Cytomegalovirus antigens

专利摘要:
The present invention relates generally to recombinant human cytomegalovirus (CMV) gB proteins and immunogenic fragments thereof, which do not include a transmembrane domain (TM); and include at least one mutation which reduces aggregation between the gB monomeric trimers and / or the adhesion of the gB monomeric trimer to the host cell.
公开号:BE1023390B1
申请号:E2015/5793
申请日:2015-12-07
公开日:2017-03-01
发明作者:Andrea Carfi；Sumana Chandramouli；Ethan Settembre
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

ANTIGENS OF CYTOMEGALOVIRUS FIELD OF THE INVENTION
The present invention relates to cytomegalovirus (CMV) proteins suitable for vaccine uses.
BACKGROUND OF THE INVENTION
Human cytomegalovirus (HCMV) is responsible for persistent and widespread human infections that vary with age and immunocompetence of the host. It can remain latent throughout the life of the host with sporadic reactivation events. Primary infection of hosts with a functioning immune system is associated with moderate symptoms, but may progress with fever, hepatitis, splenomegaly, and mononucleosis. On the other hand, when primary infection or reactivation occurs in immunocompromised or immunodeficient hosts, these often have life-threatening diseases, including pneumonia, hepatitis, retinitis and encephalitis (Sinclair and Sissons, J. Gen. Virol 87: 1763-1779, 2006). HCMV infection has been recognized for its association with three different populations: neonates with immature immune systems; transplant recipients, whose immune systems are weakened by the use of drugs, and HIV-infected patients whose immune systems are deficient due to the decline of CD4 + T cells.
HCMV can be particularly devastating in newborns, leading to abnormalities in neurodevelopment. In industrialized countries, intrauterine viral infection is the most common. It is estimated that between 0.6% and 0.7% (depending on the seroprevalence of the population examined) neonates are infected in utero (Dollard et al., Rev. Med., Virol., 17 (5) 355-363, 2007). In the United States alone, this is approximately 40,000 new infections each year. About 1.4% of CMV intrauterine infections are the result of transmission by women with established infection. New maternal infections occur in 0.7 to 4.1% of pregnancies and are transmitted to the fetus in about 32% of cases. About 90% of infected infants are asymptomatic at birth, and most will develop severe consequences of infection over time, including mental retardation and hearing loss. Other infected children have symptomatic HCMV disease, with symptoms of irreversible central nervous system involvement such as microencephaly, encephalitis, epileptic seizures, deafness, motor neuron disorders psychomotor retardation (Kenneson et al., Rev. Med Virol., 17 (4): 253-276, 2007). In summary, approximately 8,000 children in the United States develop a neurological disease each year related to the virus. Congenital infection is the driving force behind the development of an HCVV vaccine.
The CMV envelope glycoproteins, gB, gH, gL, gM and gN, represent attractive vaccine candidates since they are expressed on the viral surface and can elicit humoral protective immune responses, neutralizing the virus.
Some vaccination strategies against CMV have targeted the main surface glycoprotein, glycoprotein B (gB), which can induce a dominant antibody response (Go and Pollard, J Infect Dis., 2008; 197: 1631-1633). Glycoprotein B (gB) is a trimer protein that is highly conserved among the different strains of HCMV, as well as among other Herpes viruses, such as Herpes Simplex Virus (HSV) and Epstein Barr Virus (EBV). It belongs to the class III viral fusion proteins and plays a critical role in the viral replication cycle by allowing the fusion of the viral membrane with that of the target cell, facilitating the delivery of the viral genome into the cytoplasm. Clinical trials are underway to evaluate the efficacy of the subunit as well as candidate VLP vaccines incorporating different forms of HCMV gB. The CMV glycoprotein, gB, can induce a neutralizing antibody response, and the serum of CMV-positive patients is composed of antibodies against gB (Britt, Journal of Virology 64: 1079-1085, 1990).
WO / 2012/049317 discloses the CMV gB polypeptide comprising a fusion loop domain 1 (FL1) and a fusion loop domain 2 (FL2), wherein at least one of the domains FL1 and FL2 comprises at least one deletion or substitution of amino acids. Examples demonstrate that the percentage of gB trimers was about 70%.
An effective vaccine that targets the CMV glycoprotein gB and immunization methods that produce better immune responses are needed.
SUMMARY OF THE INVENTION
As described and illustrated herein, certain mutations can be introduced into the cytomegalovirus (CMV) gB protein (or an immunogenic fragment thereof), particularly to facilitate recombinant production and purification of the protein. recombinant gB for vaccine uses. The inventors have recognized that an ectodomain of the wild-type gB protein that naturally forms a monomeric trimer has an exposed hydrophobic surface that can make expression by recombination of the protein and subsequent secretion by the host cell very difficult. Particularly useful mutations are those which (i) at least partially mask said hydrophobic surface, (ii) reduce the overall hydrophobicity of said hydrophobic surface, or (iii) both.
Accordingly, in one aspect, the invention relates to a cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise transmembrane domain (TM); and (ii) said gB protein, or an immunogenic fragment thereof, comprises a mutation that results in a glycosylation site within the hydrophobic surface 1 (amino acid residues 154-160 and 236-243). Preferably, said glycosylation site is an N-glycosylation site comprising an N-X-S / T / C motif, wherein X is any amino acid residue (but preferably not proline).
In another aspect, the invention relates to a recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation that results in a glycosylation site, wherein said glycosylation site is (1) within the hydrophobic surface 2 (acidic residues amines 145-167 and 230-252); or (2) at a residue level. which is at most 20 angstroms from the fusion loop 1 (FL1) (amino acid residues 155-157) and / or the fusion loop 2 (FL2) (amino acid residues 240-242).
In another aspect, the invention relates to a recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation in the hydrophobic surface 1 (amino acid residues 154-160 and 236-243), wherein said mutation results in a reduction of the index overall hydrophobicity of said hydrophobic surface 1; wherein said mutation is not a deletion or substitution of an amino acid in the fusion loop 1 (FL1) or in the fusion loop 2 (FL2).
In another aspect, the invention relates to a cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a domain transmembrane (TM); (ii) said gB protein, or said immunogenic fragment thereof, comprises an ectodomain; and (iii) said gB protein, or said immunogenic fragment thereof, comprises a heterologous sequence of at least 12 residues at the C-terminus. In some aspects, the gB protein may be a fusion protein in which the heterologous sequence is fused at the C-terminus of the ectodomain. In some aspects, the heterologous sequence may be an amphipathic peptide.
