 (or a complex fragment of each of these subunits), and (b) the dimer complex consisting of gH and gL (or a complex fragment of each of these 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, said affinity purification tag binding specifically to a e liaison; and (ii) purifying said pentamer complex with an affinity chromatography matrix, said affinity chromatography matrix comprising said binding partner immobilized on a solid support.

The structure of the pentamer complex gH / gL / pUL128 / pUL130 / pUL131 is unknown; therefore, if the affinity purification tag is attached to a site that interferes with the formation of the pentamer complex, or at a site that is buried within the complex, the affinity purification will not succeed. As described and illustrated herein, it is believed that the following sites are suitable for attaching an affinity purification tag, since the tag does not appear to interfere with pentamer complex formation, and appears to be exposed on the surface of the pentameric complex. pentamer assembled: (i) C-terminal region of pUL130, (il) N-terminal region of pUL130, (iii) C-terminal region of pUL131, (iv) N-terminal region of pUL131, (v) C-terminal region pUL128, (vi) N-terminal region of pUL128, or a combination thereof.

Examples of affinity purification tags include, e.g. eg His tag (binds to a 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), the FLAG label (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (binds to an anti-FLAG antibody), Strep label (binds to streptavidin or a derivative thereof).

An illustrative embodiment is the Strep tag (or streptavidin affinity tag), a tag that binds to streptavidin or a derivative thereof, such as Strep-Tactin. The 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 II Label): Trp-Ser-His-Pro Gln-Phe-Glu-Lys. Elution of a protein attached to a Strep tag from the column may be accomplished using biotin or a derivative or homologue thereof, such as desthio-biotin. The affinity purification tag can be attached by any suitable means, directly or indirectly. For example, the tag may be attached to the N-terminus of the polypeptide sequence, or to the C-terminus of the polypeptide sequence. The operation can be performed by the recombinant expression of a fusion protein comprising the polypeptide and the tag, or by standard conjugation techniques that bind 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.

Alternatively, the tag may be noncovalently attached, 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 by non-covalence. The pentamer-antibody complex can then be purified on a protein-A or protein-G, protein-A or protein-G column being the binding partner for the antibody label.

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

In one embodiment, the tag is attached to the C-terminal residue of pUL131 (or a complex fragment thereof). In one embodiment, the tag is attached to a residue that is within 20 amino acids, 15 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, amino acids, 4 amino acids, 3 amino acids, or 2 amino acids, relative to the C-terminal residue of pUL131 (or complex-forming fragment thereof). The attachment of the label can be direct or indirect (via a linker). Suitable linkers are known to those skilled in the art and include, e.g. for example, straight or branched chain carbon linkers, heterocyclic carbon linkers, carbohydrate linkers and polypeptide linkers.

In one embodiment, cleavable linkers can be used to attach the molecule of interest to the tag. This allows the label to be separated from the purified complex, for example by adding an agent capable of cleaving the linker. A number of different cleavable linkers are known to those skilled in the art. These linkers can be cleaved, for example, by exposure of a photolabile bond to light or by acid catalyzed hydrolysis. There are also polypeptide linkers which contain a protease recognition site and which can be cleaved by the addition of a suitable protease enzyme. 5. Pharmaceutical Compositions and Administration The invention also relates to pharmaceutical compositions comprising the CMV proteins, complexes, and nucleic acids described herein. The invention also provides pharmaceutical compositions comprising nucleic acids encoding the CMV proteins, complexes, and nucleic acids described herein.

The CMV proteins, complexes, and nucleic acids described herein may be incorporated into an immunogenic composition, or a vaccine composition. These compositions can be used to elicit antibody production in a mammal (eg, human subject). The invention provides pharmaceutical compositions comprising the CMV proteins, complexes, and nucleic acids described herein, and methods for preparing a pharmaceutical composition involving the combination of the CMV proteins, complexes, and nucleic acids disclosed herein. with a pharmaceutically acceptable vehicle. The pharmaceutical compositions according to the invention typically comprise a pharmaceutically acceptable carrier, and a complete review of these vehicles is available in

Remington: The Science and Practice of Pharmacy.

The pH of the composition is generally from about 4.5 to about 9.0, such as from about 5 to about 9, from about 5.5 to about 9, from about 6 to about 9, from about 5 to about 8. , 5, from about 5.5 to about 8.5, from about 6 to about 8.5, from about 5 to about 8.5, from about 5.5 to about 8, from about 6 to about 8, of about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8 , 5, about 9, etc. A stable pH can be maintained using a p buffer. ex. Tris buffer, citrate buffer, phosphate buffer, or histidine buffer. Therefore, a composition will generally contain a buffer.

A composition may be sterile and / or pyrogen-free. The compositions can be isotonic with respect to humans.

A composition comprises an immunologically effective amount of its antigen (s). An "immunologically effective amount" is an amount that, when administered to a subject, effectively elicits an antibody response against the antigen. This quantity may vary according to the state of health and the physical condition of the individual to be treated, his age, the capacity of his immune system to synthesize antibodies, the degree of protection sought, the formulation of the vaccine, in the opinion of the attending physician on the medical case, and other relevant factors. This quantity should be in a relatively wide range that can be determined by routine testing. The antigen content of the compositions according to the invention will generally be expressed in terms of protein weight per dose. A dose of 10 to 500 μg (eg, 50 μg) of antigen may be useful.

The immunogenic compositions may contain an immunological adjuvant. By way of examples, the adjuvants may comprise: 1. compositions containing a mineral; 2. oily emulsions; 3. saponin formulations; 4. virosomes and viroid particles; 5. bacterial or microbial derivatives; 6. bioadhesives and mucoadhesives; 7. liposomes; 8. formulations based on polyoxyethylene ethers and polyoxyethylene esters; 9. polyphosphazenes (pcpp); 10. muramyl peptides; 11. imidazoquinolone compounds; 12. thiosemicarbazone compounds; 13. tryptanthrin compounds; 14. human immunomodulators; 15. lipopeptides; 16. benzonaphthyridines; 17. microparticles; 18. immunostimulatory polynucleotides (such as RNA or DNA, eg, oligonucleotides containing cpg motifs).

For example, the composition may comprise an aluminum salt or oil-in-water emulsion aid (eg, an oil-in-water emulsion comprising a squalene, such as MF59 or ASO3). Suitable aluminum salts include hydroxides (eg, oxyhydroxides), phosphates (eg, hydroxyphosphates, orthophosphates) (see, for example, Chapters 8 & 9 of Vaccine Design ... (1995) Eds Powell & Newman, ISBN: 030644867X, Plenum), or their mixtures. The salts may take any suitable form (eg, gel, crystalline forms, amorphous, etc.), with the adsorption of the antigen on the salt being an example. The concentration of Al +++ in a composition to be administered to a patient may be less than 5 mg / ml. ex. <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is between 0.3 and 1 mg / ml. A maximum of 0.85 mg / dose is preferred. Adjuvants of the aluminum hydroxide and aluminum phosphate type are suitable in the context of the present invention.

A suitable immunological adjuvant comprises a compound of Formula (I) as defined in WO2011 / 027222, or a pharmaceutically acceptable salt thereof, adsorbed on an aluminum salt. Many other adjuvants can be used, including any of those described in Powell &amp; Newman (1995).

The compositions may comprise an antimicrobial, especially when packaged in multidose format. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly used in vaccines, but sometimes it may be desirable to use either a mercury-free preservative or no preservative at all.

The compositions may comprise a detergent, e.g. ex. polysorbate, such as polysorbate 80. Detergents are generally present at low levels, e.g. ex. <0.01%.

The compositions may include sodium salts (eg, sodium chloride) to enhance tone. A concentration of 10 + 2 mg / ml NaCl is typical, e.g. ex. about 9 mg / ml.

The compositions according to the invention will generally be administered directly to the subject. Direct administration may be by parenteral injection (eg subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial space of tissue), or by any other suitable route. For example, intramuscular administration may be practiced, e.g. ex. in the thigh or upper arm. Injection can be via a needle (eg, hypodermic needle), but alternatively needle-free injection can be used. A typical volume for an intramuscular dose is 0.5 ml.

The dose may be administered in a single dose regimen or a multidose regimen. Multiple doses may be used according to a primo-immunization and / or booster regimen. In a multidose schedule, the various doses can be administered by the same or different routes, e.g. eg, parenteral primo-immunization and mucosal boosting, mucosal primo-immunization and parenteral boosting, etc. Multidoses will typically be administered at an interval of at least 1 week (eg, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, approximately 16 weeks). weeks, etc.).

The subject may be a human subject, and may also be, p. eg, a cow, pork, chicken, cat or dog, as pathogens covered by this may be problematic in a wide range of species. When the vaccine is for prophylaxis, the human subject is preferably a child (eg, toddler or infant), adolescent, or adult; when the vaccine is for therapeutic purposes, the human subject is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults, eg eg, to evaluate safety, dosage, immunogenicity, etc.

Vaccines according to the invention may be prophylactic (ie to prevent disease) or therapeutic (ie to reduce or eliminate symptoms of a disease).

The nucleic acid molecules described herein can be formulated and administered in the form of a nucleic acid vaccine according to the standards known in the state of the art.

Isolated and / or purified CMV proteins, complexes, and nucleic acids described herein may be administered alone, or as a first-in-immunization or booster in mixed modality strategies, such as RNA primo-immunization. followed by a protein booster. The advantages of the primo-immunization / RNA / protein booster strategy, as compared to the protein / protein immunization / boosting strategy, include, for example, increased antibody titers, a more balanced profile of IgG1: IgG2a subtypes, and induction of a TH1 type CD4 + T cell-mediated immune response similar to that obtained with virus particles, and reduced production of non-neutralizing antibodies. RNA priming immunization can increase the immunogenicity of the compositions regardless of the presence or absence of an adjuvant.