The present invention also relates to immunogenic compositions comprising CMV gB proteins and immunogenic fragments thereof, as described herein. The immunogenic compositions may comprise an immunological adjuvant, and / or other CMV antigen.
The present invention also relates to nucleic acids encoding CMV gB and immunogenic fragments thereof, as described herein. The nucleic acid can be used in the form of a nucleic acid-based vaccine (e.g., a self-replicative RNA molecule encoding gB or an immunogenic fragment thereof). The nucleic acid can also be used for the recombinant production of the gB protein. The invention also relates to a host cell comprising the nucleic acids described herein. The nucleic acids can express a gB protein (or an immunogenic fragment thereof), and preferably form a monomeric trimer. Preferably, the monomeric trimer can be secreted by the host cell. Preferred host cells are mammalian host cells, such as CHO cells or HEK-293 cells. The invention also relates to a cell culture comprising the host cell described herein. Preferably, the culture is at least 20 liters in size, and / or the yield of gB protein (or an immunogenic fragment thereof) is at least 0.1 g / L. The invention also relates to a method of inducing an immune response against cytomegalovirus (CMV), comprising administering to a subject requiring it an immunologically effective amount of the gB protein (or an immunogenic fragment thereof). as described in this document. The invention also relates to a method of inhibiting the entry of cytomegalovirus (CMV) into a cell, comprising contacting the cell with the gB protein (or an immunogenic fragment thereof) described herein. .
The present invention also relates to the use of the compositions described herein to induce an immune response against cytomegalovirus (CMV), and the use of the compositions described herein in the manufacture of a medicament for inducing an immune response. immune response against cytomegalovirus (CMV).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of the CMV gB protein of the Merlin strain. TM: transmembrane domain; Cyto: cytoplasmic domain; SP: signal peptide; MPR: proximal membrane region; I: Domain I; II: Domain II; III: Domain III; IV: Domain IV; V: Domain V. The numbers indicated refer to the position of the amino acid residues of the CMV gB of the Merlin strain and presented in SEQ ID NO: 1.
Figure 2 shows the result of steric exclusion chromatography. The figure shows that gB-698, with mutational loop mutations but without glycosylation, formed dimeric trimers, whereas the mutant gB-698glyc did not form dimeric trimers even at a high concentration of protein.
Figure 3 shows the crystal structure of ANgB.
Figure 4 shows the Western blot of cell culture supernatant using anti-His antibody. Routes: 1: wild type (WT); 2: R236N; 3: G237N; 4: T158N / Y160T; 5: Y160E; 6: R236E / S238E; 7: R236E / S238E / T239E; 8: NGT inserted before W240; 9: I156H / H157R / W240N / Y242T. The left pane shows samples that have been reduced with 50 mM DTT and boiled at 95 ° C for 5 minutes. The right panel shows samples under boiling and non-reducing conditions. All constructs except the ectodomain of gB and R236E / S238E had detectable expression under boiling and reducing conditions (left panel).
DETAILED DESCRIPTION OF THE INVENTION
1. GENERAL
As described and illustrated herein, the inventors have discovered that certain mutations can be introduced into the cytomegalovirus (CMV) gB protein (or an immunogenic fragment thereof, eg, the ectodomain) to facilitate the recombinant production of this protein.
In general, the CMV gB protein forms a monomeric trimer (comprising three gB proteins, which are also referred to as subunits) that can be used as an antigen for CMV. However, the monomer trimer includes an exposed hydrophobic surface, which can cause considerable problems both in the production and purification of the antigens. For example, the hydrophobic surface may result in aggregation of the recombinantly produced protein gB (for example, two monomer trimers may form a dimer trimer via the hydrophobic surface, which may result in production problems). The hydrophobic surface may also cause adhesion of the gB monomeric trimer to the host cell (e.g., cell membrane, ER membrane, other hydrophobic proteins, aggregated proteins, etc.). The inventors have discovered that modification of this hydrophobic surface could greatly facilitate the production and subsequent purification of the gB antigen.
As described herein, the inventors have solved the crystal structure of CMV gB, in a monomeric trimer form, complexed to an anti-gB (Fab) antibody. Based on the crystal structure, the inventors have identified several categories of mutations that can reduce aggregation between monomer trimers, and / or monomer trimer adhesion to the host cell (e.g., cell membrane, the ER membrane, other hydrophobic proteins, aggregated proteins, etc.). In particular, the mutations also allow, in general, the secretion of the monomeric trimer by a host cell, thus significantly improving the efficiency of the production processes.
Based on the crystal structure, the inventors have discovered that several categories of mutations can be introduced.
First, a glycosylation site can be introduced into a narrower hydrophobic surface, referred to as "hydrophobic surface 1" (which includes amino acid residues 154-160 and 236-243). Without wishing to be bound by theory, it is believed that the attachment of a glycan moiety may create a physical barrier (as well as a more hydrophilic surface), which reduces the undesirable aggregation of monomeric trimers, and / or undesirable adhesion. from a monomeric trimer to the host cell (e.g., cell membrane, ER membrane, other hydrophobic proteins, aggregated proteins, etc.) via the hydrophobic surface. The mutation allows the gB protein (or an immunogenic fragment thereof) to be glycosylated by covalently bonding a sugar moiety (glycan) to the hydrophobic surface 1. Preferably, the glycosylation site is a N-site. glycosylation comprising an NXS / T / C motif, wherein X is any amino acid residue (but preferably not proline, since proline may reduce the glycosylation efficiency). Such a glycosylation site can be created, for example, by substituting a residue with an N, or by inserting an N-residue.
Second, a glycosylation site can be introduced (i) into a larger hydrophobic surface, referred to as "hydrophobic surface 2" (which includes amino acid residues 145-167 and 230-252), or ( ii) at a residue that is at most 20 angstroms from one of two hydrophobic fusion loops: fusion loop 1 (FL1) (amino acid residues 155-157), or the fusion loop 2 (FL2) (amino acid residues 240-242), or both. For example, based on the crystal structure, it has been found that the C-terminal region of the ectodomain is conformationally close to highly hydrophobic FL1 or FL2. For example, residues 696-698 (Y, E, and E, respectively, in Merlin strain gB) are expected to be at most 20 angstroms of FL1 and / or FL2. Therefore, introducing a glycan moiety at the C-terminal region of the ectodomain (for example, creating a glycosylation site at residues 696-698, for example, by replacing a residue by N, or by inserting an NXS / T / C sequence) can also create a physical barrier to reduce aggregation and / or adhesion of the monomeric trimers.