In the primo-immunization / RNA / protein booster strategy, the RNA and the protein are directed against the same target antigen. Examples of suitable modes of administration of RNA are viroid replicon particles (VRP), alphavirus RNA, lipid nanoparticle-encapsulated replicons (LNP), or formulated RNAs, such as that replicons formulated with cationic nanoemulsions (CNEs). Suitable cationic water-in-water nanoemulsions, including p. ex. a lipid core (e.g., comprising squalene) and a cationic lipid (e.g., DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.) are described in WO2012 / 006380. WO2012 / 051211 discloses that antibodies directed against the pentamer complex are produced in mice that have been immunized with VRPs and formulated RNAs (CNE and LNP) that encode the protein components of the pentamer complex. These antibodies have been shown to be able to neutralize CMV infection in epithelial cells. The primary / immunization / RNA / protein recall strategy may initially involve (eg between 0-8 weeks) one or more primary immunizations with RNA (which may be administered as VRP, LNP, CNE, etc. which encodes one or more of the protein components of the CMV protein complex according to the invention, and then one or more subsequent booster immunizations (e.g., between 24-58 weeks) with: a CMV isolated protein complex according to the invention; invention, optionally formulated with an adjuvant or a purified CMV protein complex according to the invention, optionally formulated with an adjuvant.

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

Kits for administering nucleic acid (eg, RNA), purified proteins, and purified complexes described herein, and instructions for use are also provided. The invention also provides a pre-filled delivery device with a composition or vaccine described herein.

The pharmaceutical compositions described herein may be administered together with one or more other therapeutic agents. These other therapeutic agents may include, inter alia, antibiotics or antibacterials, antiemetics, antifungals, anti-inflammatories, antivirals, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites , antitumor antibiotics, antimitotics, topoisomerase inhibitors, cytostatic agents, antiinvasive agents, antiangiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anti-cancer agents, interferons a, beta interferons, ribavirin, hormones and other Toll-like receptor modulators, immunoglobulins (Ig), and Ig-modulating antibodies (such as anti-IgE (omalizumab)).

In some embodiments, the compositions described herein may be used as a medicament, e.g. eg, for use in inducing or enhancing an immune response in a subject in need, such as a mammal.

In some embodiments, the compositions described herein may be used in the manufacture of a medicament for inducing or enhancing an immune response in a subject in need, such as a mammal.

One way of verifying the effectiveness of the therapeutic treatment involves monitoring for infection with a pathogen after administration of the compositions or vaccines described herein. One way of verifying the effectiveness of prophylactic treatment involves the surveillance of immune responses, systemically (eg, monitoring the level of production of IgG1 and IgG2a) and / or mucosally (eg, monitoring of the level of production). IgA), directed against the antigen. Typically, antigen-specific serum antibody responses are determined after immunization but before challenge, whereas mucosal antibody responses are determined after immunization and after challenge. . 6. Definitions

The term "complex fragment" of a CMV protein (such as gL) refers to any portion or portion of the protein that retains the ability to complex with other CMV proteins. These complexes include, e.g. eg, dimer complex gH / gL, trimer complex gH / gL / gO, or pentamer complex gH / gL / pUL128 / pUL130 / pUL131.

As used herein, "dimer complex gH / gL" or "dimer gH / gL" refers to a protein complex formed by gH and gL. The subunits gH and gL of CMV can form either a "monomer" dimer (dimer consisting of a gH subunit and a gL subunit) or a "dimer" dimer (two "monomer" dimers that associate with each other). to one another to give a complex consisting of two copies of the gH subunit and two copies of the gL subunit). Both forms of dimers are generally referred to as "dimer complex gH / gL", or "dimer gH / gL". Although generally referred to as "dimer gH / gL" in the description, the gH and gL subunits do not need to be of the full length type; the term also encompasses "monomer" dimers and "dimeric" dimers formed by gH and gL complex fragments.

As used herein, "pentamer complex" or "pentamer" refers to a CMV complex that comprises five different subunits: gH, gL, pUL128, pUL130, and pUL131. Although generally referred to as "pentamer gH / gL / pUL128 / pUL130 / pUL131" (or pentamer complex comprising gH, gL, pUL128, pUL130, and pUL131) in the description, each of these five subunits does not require be of the full-length type; the term also encompasses pentamers formed by complex fragments of gH, gL, pUL128, pUL130, and PÜL131.

The term "substantially equal" here refers to any numerical value having a variance of +/- 20% with respect to the basic numerical value.

The term "about" as used herein means +/- 5% of a base value.

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

An amino acid residue of a query sequence "corresponds to" a position indicated in 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 corresponds to the indicated position. These alignments can be performed manually or using well-known sequence alignment programs such as ClustalW2, or "BLAST 2 Sequences" using the default settings.

An amino acid residue includes a "bulky side chain" when the side chain comprises a branched or cyclic substituent. As examples of amino acid residues having a bulky side chain, there is tryptophan, tyrosine, phenylalanine, homophenylalanine, leucine, isoleucine, histidine, 1-methyl tryptophan, α-methyltyrosine, α-methylphenylalanine, α-methyl-leucine, α-methyl-isoleucine, α-methylhistidine, cyclopentylalanine, cyclohexylalanine, naphthylalanine, etc .;

EXAMPLES

The inventors have found that when a pentamer complex (gH / gL / pUL128 / pUL130 / pUL131) is expressed by recombinant techniques and purified, a considerable amount of contaminating gH / gL dimer is formed. In a typical experiment, the regular presence of contaminating gH / gL dimers, representing about 10 to 20% of the total amount, was observed. The CMV gH / gL subunits can form either a "monomer" complex (dimer of gH and gL) or a "dimer" complex (dimer of heterodimers). Both complexes exist with the pentamer complex and must be removed.

The inventors have identified several strategies for limiting or eliminating the excess gH / gL dimers described herein during pentamer production for vaccine development. 2. Example 1: modified gL

The inventors have identified 3 point mutants on the gL subunit that interfere with the formation of gH / gL complexes (for both "monomer" and "dimeric" forms of gH / gL dimers). These mutants do not substantially interfere with pentamer formation and can be used to purify large amounts of pentamers. The purified pentamer complex does not include gH / gL contamination (at least not detectable by standard methods).

The sequence of wild-type gL used for the production of pentamers is as follows (signal peptide in bold, Cys47, Cys54, and Cys144 underlined):

MCKRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNV

TGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA

VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNWVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL

YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR

The gL mutants used to avoid gH / gL contamination are as follows (signal peptide in bold, Cys47-Ser, Cys54-Ser, and Cysl44-Ser underlined): gL Cys47-Ser

MCKRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAESPELTRRCLLGEVFEGDKYESWLRPLVNV

TGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA

VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNWVAIRNEATRTNRAVRLPVSTAÄAPEGITLFYGL

YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR gL Cys54-Ser

MCKRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRSLLGEVFEGDKYESWLRPLVNV

TGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA

VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL

YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR gL Cysl44-Ser

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNV

TGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSESGDGSPA

VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNWVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL

YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR

293EBNA cells were transfected with plasmids encoding individual subunits bearing point mutations in gL. Proteins were purified from mammalian cell supernatants by Strep-Tactin resin affinity chromatography

Superflow Plus (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 the elution buffer. Strep-labeled proteins were overexpressed and purified by a similar strategy and eluted in 25 mM Hepes pH 7.5 buffer, 150 mM NaCl. The HCMV complexes were incubated for 2 hours on ice and purified by SEC.

EM grids were prepared by depositing a thin layer of continuous carbon on a carbon-hole layer on a 400 mesh copper grid (Electron Microscopy Sciences). Five microliters of purified sample (approximately 30 ng) were placed on a grid having recently been subjected to a glow discharge. After 30 seconds of incubation, the grid was deposited on droplets of 75 μl of a freshly prepared solution of 2% (w / v) uranyl formate and gently stirred during the five subsequent 10 s staining steps. . The samples were imaged using a Tecnai Spirit T12 transmission electron microscope operating at 120 keV at a nominal magnification of x49,000 (1.57 À / pixel at the detector) using a defocus range of - 0.8 to -1.2 pm. The images were recorded in low dose conditions by a Gatan CCD camera 4096 χ 4096 pixels. For the TCT reconstruction data set, the images were collected at -56 ° and 0 °. Particle removal was performed in semi-automatic mode using an e2boxer (Tang, G., Peng, L., Baldwin, PR, Mann, DS, Jiang, W., Rees, I., and Ludtke, SJ (2007) EMAN2: An Extensible Image Processing Suite for Electron Microscopy Journal of Structural Biology 157, 38-46

A particle window of 224 χ 224 pixels was used for all datasets. All datasets have

were filtered by a bandpass filter at either a 200A high-pass cutoff or a 20A low-pass cutoff. Iterative multivariate statistical analysis (MSA) and multi-reference alignment (MRA) under Imagic (van Heel, M., Harauz, G., Orlova, EV, Schmidt, R., and Schatz, M. (1996) A new generation of the IMAGIC image processing system, Journal of Structural Biology 116, 17-24) extracted particles generated 2D views representative of the HCMV complexes. On average, about 20 particles were included per class.

The pentamer complex obtained using the procedure described had properties substantially identical to the wild type. It was able to bind to neutralizing Fabs, was analyzed by SDS-PAGE and SEC at similar apparent molecular weight, and could not be distinguished by electron microscopy. See, FIG. 1A-1D.

Significantly, these mutants also elicited an immune response similar to that of the wild-type in Balb C mice. See, FIG. 2. 3. Example 2: Affinity Purification

In this example, we show that by attaching a Strep tag to the C-terminal region of pUL130, and then proceeding with affinity purification, gH / gL contamination has been substantially eliminated.

293EBNA cells were transfected with plasmids encoding individual subunits. The proteins were purified from mammalian cell supernatants by Strep-Tactin Superflow Plus resin affinity chromatography (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 the elution buffer.

The purification can be carried out in a single step and the pentamer complex obtained was substantially homogeneous, and substantially free of gH / gL dimers (data not shown). 4. Example 3: Exchange of ions

In this Example, we show that, by ion exchange chromotography, gH / gL contamination was substantially eliminated.

More specifically, an ion exchange column has been used. The pentameric complex containing the gH / gL contamination was initially dialyzed in 20mM MES buffer pH 6.0, 50 mM NaCl. This sample was then loaded onto a MonoS PC 1.6 / 5 column. The pentamer complex was retained on the column while the excess of gH / gL was present in the unbound fraction. The pentamer complex was purified without contamination gH / gL from the MonoS column with a 20 mM MES pH 6.0 buffer, 250 mM NaCl (data not shown).