Third, a mutation can be introduced into the hydrophobic surface 1 (which includes residues 154-160 and 236-243), wherein the mutation results in a reduction in the overall hydrophobicity index of said hydrophobic surface 1. The creation a hydrophilic surface can reduce the aggregation and / or adhesion of the monomeric trimers.
Fourth, because the C-terminal region of the ectodomain is in conformational proximity to the hydrophobic surface 1, a heterologous sequence may be added to the C-terminal region of the ectodomain to "mask" the hydrophobic surface. It is believed that the heterologous sequence may serve as a lipid to cover the hydrophobic surface, thereby reducing the aggregation and / or adhesion of the monomeric trimers. Preferably, the heterologous sequence comprises an amphipathic peptide. An amphipathic peptide comprises a hydrophobic moiety that can interact with the hydrophobic surface of the monomeric trimer, and the peptide also comprises a hydrophilic surface that can be exposed to an aqueous solution.
These four types of mutations can be used alone, or in any combination, to produce a recombinant gB protein. For example, the gB may comprise two mutations, one creating a glycosylation site in said hydrophobic surface 1 or said hydrophobic surface 2, and the other replacing a hydrophobic residue with a more hydrophilic residue.
Accordingly, the invention relates to modified gB proteins and immunogenic fragments thereof comprising a glycosylation site and / or one or more mutations that reduce the aggregation and / or adhesion of gB monomeric trimers.
In general, the gB proteins and immunogenic fragments described herein do not include a transmembrane domain (TM) (in other words, the TM domain of gB is deleted).
2. DEFINITIONS gB is a B-envelope glycoprotein fulfilling a large number of roles, one of them being the involvement in the fusion of cytomegalovirus with host cells. It is encoded by the UL55 gene of the HCMV genome. The size of the native form of gB depends on the size of the Open Reading Frame (ORF) which may vary slightly depending on the strain. For example, the ORF of strain AD169, which is 2717 bp in length, encodes a full length gB of 906 amino acids, whereas the ORF of the Towne strain encodes a full length gB 907 amino acids. Although the present invention is applicable to the gB proteins from any CMV strain, for ease of understanding, when reference is made to the amino acid positions in the present application, the numbering is given relative to the sequence of acids. amino acids of the gB protein of SEQ ID NO: 1 from the clinical isolate of the Merlin strain, unless otherwise specified. The present invention is however not limited to the Merlin strain. Comparable amino acid positions in a gB protein of any other CMV strain can be determined by those skilled in the art by aligning the amino acid sequences using readily available and well known alignment algorithms (such as BLAST, using the default settings; ClustalW2, using the default settings; or the algorithm described by Corpet, Nucleic Acids Research, 1998, 16 (22): 10881-10890, using the default settings). Therefore, when referring to a "CMV gB protein", one must understand a CMV gB of any strain (besides the Merlin strain). It may be necessary to adjust the true number for the gB proteins of other strains depending on the sequence alignment itself.
For example, the melting loop 1 (FL1) is defined as consisting of amino acid residues 155-157, and the fusing loop 2 (FL1) is defined as consisting of amino acid residues 240-242. These numbers are relative to the amino acid sequence of the gB protein of SEQ ID NO: 1. The FL1 and FL2 sequences / positions of gB proteins of other CMV strains, or other mutants or variants of gB, or gB fragment can be verified using standard sequence alignment programs that align a query sequence with SEQ ID NO: 1, and identify residues that mate with residues 155-157 and 240-242 of SEQ ID NO : 1.
The specific positions of the amino acid residues are also numbered according to SEQ ID NO: 1. For example, "Y160" refers to position 160 of SEQ ID NO: 1 (which is a Y), as well as corresponding residues of other gB sequences (or variants or fragments) that are Y160-like of SEQ ID NO: 1, when the sequence is aligned with SEQ ID NO: 1. For simplicity, reference is made to any residue of a gB (or variant or fragment) sequence which corresponds to Y160 of SEQ ID NO: 1 as to Y160, although the true position of this residue may or may not be 160, and the residue itself may or may not be Y For example, a conservative substitution (e.g., F) may be aligned with Y160 of SEQ ID NO: 1. A conservative substitution is typically identified as "positive" or "+" by BLAST 2.
Similarly, the mutations are also identified according to the numbering of SEQ ID NO: 1. For example, Y160T means that any residue of a gB sequence (or a variant or fragment) that corresponds to Y160 of SEQ ID NO: 1 is mutated in T.
An amino acid residue of a query sequence "corresponds to" a designated position of a reference sequence (eg, Y160 of SEQ ID NO: 1) when, by aligning the query amino acid sequence with the sequence of reference, the position of the residue corresponds to the designated position. These alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, or "Blast 2 Sequences" using the default settings.
The native form of gB Merlin contains in the N-terminal to C-terminal direction of the protein (see Figure 1) (i) a signal peptide, known to be involved in the intracellular trafficking of the polypeptide comprising targeting the polypeptide to the secretion, followed by (ii) a region referred to as a leader sequence, (iii) an extracellular domain which also comprises a furin-like endoproteolytic cleavage site between amino acid residues 456 and 460, (iv) a transmembrane domain and (v) a cytoplasmic C-terminal domain. The ectodomain comprises two hydrophobic fusion loops: the fusion loop 1 (FL1) (amino acid residues 155-157) or the fusion loop 2 (FL2) (amino acid residues 240-242).
"Hydrophobic surface 1" consists of amino acid residues 154-160 and 236-243. "Hydrophobic surface 2" consists of amino acid residues 145-167 and 230-252. Here again, the amino acid residues are identified by the position as a function of the gB protein of the Merlin strain (SEQ ID NO: 1). The corresponding amino acid residues of other gB sequences or fragments can be verified by aligning the query sequence against SEQ ID NO: 1.
CMV gt Ectodomain refers to a CMV gB fragment that comprises substantially the extracellular portion of the mature CMV gB protein, with or without the signal peptide, and does not include the transmembrane domain and the C-terminal domain of the CMV. Natural CMV gB protein. In a preferred embodiment, the ectodomain comprises amino acid residues 69 to 698.
The transmembrane domain (TM) refers to the region that covers the cell membrane. The minimum region is amino acid residues 750-766.
A monomeric trimer is formed of three gB proteins (which are also referred to as subunits). A dimer trimer is formed by dimerization of two monomeric trimers. Thus, a dimer trimer comprises six gB subunits.
An immunogenic fragment of gB refers to a fragment that retains at least one predominant immunogenic epitope of full-length gB. Several antigenic domains (AD) of gB have been described. See, for example, Wiegers et al., J Virol. 2014 Oct 15. pii: JVI.02393-14; Epub in press; Pötzsch et al., PLoS Pathog 7 (8): el002172. doi: 10.1371 / journal.ppat.1002172 (Aug 2011); Spindler et al., J Virol. 2013 Aug; 87 (16): 8927-39. doi: 10.1128 / JVI.00434-13; Ohlin et al., J. Virol. 67 (2): 703-710, 1993. AD-1 is approximately 80 residues in length between positions 560 and 640 of strain AD169 gB. This is the immunodominant region of gB since virtually all sera from patients infected with HCMV recognize AD-1. AD-2, located at the amino-terminus of the protein, comprises at least two distinct sites between ADB's gA and ADB. Site I (amino acids 50-54 of ADI69) differs between strains and is recognized by strain-specific antibodies, some of which neutralize in a complement-dependent manner. Site II (amino acids 69-78 of AD169) is common to all strains of HCMV and induces largely neutralizing antibodies. An additional linear amino acid sequence, AD-3 (a, 783-906), recognized by gB-specific antibodies in human serum, includes epitopes at the intraluminal / intraviral portion of the molecule. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 gB of strain ADI69. AD-5 is formed by a continuous sequence comprising A.a. 133-343 of strain AD169. In preferred embodiments, the immunogenic fragment described herein comprises an antigenic domain selected from the group consisting of AD-1, AD-2, AD-3, AD-4, AD-5, and a combination of these.
A heterologous sequence refers to an amino acid sequence or a nucleotide sequence that is not found in the natural CMV gB protein, nor in a nucleic acid encoding a CMV gB protein.
An amphipathic peptide refers to peptides containing hydrophilic amino acid residues and hydrophobic amino acid residues, where the spatial separation of these residues, such as by the secondary structure of the peptide, results in their ability to separate at the an interface between a polar medium and a non-polar medium such as a lipid interface, an air / water interface, a hydrophilic solvent / hydrophobic solvent interface and an air / packaging material interface. Typically, the amphipathic peptides have an amphipathicity defined by a mean hydrophobic moment of between about 0 and about 0.9, according to the Eisenberg curve (Eisenberg et al., J. Mol Biol 179: 125-142, 1984). .
3. RECOMBINANT gB PROTEINS
The CMV gB protein comprises an N-terminal extracellular domain of approximately 725 amino acids, followed by a transmembrane region and a C-terminal domain. The most well-known neutralizing epitopes on gB associate with the ectodomain of gB, as do two hydrophobic regions, referred to as fusion loops 1 (FL1) and 2 (FL2). It is assumed that gB inserts these fusion loops into the target cell membrane and, by means of a conformational modification of its structure, puts the viral and cellular membrane in juxtaposition to facilitate fusion of the viral / cellular membranes.
As described herein, the inventors have solved the crystal structure of CMV gB, in a monomeric trimer form, complexed to an anti-gB antibody (Fab fragment). Based on the crystal structure, the inventors have identified an exposed hydrophobic surface which can result in the aggregation of monomeric trimers of gB (e.g., the formation of dimeric trimers, which comprise six gB subunits), as well as the undesirable adhesion of a monomeric trimer to the host cell (eg, cell membrane, ER membrane, other hydrophobic proteins, aggregated proteins, etc.). A narrower hydrophobic surface (hydrophobic surface 1) consists of residues 154-160 and 236-243. A larger hydrophobic surface (hydrophobic surface 2) consists of residues 145-167 and 230-252.
Based on this information, the inventors have identified several categories of mutations that can reduce aggregation between monomeric trimers, and / or adhesion of the monomeric trimer to the host cell (e.g., cell membrane, ER membrane, other hydrophobic proteins, aggregated proteins, etc.). In particular, mutations generally allow the secretion of the monomeric trimer by a host cell, when heterologously expressed in mammalian cells), thus significantly improving the efficiency of the production process.
In particular, four strategies have been identified. The first is to introduce a glycosylation site within the hydrophobic surface 1 (residues 154-160 and 236-243). The second consists of introducing a glycosylation site either inside the hydrophobic surface 2 (residues 145-167 and 230-252), and / or at a residue which is at most 20 angstroms from the loop. melting point 1 (FL1) (residues 155-157) and / or melting loop 2 (FL2) (residues 240-242). For example, residues 696, 697, and 698 are at most 20 angstroms of FL1 and / or FL2 and can be used to introduce a glycosylation site. The third is to reduce the overall hydrophobicity index of the hydrophobic surface 1 or 2. The fourth is to include a heterologous sequence at the C-terminal region of the ectodomain to at least partially mask the hydrophobic surface 1 or 2.
These mutations can be used individually and in combination to overcome the hydrophobicity of the exposed hydrophobic surface. For example, the mutants can be evaluated by size exclusion chromatography and potentially by electron microscopy to verify the formation of soluble monomeric trimers of gB.
In general, the recombinant human CMV gb protein (or its immunogenic fragment) described herein does not include a transmembrane domain (TM). The TM domain is at least composed of amino acid residues 750-766. However, since residues adjacent to this minimal TM region are generally hydrophobic, in certain preferred embodiments, the gB protein (or an immunogenic fragment thereof) does not include (i) amino acid residues 700-766 (ii) amino acid residues 700-776, (iii) amino acid residues 698-766, or (iv) amino acid residues 698-776. The deletion of these amino acid residues is believed to facilitate the recombinant production of the gB proteins described herein. Alternatively, in some embodiments, the gB protein (or an immunogenic fragment thereof) does not include two hydrophobic residue extensions, the first consisting of residues 725 to 744, and the second consisting of residues 751 to 773.