The description will be better understood in light of the teachings of the references cited herein. Embodiments in the description illustrate embodiments according to the invention and should not be interpreted as limiting its scope. Those skilled in the art will readily understand that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. In the case where the literature incorporated by reference contradicts or is inconsistent with the present description, the present description will prevail. The citation of references herein is not an admission that these references would represent the prior art with respect to the present invention. Those skilled in the art will understand, recognize, or be able to establish, by routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be encompassed by the claimed embodiments.

Particular embodiments of the invention include: 1. A gL protein isolated from cytomegalovirus (CMV1, or a complex-forming fragment thereof, carrying an amino acid modification such that, in the presence of substantially equal molar amounts of gH, pUL128, pUL130, pUL131, and said CMV gL or complex moiety thereof: (i) at least 90% of the gL molecules, or complex fragment thereof, form a complex pentamer comprising gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof (ii) not more than 10% of the gL molecules, or complex-forming moiety thereof, form a dimer complex consisting of by gH and said gL or a complex-forming fragment thereof, or (iii) said amino acid modification reduces the amount of dimeric complexes, consisting of gH, and said gL or a complex-forming fragment thereof, of at least 50%, compared to a pro gL, or a complex-forming moiety thereof, without said amino acid modification. 2. The gL protein according to embodiment 1, wherein at least 90% of the gL molecules, or the complex-forming moiety thereof, form a pentamer complex comprising gH, p12112, pUL130, pUL131, and said gL or a complex-forming fragment thereof. 3. The gL protein according to embodiment 1 or 2, wherein at least 95% of the gL molecules, or complex fragment thereof, form a pentamer complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex moiety thereof. 4. The gL protein according to embodiment 1, wherein not more than 10% of the gL molecules, or the complex-forming fragment thereof, form a dimer complex consisting of gH and gL or a complex-forming fragment thereof. this. 5. The gL protein according to embodiment 1 or 4, wherein not more than 5% of the gL molecules, or the complex-forming moiety thereof, form a dimer complex consisting of gH, and gL or a complex fragment of these. 6. The gL protein according to embodiment 1, wherein said amino acid modification reduces the amount of dimeric complexes, consisting of gH, and gL or a complex-forming moiety thereof, by at least 50%, with respect to a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 7. The gL protein according to embodiment 1 or 6, wherein said amino acid modification reduces the amount of dimeric complexes, consisting of gH, and gL or a complex moiety thereof, of at least 75. %, with respect to a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 8. The gL protein according to any one of embodiments 1 and 6-7, wherein said amino acid modification reduces the amount of dimeric complexes consisting of gH, and gL or a complex moiety thereof, at least 90%, based on a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 9. The gL protein according to any one of embodiments 1-8, wherein said amino acid modification is performed on an amino acid residue corresponding to Cys47 of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID No: 1. 10. The protein gL according to embodiment 9, wherein the amino acid residue corresponding to Cys47 of SEQ ID No: 1 is absent, or is a residue of amino acid other than Cysteine. 11. The gL protein according to embodiment 10, wherein the amino acid residue corresponding to Cys47 of SEQ ID NO: 1 is Glycine, Serine or Alanine. 12. The gL protein according to any one of embodiments 9-11, wherein the amino acid residue corresponding to Cys54 of SEQ ID NO: 1 is absent, or is an amino acid residue other than Cysteine. 13. The gL protein according to embodiment 12, wherein the amino acid residue corresponding to Cys54 of SEQ ID NO: 1 is Glycine, Serine or Alanine. 14. The gL protein according to any one of embodiments 9-13, wherein the amino acid residue corresponding to Cys144 of SEQ ID NO: 1 is absent, or is an amino acid residue other than Cysteine. 15. The gL protein according to embodiment 14, wherein the amino acid residue corresponding to Cys144 of SEQ ID NO: 1 is Glycine, Serine or Alanine. The gL protein according to any one of embodiments 1-15, wherein said amino acid modification takes place on an amino acid residue adjacent to the Cys47 position of SEQ ID No: 1, Cys54 of SEQ ID No: 1, or Cys144 of SEQ ID No: 1. 17. The protein gL according to embodiment 16, wherein the amino acid residue adjacent to the Cys47 position of SEQ ID No: 1 comprises a cumbersome side chain. 18. The gL protein according to embodiment 16 or 17, wherein the amino acid residue adjacent to the Cys54 position of SEQ ID No: 1 comprises a bulky side chain. 19. The gL protein according to embodiment 16-17, wherein the amino acid residue adjacent to the Cys144 position of SEQ ID NO: 1 comprises a cumbersome side chain. 20. An isolated trimer complex comprising the gL protein according to any one of embodiments 1-19, and which further comprises the following CMV proteins: gH or a complex fragment of gH, and gO or a complex fragment of gO. 21. An isolated pentamer complex comprising the gL protein according to any one of embodiments 1-19, and which further comprises the following CMV proteins: pUL128 or a pUL128 complex fragment, pUL130 or a complex fragment of pUL130, pUL131 or a complex fragment of pUL131, and gH or a gH complex fragment. 