The gB proteins of the invention may be variants of gB which have different degrees of identity with SEQ ID NO: 1 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 described in SEQ ID NO: 1. In certain embodiments, variants of the gB protein: i) do not form a significant amount of dimer trimer; (ii) comprise at least one epitope of SEQ ID NO: 1; and / or (iii) can elicit antibodies in vivo (preferably neutralizing antibodies) that exhibit a cross-immunological reaction with a CMV virion. A substantial amount of dimer trimer means that, for example, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, or at least 5%, of Total gB units are in a dimer trimer form. A. Glycosylation
In one aspect, the present invention relates to gB proteins, or immunogenic fragments thereof, which comprise a glycosylation site in the hydrophobic surface 1. In particular, the invention relates to a cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation that results in a glycosylation site within the hydrophobic surface 1. Preferably, said glycosylation site is a N-site. glycosylation comprising an NXS / T / C unit, wherein X is any amino acid residue (preferably not proline). More preferably, said glycosylation site is an N-glycosylation site comprising an N-X-S / T motif, wherein X is any amino acid residue (preferably not proline). Accordingly, when recombinantly produced in a suitable host cell (e.g., a host cell that comprises a glycosylation enzyme), the gB protein or immunogenic fragment thereof comprises a glycan moiety bound to a residue inside the hydrophobic surface 1.
In another aspect, the present invention relates to gB proteins, or immunogenic fragments thereof, which comprise a glycosylation site in the hydrophobic surface 2, or at a residue that is in the conformational vicinity of FL1 and / or FL2. In particular, the invention relates to a recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation that results in a glycosylation site, wherein said glycosylation site is (1) within the hydrophobic surface 2 (acidic residues amines 145-167 and 230-252); or (2) at a residue that is at most 20 angstroms from the fusion loop 1 (FL1) (amino acid residues 155-157) and / or the fusion loop 2 (FL2) (acidic residues Amines 240-242). Accordingly, when recombinantly produced in a suitable host cell (e.g., a host cell that comprises a glycosylation enzyme), the gB protein or immunogenic fragment thereof comprises a glycan moiety bound to a residue within the hydrophobic surface 2, or a residue which is at most 20 angstroms of the melting loop 1 (FL1) and / or the melting loop 2 (FL2).
As described herein, binding of a glycan creates a physical barrier (as well as a more hydrophilic surface) to reduce aggregation / adhesion via the hydrophobic surface. The glycosylation site may be within the narrower hydrophobic surface 1 or the larger hydrophobic surface 2 described herein. Alternatively, the glycosylation site may be at a residue which is in conformational proximity to highly hydrophobic FL1, and / or FL2 (for example, at most 30 angstroms, or at most 25 angstroms, or more than 20 angstroms, or not more than 15 angstroms, or not more than 14 angstroms, or not more than 13 angstroms, or not more than 12 angstroms, or not more than 11 angstroms, or not more than 10 angstroms, or not more than at 9 angstroms, or at most 8 angstroms, or at most 7 angstroms, or at most 6 angstroms, or at most 5 angstroms, of one of the FL1 and / or FL2 atoms). For example, based on the crystal structure, the C-terminal region of the ectodomain is in conformational proximity to FL1 and / or FL2. For example, residues 696, 697, and 698 are all at most 20 angstroms of FL1 and / or FL2 and can be used to introduce a glycosylation site.
The glycosylation sites can be introduced at desired locations by suitable modification of the amino acid sequences of the gB protein. Preferably, N-linked glycosylation sites, including the N-X-S / T / C motif, are introduced. Preferably, the unit is N-X-S / T. Preferably, X is not proline.
For example, N-linked glycosylation can be introduced into the hydrophobic surface by modification of the amino acid sequence of the gB protein to include an NXS / T / C motif for N-linked glycosylation. This can be accomplished by insertion. of the NXS / T / C motif in the gB sequence, or by replacing one or more amino acids to produce the glycosylation site, or any combination of addition and mutation resulting in the NXS / T / C motif. For example, N can be added, while the S / T / C position can be mutated; or N can be mutated from another residue, while the S / T / C position is added. When the protein is expressed in suitable cells, for example, mammalian cells, the N-linked glycans will be linked to the N-residue to create N-glycosylated gB.
Similarly, sites for O-linked glycosylation can also be added. In O-linked glycosylation, the carbohydrate moiety is bonded to the oxygen atom of the hydroxyl group of serine and threonine. In addition, O-linked glycosylation may occur at tyrosine, 5-hydroxylysine, and 4-hydroxyproline.
In some embodiments, the mutation comprises an insertion of the NXS / T / C sequence (eg NXS, NXT, NXC, NGS, NGT, NAS, NAT, etc., where x is any amino acid, but preferably not proline). In some embodiments, the mutation comprises an insertion of the NXS / T / C sequence (eg NXS, NXT, NXC, NGS, NGT, NAS, NAT, etc., where x is any amino acid, but preferably not proline) in fusion loop 1 (FL1) (amino acid residues 155-157), fusion loop 2 (FL2) (amino acid residues 240-242), or both. In some embodiments, the mutation comprises an insertion of the NXS / T / C sequence (eg NXS, NXT, NXC, NGS, NGT, NAS, NAT, etc., where x is any amino acid, but preferably not proline) without mutation of other residues in FL1 and FL2.
In some embodiments, the mutation comprises the mutation of 236RGSTW (SEQ ID NO: 12) to 236RGSTNGTW (SEQ ID NO: 13); 240WLYR (SEQ ID NO: 14) in 240WLYNGTR (SEQ ID NO: 15), or a combination thereof.
In some embodiments, the gB protein or immunogenic fragment thereof comprises a mutation that is selected from the group consisting of (i) R236N, (ii) G237N, (iii) T158N, (iv) W240N and Y242T, (v) W240N and Y242S, (vi) W240N and Y242C, and a combination thereof. In some embodiments, the gB protein or immunogenic fragment thereof comprises a mutation that is selected from the group consisting of (i) R236N, (ii) G237N, (iii) T158N, and a combination of those -this. In some embodiments, the mutation comprises the T158N mutation.
In some embodiments, the mutation comprises mutations (i) W240N; and (ii) Y242T, Y242S, or Y242C. The combination of the two mutations creates a glycosylation site.
In addition to glycosylation, the gB protein and immunogenic fragments thereof described herein may also include one or more mutations that reduce the overall hydrophobicity index of the hydrophobic surface, as described below, and / or comprises a C-terminal heterologous sequence, as described below. B. Reduction of the overall hydrophobicity index
In another aspect, the invention relates to a recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation in the hydrophobic surface 1 (amino acid residues 154-160 and 236-243), or the hydrophobic surface 2 (amino acid residues 145-167 and 230-252); wherein said mutation results in a reduction in the overall hydrophobicity index of said hydrophobic surface 1 or 2.
In preferred embodiments, the mutation is not a deletion or substitution of an amino acid in fusion loop 1 (FL1) (amino acid residues 155-157) and fusion loop 2 (FL2) ( amino acid residues 240-242). So, in
certain embodiments, the gB proteins comprising a deletion or substitution of an amino acid residue selected from the group consisting of amino acid residues 155, 156, 157, 240, 241 and 242 are excluded. The hydrophobicity of a particular amino acid sequence can be determined using a hydrophobicity scale, such as the Kyte and Doolittle scale (Kite et al., 1982. J. Mol., Bio 157: 105- 132). The hydrophobicity of an amino acid sequence or a fragment thereof is dictated by the type of amino acids that make up that sequence or a fragment thereof. Amino acids are commonly classified into distinct groups according to their side chains. For example, some chains are considered non-polar, in other words hydrophobic, while others are considered polar. For the purposes of the present invention, it is considered that alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F) and tryptophan (W) are part of the hydrophobic amino acids, whereas it is considered that serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y), cysteine (C), lysine (K), arginine (R), histidine (H), aspartic acid (D) and glutamic acid (E) ), are part of the polar amino acids. Regardless of their hydrophobicity, amino acids are also classified into subgroups on the basis of common properties shared by their side chains. For example, phenylalanine, tryptophan and tyrosine are together classified as aromatic amino acids and will be considered aromatic amino acids within the meaning of the present invention. Aspartate (D) and qlutamate (E) are among the acidic or negatively charged amino acids, while lysine (K), arginine (R) and histidine (H) are among the amino acids basic or positive charge, and they will be considered as such in the sense of the present invention. Scales of hydrophobicity are available that utilize the hydrophobic and hydrophilic properties of each of the 20 amino acids and assign a hydrophobicity score to each amino acid, thereby creating a hydrophobicity rating. By way of example only, the Kyte and Doolittle scale can be used (Kyte et al., 1982. J. Mol Bio 157: 105-132). This scale allows the skilled person to calculate the average hydrophobicity in a segment of a predetermined length. Accordingly, the hydrophobic regions in an amino acid sequence can be identified by those skilled in the art as potential mutation targets according to the present invention. The ability of the mutation of said regions to induce an improved product profile of the resulting mutant protein, in other words to promote the proportion of monomeric trimers in the population, can be tested as described below. The mutation of a hydrophobic region may be in an addition, deletion, or substitution of the amino acid in the hydrophobic surface (for example, by substituting the hydrophobic amino acids with polar amino acids).
The fact that a mutation can reduce the overall hydrophobicity index of the hydrophobic surface can also be determined by, for example, analyzing the effect resulting from said mutation on the product profile. For example, upon recombinant expression in said host cells, a mutation should result in an improved profile enriched with monomeric trimers (eg, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the recombinantly produced gB (or an immunogenic fragment thereof) ) is in a form of monomeric trimer).
In some embodiments, the mutation comprises replacing a hydrophobic amino acid residue in hydrophobic surface 1 or 2 with an amino acid residue that includes a charged side chain or a polar side chain. In some embodiments, the hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. In some embodiments, the amino acid residue comprises a charged side chain is selected from the group consisting of D, E, K, R, and H. In some embodiments, the amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N , Q, and Y.
In some embodiments, the mutation comprises deleting a hydrophobic amino acid residue within hydrophobic surface 1 or 2. In some embodiments, the hydrophobic amino acid residue is selected from the group consisting of: , V, L, I, P, M, F, G, and W.
In some embodiments, the mutation comprises inserting an amino acid residue that includes a charged side chain or a polar side chain into the hydrophobic surface 1 or 2. In some embodiments, the hydrophobic amino acid residue is selected. in the group consisting of: A, V, L, I, P, M, F, G, and W. In some embodiments, the amino acid residue comprising a charged side chain is selected from the group consisting of D, E , K, R, and H. In some embodiments, the amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q, and Y.
In some embodiments, the mutation comprises replacing Y160 with an amino acid residue that includes a charged side chain or a polar side chain.
In some embodiments, the amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. In some embodiments, the gB protein or an immunogenic fragment thereof includes a Y160E mutation.
In some embodiments, the amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, and Q. In some embodiments, the gB protein or an immunogenic fragment thereof includes a Y160T mutation.
In some embodiments, the gB protein or an immunogenic fragment thereof includes the mutation of 1S8TTY160 to 158NTT160.
In some embodiments, the mutation comprises replacing S238 with an amino acid residue that includes a charged side chain.
In some embodiments, the amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. In some embodiments, the gB protein or an immunogenic fragment thereof includes an S238E mutation.
In some embodiments, the mutation comprises replacing T239 with an amino acid residue that includes a charged side chain.
In some embodiments, the amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. In some embodiments, the gB protein or an immunogenic fragment thereof includes a T239E mutation.
In some embodiments, the gB protein or an immunogenic fragment thereof comprises an S238E mutation and a T239E mutation.
In some embodiments, the gB protein or an immunogenic fragment thereof comprises a R236E mutation or an R236D mutation. In some embodiments, the gB protein or an immunogenic fragment thereof comprises a R236E mutation.
In some embodiments, the gB protein or an immunogenic fragment thereof comprises mutations selected from the group consisting of: (i) R236E and S238E; (ii) R236E and T239E; and (iii) R236E, S238E, and T239E.
In some embodiments, the gB protein or an immunogenic fragment thereof comprises a mutation at I156, H157, or a combination thereof to reduce hydrophobicity. In some embodiments, the residue is replaced by a corresponding gB residue of another species of herpesvirus, for example HSV-1, HSV-2, or VZV. In one exemplary embodiment, the gB protein or an immunogenic fragment thereof comprises an I156H mutation. In another exemplary embodiment, the gB protein or an immunogenic fragment thereof comprises a Y242T mutation. C. C-terminal heterologous sequence
In another aspect, the invention relates to a cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a domain transmembrane (TM); (ii) said gB protein, or said immunogenic fragment thereof, comprises an ectodomain (amino acid residues 69-698); and (iii) said gB protein, or said immunogenic fragment thereof, comprises a heterologous sequence of at least 12 residues at the C-terminus. In some aspects, the gB protein may be a fusion protein in which the heterologous sequence is fused at the C-terminus of the ectodomain. In some aspects, the heterologous sequence may be an amphipathic peptide.