22. A nucleic acid comprising a sequence that encodes the gL protein according to any one of Embodiments 1-19. 23. The nucleic acid according to embodiment 22, further comprising a sequence that encodes a CMV protein selected from the group consisting of: pUL128 or a pUL128 complex fragment, pUL130 or a pUL130 complex fragment, pUL131 or a complex fragment of pUL131, and gH or a complex fragment of gH, and a combination thereof. 24. The nucleic acid according to embodiment 22 or 23, wherein said nucleic acid is a DNA molecule. 25. The nucleic acid according to embodiment 22 or 23, wherein said nucleic acid is an RNA molecule. 26. The nucleic acid according to embodiment 25, wherein said RNA molecule is a self-replicating RNA molecule. 27. A host cell comprising the nucleic acid according to any one of embodiments 22-26. 28. A composition comprising (i) the gL protein according to any one of Embodiments 1-19, the complex according to Embodiment 20 or 21, or the nucleic acid according to any one of embodiments 22 -26, and (ii) a pharmaceutically acceptable carrier. 29. The composition of embodiment 28, further comprising an adjuvant. 30. The gL protein according to any one of embodiments 1-19, the complex according to embodiment 20 or 21, the nucleic acid according to any one of embodiments 22-26, or the composition according to Embodiment 28 or 29, for use in inducing an immune response in a subject. 31. The use according to embodiment 30, wherein the immune response comprises the production of neutralizing antibodies. 32. The use according to embodiment 31, wherein said neutralizing antibodies are complement-independent. 33. A method of inducing an immune response against CMV in a subject in need, comprising administering to said subject an immunologically effective amount of (i) gL protein according to any of the embodiments 1-19, (ii) the complex according to embodiment 20 or 21, (iii) the nucleic acid according to any one of embodiments 22-26, or (iv) the composition according to the method of Embodiment 28 or 29. 34. The method according to embodiment 33, wherein the immune response comprises the production of neutralizing antibodies. 35. The method according to embodiment 34, wherein said neutralizing antibodies are complement-independent. 36. A method of purifying the pentameric CMV complex from a sample, wherein said pentamer complex comprises the following CMV proteins: gH or a gH complex fragment, gL or a complex fragment of gL, pUL128 or a complex fragment of pUL128, pUL130 or a complex fragment of pUL130, and pUL131 or a complex fragment of pUL131, comprising: (i) using a sample comprising (a) said pentamer complex, and (b) dimeric complexes consisting of: gH or a complex fragment of gH, and gL or a complex fragment of gL; (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction which comprises said pentamer complex from the ion exchange column; wherein (i) not more than 10% of the protein complexes collected from said fraction are said dimeric complexes; or (ii) at least 90% of the protein complexes collected from said fraction are said pentamer complexes. 37. The method according to embodiment 36, wherein not more than 10% of the protein complexes collected from said fraction are dimeric complexes. 38. The method according to embodiment 36 or 37, wherein not more than 5% of the protein complexes collected from said fraction are dimeric complexes. 39. The method according to any one of embodiments 36-38, wherein at least 90% of the protein complexes collected from said fraction are pentamer complexes. 40. The method according to any one of embodiments 36-39, wherein at least 95% of the protein complexes collected from said fraction are pentamer complexes. 41. The method according to any one of embodiments 36-40, wherein said ion exchange column is a Mono-S column. 42. A method of purifying the CMV pentameric complex from a sample, wherein said pentamer complex comprises the following CMV proteins: gH or a complex fragment of gH, gL or a complex fragment of gL, pUL128 or a complex fragment of pUL128, pUL130 or a complex fragment of pUL130, and pUL131 or a complex fragment of pUL131, comprising: (i) using a sample comprising: (a) said pentamer complex, and (b) ) dimeric complexes consisting of gH or a complex fragment of gH, and gL or a complex fragment of gL, in which 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, said affinity purification tag it binds specifically to a link partner; (ii) purifying said pentamer complex with an affinity chromatography matrix, said affinity chromatography matrix comprising said binding partner immobilized on a solid support; wherein (i) not more than 10% of the protein complexes obtained by affinity purification are said dimer complexes; or (ii) at least 90% of the protein complexes obtained by affinity purification are said pentamer complexes. 43. The method according to embodiment 42, wherein not more than 10% of the protein complexes obtained by affinity purification are said dimer complexes. 44. The method according to embodiment 42 or 43, wherein not more than 5% of the protein complexes obtained by affinity purification are said dimer complexes. 45. The method of any one of embodiments 42-44, wherein at least 90% of the protein complexes obtained by affinity purification are said pentamer complexes. 46. The method of any one of embodiments 42-45, wherein at least 95% of the protein complexes obtained by affinity purification are said pentamer complexes. 47. The method according to 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, region C-terminus 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. 