The inventors discovered from the crystalline structure that the C-terminal region of the ectodomain was in proximity to the hydrophobic surface. It has further been found that when the TM domain of gB is deleted and the cytoplasmic domain is fused directly to the extracellular domain, the gB protein can form a soluble monomeric trimer. It is believed that additional amino acid residues at the C-terminal region may serve as a physical barrier to at least partially cover or mask the exposed hydrophobic surface, thereby reducing the aggregation and / or adhesion of the monomeric trimers.
Preferably, the heterologous sequence is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids. Preferably, the heterologous sequence will be at least 12 amino acids in length. In a preferred embodiment, the heterologous sequence is about 20 amino acids in length. In some embodiments, the length of the heterologous sequence is not greater than 50, 45, 40, 35, 30, or 25 amino acids.
Preferably, the heterologous sequence comprises an amphipathic peptide. An amphipathic peptide comprises a hydrophobic moiety that can interact with the hydrophobic surface of the monomeric trimer, and the peptide also comprises a hydrophilic surface that can be exposed to an aqueous solution.
Examples of amphipathic peptides can be found, for example, in sequences derived from apolipoproteins. Apolipoproteins are lipid binding proteins that are divided into six major classes (A, B, C, D, E and H) and several subclasses. The design and synthesis of amphipathic peptides that mimic the properties of apolipoproteins are known, see, for example,
Mishra et al. Biochemistry 1996, August 27; 35 (34): 11210-20. Specific examples include peptides include, for example, DWLKAFYDKVAEKLKEAFLA (SEQ ID NO: 3); ELLEKWKEALAALAEKLK (SEQ ID NO: 4); FWLKAFYDKVAEKLKEAF (SEQ ID NO: 5); DWLKAFYDKVAEKLKEAFRLTRKRGLKLA (SEQ ID NO: 6), and DWLKAFYDKVAEKLKEAF (SEQ ID NO: 7).
The mutation strategies described herein may be used alone or in combination, for example (i) introducing a glycosylation site and reducing the overall hydrophobicity index of the hydrophobic surface; (ii) introducing a glycosylation site and introducing a C-terminal heterologous sequence; (iii) reducing the hydrophobicity index of the hydrophobic surface and introducing a C-terminal heterologous sequence; and (iv) introducing a glycosylation site, reducing the overall hydrophobicity index of the hydrophobic surface, and introducing the C-terminal heterologous sequence.
Specific mutations described herein include: 236RGSTW2 <(SEQ ID NO: 12) mutated to 236NGSTW240 (SEQ ID NO: 16); 236RGSTW240 (SEQ ID NO: 12) mutated to 236RNSTW24 ° (SEQ ID NO: 17); 236RGSTW240 (SEQ ID NO: 12) mutated at 23 "EGETW240 (SEQ ID NO: 18); 236RGSTW24 ° (SEQ ID NO: 12) mutated to 236EGEEW240 (SEQ ID NO: 19). 236RGSTW24o (seq id NO: 12) mutated to "RGSTNGTW2" (SEQ ID NO: 13); 240WLYR243 (SEQ ID NO: 14) mutated to WLYNGTR246 (SEQ ID NO: 15); i58TTYi60 mutated to i58ntti "°; 158TTY160 mutated at i58Tte16 °; and 156IH157 and 240WLY242 mutated to HR137 and 24on1T242.
Mutations relating to glycosylation sites and hydrophobicity are not limited to the mutations described above. Other mutations that are not described herein, as well as combinations of mutations described herein, can be made. The mutants obtained can be analyzed, for example, by electron scanning microscopy (SEM), computer modeling, sedimentation (eg analytical ultracentrifugation (UCA)), chromatography etc., to evaluate the production of monomeric trimer. For example, size exclusion chromatography (SEC), for example UV-based steric exclusion chromatography (SEC-UV), may be used. Alternatively, the sample may be treated with a crosslinking agent to form covalent bonds between two proteins. After crosslinking, loading the sample on a gel under denaturing conditions, for example SDS-PAGE, and staining the gel for the presence of proteins, for example with Coomassie blue or nitrate d silver, will present the aggregates, if any, which are separated according to their molecular mass. For example, the CMV strain ADI 6 9 gb (with the deleted transmembrane domain) has a predicted molecular weight of 92 kDa. In the case of the formation of a monomeric trimer, the expected average molecular weight should be about 276 kDa. D. Other Modifications Other modifications may also be introduced to facilitate the recombinant production of the gB protein, or immunogenic fragments thereof.
In general, the original C-terminal cytoplasmic domain of the gB protein can be deleted to a varying degree. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, more suitably at least 80%, at least 90%, or 100% of the amino acids of the cytoplasmic domain are deleted.
In some embodiments, the recombinant human CMV gB protein (or an immunogenic fragment thereof) comprises a mutation of the furin cleavage site. The ectodomain comprises a furin cleavage site at residues 457-460 (RTKR (SEQ ID NO: 20) for Merlin strain SEQ ID NO: 1). Such a mutation may be, for example, R457S, R460S, or R457S / R460S double mutations. This or these mutations can destroy the furin cleavage site, thereby promoting the production of intact gB or immunogenic fragment of intact gB (eg, theododomain). There is another potential cleavage site of furine at residues 774-777 (RQRR) (SEQ ID NO: 21), which can also be mutated if present in the gB protein or its immunogenic fragment described in US Pat. this document.
In some embodiments, the recombinant human CMV gB protein (or an immunogenic fragment thereof) comprises a mutation at C246. Such a mutation may be, for example, C246S, C246A, or C246G. It appears that C246 is an unpaired cysteine, and mutation of this unpaired cysteine can reduce the undesired formation of intermolecular disulfide bonds. There is another potential unmatched cysteine at the C-terminal region (residue 779). If necessary, this cysteine can also be mutated.