48. The method of any one of embodiments 42-47, wherein said affinity purification tag is a Strep tag. 49. The method of any one of embodiments 42-48, further comprising removing said affinity purification tag of the purified pentamer complex. 50. 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 protein pUL130. 51. A recombinant cytomegalovirus (CMV) pUL128 protein, or a complex fragment thereof, comprising an affinity purification tag that is attached to the N-terminus or C-terminus of said protein pUL128. 52. 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 protein pUL131. 53. The recombinant protein according to any one of embodiments 50-52, wherein said affinity purification tag is a Strep tag. 54. The recombinant protein according to any one of embodiments 50-53, wherein said affinity purification tag is covalently attached to the N-terminus or C-terminus of said protein. 55. An isolated pentamer complex comprising the pUL130 protein, or a complex fragment thereof, according to any of embodiments 50 and 53-54, and further comprising the CMV: gH proteins or a fragment forming gH complex, gL or a complex fragment of gL, pUL128 or a complex fragment of pUL128, and pUL131 or a complex fragment of pUL131. 56. An isolated pentamer complex comprising the pUL128 protein, or a complex fragment thereof, according to any of embodiments 51 and 53-54, and further comprising the CMV: gH proteins or a fragment forming gH complex, gL or a complex fragment of gL, pUL130 or a complex fragment of pUL130, and pUL131 or a complex fragment of pUL131. 57. An isolated pentamer complex comprising the pUL131 protein, or a complex fragment thereof, according to any one of embodiments 52-54, and further comprising the CMV: gH proteins or a complex fragment thereof. gH, gL or a complex fragment of gL, pUL130 or a complex fragment of pUL130, and pUL128 or a complex fragment of pUL128. 58. A gL protein isolated from cytomegalovirus (CMV), or a complex-forming fragment thereof, carrying an amino acid modification such that in the presence of substantially equal molar amounts of gH protein, and said CMV gL. or a complex-forming fragment thereof: (i) at least 90% of the gL molecules, or the complex-forming moiety thereof, form a "monomer" dimer consisting of: a copy of gH, and a copy said gL or complex moiety thereof; (ii) not more than 10% of the gL molecules, or the complex fragment thereof, form a "dimer" dimer consisting of: two copies of gH, and two copies of said gL or complex fragment thereof; this ; or (iii) said amino acid modification reduces the amount of "dimer" dimers, consisting of two copies of gH, and two copies of said gL or complex moiety thereof, of at least 50%, by relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 59. The gL protein according to embodiment 58, wherein at least 90% of the gL molecules, or the complex-forming moiety thereof, form a "monomer" dimer consisting of: a copy of gH, and a copy of said gL or complex fragment thereof. 60. The gL protein according to embodiment 58 or 59, wherein at least 95% of the gL molecules, or the complex-forming moiety thereof, form a "monomer" dimer consisting of: a copy of gH, and a copy of said gL or complex fragment thereof. 61. The gL protein according to embodiment 58, wherein not more than 10% of the gL molecules, or the complex-forming moiety thereof, form a "dimer" dimer consisting of: two copies of gH, and two copies said gL or complex fragment thereof. 62. The gL protein according to embodiment 58 or 61, wherein not more than 5% of the gL molecules, or the complex-forming moiety thereof, form a "dimer" dimer consisting of: two copies of gH, and two copies of said gL or complex fragment thereof. 63. The gL protein according to embodiment 58, wherein said amino acid modification reduces the amount of "dimer" dimers, consisting of two copies of gH, and two copies of said gL or complex fragment thereof. ci, of at least 50%, relative to a gL protein, or to a complex forming moiety thereof, without said amino acid modification. 64. The gL protein according to embodiment 58 or 63, wherein said amino acid modification reduces the amount of "dimer" dimers, consisting of two copies of gH, and two copies of said gL or these, by at least 75%, with respect to a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 65. The gL protein according to any of embodiments 58 and 63-64, wherein said amino acid modification reduces the amount of "dimer" dimers, consisting of two copies of gH, and two copies of said gL. or the complex-forming fragment thereof, at least 90%, relative to a gL protein, or a complex-forming fragment thereof, without said amino acid modification. 66. The gL protein according to any one of embodiments 58-65, wherein said amino acid modification is performed on an amino acid residue corresponding to Cys144 of SEQ ID NO: 1. 67. The gL protein according to embodiment 66, wherein the amino acid residue corresponding to Cys144 of SEQ ID NO: 1 is absent, or is an amino acid residue other than Cysteine. 68. The gL protein according to embodiment 67, wherein the amino acid residue corresponding to Cys144 of SEQ ID NO: 1 is Glycine, Serine or Alanine. 69. The gL protein according to any one of embodiments 58-65, wherein said amino acid modification is performed on an amino acid residue adjacent to the Cys144 position of SEQ ID NO: 1. 70. The gL protein according to embodiment 69, wherein said amino acid residue adjacent to the Cys144 position of SEQ ID No: 1 comprises a cumbersome side chain. 71. An isolated "monomeric" dimer consisting of a copy of the gL protein according to any of embodiments 58-70, and a copy of gH or a gH complex fragment. Sequences SEQ ID NO: 1 (gL resulting from the Merlin strain of HCMV = GI: 39842115)

MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNV TGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR SEQ ID NO: 2 (gL end of the Towne strain of HCMV = GI: 239909463)

MCRRPDCGFSFSPGPVALLWCCLLLPIVSSATVSVAPTVAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNV TRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVWAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL YNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR SEQ ID NO: 3 (gL end of the AD169 strain of HCMV = GI: 2506510)

MCRRPDCGFSFSPGPWLLWCCLLLPIVSSVAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNV

TRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPA

VYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNWVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGL

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

AAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAF

LDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLG

FELVPPSLFNVWAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPP

ELKQTRVNLPAHSRYGPQAVDAR

Claims (15)

1. An isolated cytomegalovirus (CMV) g protein, or complex-forming fragment thereof, carrying an amino acid modification such that, in the presence of substantially equal molar amounts of gH, pUL128, pUL130, pUL131, and said CMV gil or complex-forming fragment thereof: (i) at least 90% of the gL molecules, or complex-forming fragment thereof, form a pentamer complex comprising gH, pUL128, pUL130, pUL131, and said gL or a complex-forming fragment thereof; (ii) not more than 10% of the gL molecules, or the complex moiety thereof, form a dimer complex consisting of: gH and said gL or a complex moiety thereof; or (iii) said amino acid modification reduces the amount of dimeric complexes, consisting of: gH and said gL or a complex-forming fragment thereof, of at least 50%, relative to a gL protein, or a complex-forming moiety thereof without said amino acid modification. 2. The gL protein according to claim 1, wherein at least 95% of the gL molecules, or complex fragment thereof, form a pentamer complex comprising gH, pUL128, pUL130, pUL131, and said gL or complex fragment of these. 3. The gL protein according to claim 1 or 2, wherein not more than 5% of the gL molecules, or the complex-forming moiety thereof, form a dimer complex consisting of gH, and gL or a complex-forming fragment thereof. this. 4. The gL protein according to any one of claims 1 to 3, wherein said amino acid modification is performed on an amino acid residue corresponding to Cys47 in SEQ ID No: 1, Cys54 in SEQ ID No: 1 , Cysl44 in SEQ ID No: 1, or a combination thereof. 5. The gL protein according to claim 4, wherein the amino acid residue corresponding to Cys47 of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID NO: 1, or a combination thereof , is absent, or is an amino acid residue other than Cysteine. 6. The gL protein according to any one of claims 1 to 3, wherein said amino acid modification is carried out on an amino acid residue adjacent to the Cys47 position of SEQ ID No: 1, Cys54 of SEQ ID No: 1, Cysl44 of SEQ ID No: 1, or a combination thereof. The gL protein of claim 6, wherein the amino acid residue adjacent to the Cys47 position of SEQ ID NO: 1, Cys54 of SEQ ID No: 1, Cys144 of SEQ ID NO: 1, or a combination thereof. of these, includes a bulky side chain. An isolated trimer complex comprising the gL protein of any one of claims 1 to 7, and which further comprises the following CMV proteins: gH or a gH complex fragment, and gO or a gO complex fragment. An isolated pentamer complex comprising the gL protein according to any one of claims 1 to 7, and which further comprises the CMV proteins: pUL128 or a pUL128 complex fragment, pUL130 or a pUL131 or pUL131 complex fragment. a complex fragment of pUL131, and gH or a complex fragment of gH. Nucleic acid comprising a sequence which encodes the gL protein according to any one of claims 1 to 7. 11. Host cell comprising the nucleic acid of claim 10. A composition comprising (i) the gL protein of any one of claims 1 to 7, the complex of claim 8 or 9, or the nucleic acid of claim 10, and (ii) a pharmaceutically acceptable carrier. The protein according to any of claims 1 to 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 the pentameric CMV complex from a sample, wherein said pentamer complex comprises the following CMV proteins: gH or a complex fragment of gH, gL or a complex fragment of gL, pULl28 or a pUL128 complex fragment, pUL130 or a pUL130 complex fragment, and pUL131 or a pUL131 complex fragment, comprising: (i) using a sample comprising (a) said pentamer complex, and (b) dimer complexes consisting of: gH or a complex fragment of gH, and gL or a complex fragment of gL; (ii) passing said sample through an ion exchange chromatography column; and (iii) collecting the fraction which comprises said pentamer complex from the ion exchange column; wherein (i) not more than 10% of the protein complexes collected from said fraction are said dimeric complexes; or (ii) at least 90% of the protein complexes collected from said fraction are said pentamer complexes. 15. A method of purifying the pentameric CMV complex from a sample, wherein said pentamer complex comprises the following CMV proteins: gH or a gH complex fragment, gL or a complex fragment of gL, pUL128 or a pUL128 complex fragment, pUL130 or a pUL130 complex fragment, and pUL131 or a pUL131 complex fragment, comprising: (i) using a sample comprising: (a) said pentamer complex, and (b) dimeric complexes consisting of: gH or a complex fragment of gH, and gL or a complex 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 pUL128, an N-terminal region of pUL128, or a combination thereof, said affinity purification tag specifically binding to a binding partner; (ii) purifying said pentamer complex with an affinity chromatography matrix, said affinity chromatography matrix comprising said binding partner immobilized on a solid support; wherein (i) not more than 10% of the protein complexes obtained by affinity purification are said dimer complexes; or (ii) at least 90% of the protein complexes obtained by affinity purification are said pentamer complexes.