Optionally, to facilitate expression and recovery, the gB protein (or an immunogenic fragment thereof) may comprise a signal peptide at the N-terminus. A signal peptide can be selected from many signal peptides known in the art, and is typically selected to facilitate production and processing in a system selected for recombinant expression of the gB protein (or its immunogenic fragment). In general, the signal peptides are from 5 to 30 amino acids in length, and are typically present at the N-terminus of a newly synthesized protein. The signal peptide core generally contains a long stretch of hydrophobic amino acids that tends to form a single alpha helix. In addition, a large number of signal peptides start with a short hydrophilic extension (usually positively charged) of amino acids, which may help to induce the correct topology of the polypeptide during translocation. At the end of the signal peptide (C-terminus), there is typically a hydrophilic amino acid extension that is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during or after the end of the translocation to generate a free signal peptide and a mature protein.
In some embodiments, the signal peptide is the naturally occurring peptide in native gB proteins. For the Merlin and AD169 strains, the signal peptide is located at residues 1 to 22 of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The signal peptide of other strains can be identified by sequence alignment. Alternatively, the signal peptide may be a heterologous sequence in that the sequence is derived from a protein distinct from gB. Examples of signal peptides suitable for use in the context of the gB protein (or an immunogenic fragment thereof) described herein include signal peptides of tissue plasminogen activator (tPA), the protein gD Herpes Simplex virus (HVS), human endostatin, HIV gp120, CD33, human Her2Neu, gp67, or Epstein-Barr virus (EBV) gp350. The signal peptide may be non-native and may include mutations, e.g., substitutions, insertions, or deletions of amino acids. In particular, the mutations can be introduced at the C-terminal portion of the signal peptide.
Optionally, the CMV gB proteins (or their immunogenic fragment) of the invention may comprise the addition of an amino acid sequence which constitutes a marker, which may facilitate detection (e.g., an epitope tag for detection by monoclonal antibodies) and / or purification (e.g., a polyhistidine label to allow purification on a nickel chelator resin) of the proteins. Examples of affinity purification markers include, for example, a His tag (hexahistidine (SEQ ID NO: 8), binds to a metal ion), the maltose binding protein (MBP) (binds to amylose) glutathione-S-transferase (GST) (binds to glutathione), a FLAG marker (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 9), binds to an anti-antibody -flag), a Strep marker (Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 10), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ( SEQ ID NO: 11), binds to streptavidin or a derivative thereof), an HA tag, a MYC tag, or a combination thereof.
In one embodiment, cleavable linkers may be used. 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 irradiation of a photolabile linkage or by acid catalyzed hydrolysis. There are also polypeptide linkers that incorporate a protease recognition site and that can be cleaved by the addition of a suitable protease enzyme.
In other embodiments, it may be more desirable to express gB (or an immunogenic fragment thereof) without an exogenous marker sequence, for example, for reasons of clinical tolerance or efficacy.
The CMV recombinant gB protein (or an immunogenic fragment thereof) described herein may also contain a trimerization marker to enhance trimerization. For example, a foldon marker of T4 fibritin or a trimerization domain GCN4 may be inserted at the C-terminus of the CMV gB protein (or an immunogenic fragment thereof).
The present invention further relates to a CMV complex comprising the recombinant gB protein (or a fragment thereof) described herein. In some embodiments, the complex is a monomeric trimer consisting of three subunits of the gB protein.
In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the recombinantly produced gB (or an immunogenic fragment thereof) is in a monomeric trimer form. In some embodiments / not more than 50%, not more than 45%, not more than 40%, not more than 35%, not more than 30%, not more than 25%, not more than 20%, not more than of 15%, not more than 10%, not more than 5% of the recombinantly produced gB (or an immunogenic fragment thereof) is in a dimer trimer form.
4. RECOMBINANT EXPRESSION OF gB
The present invention also relates to nucleic acids encoding CMV gB and immunogenic fragments thereof, as described herein. The nucleic acid, such as DNA, can be used for the recombinant production of the gB protein. The invention also relates to a host cell comprising the nucleic acids described herein. When the host cell is cultured in a suitable condition, the nucleic acids can express a gB protein (or an immunogenic fragment thereof). Preferably, said gB protein (or an immunogenic fragment thereof) forms a monomeric trimer. Preferably, the monomeric trimer can be secreted by the host cell.
Suitable host cells include, for example, insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., human beings cells). humans, non-human primates, horses, cows, sheep, dogs, cats, rodents (eg, hamsters)), avian cells (eg, chickens, ducks, and geese) ), bacteria (e.g., E. coli, Bacillus subtilis, and Streptococcus spp.), yeast cells (e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorph, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica),
Tetrahymena (e.g., Tetrahymena thermophila) or combinations thereof.
For mutants that include a glycosylation site, the host cells must be cells that have enzymes that mediate glycosylation. Bacterial hosts are generally not suitable for these mutants unless the strain is modified to introduce glycosylation enzymes; on the other hand, a eukaryotic host, such as an insect cell, an avian cell, or a mammalian cell, may be used.
Suitable insect cell expression systems, such as baculoviral systems, are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA. For example, for expression in insect cells a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express the recombinant protein. Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clone isolate derived from the parental Trichoplusia nor BTI-TN cell line). -5B1-4 (Invitrogen)).
Avian cell expression systems are also known to those skilled in the art and described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent EP 0787180B; European Patent Application ηβ EP03291813.8; WO 03/045964, and WO 03/076601. Suitable avian cells include, for example, embryonic stem cells (e.g., EBx® cells), chicken embryo fibroblasts, embryonic chicken germ cells, duck cells (e.g., AGE1.CR cell lines). and AGEl.CR.pIX (ProBioGen) which are described, for example, in Vaccine 27: 4975-4982 (2009) and WO 2005/042728), EB66 cells, and the like.
Preferably, the host cells are mammalian cells (e.g., cells of humans, nonhuman primates, horses, cows, sheep, dogs, cats, and rodents (e.g., hamsters Suitable mairanalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293 cells, typically transformed with adenovirus type 5 sheared DNA), NIH cells. 3T3, 293-T cells, VERO cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL -75), macaque fetal lung cells (ATCC CL-160), Madin-Darby bovine kidney cells ("MDBK"), Madin-Darby canine kidney cells ("MDCK") (e.g., MDCK (NBL2)) ATCC CCL34 or MDCK 33016, DSM ACC 2219), kidney hamster (BHK) kidney cells, such as s BHK21-F, HKCC, and the like.
In some embodiments, the host cell is a CHO cell. In some embodiments, the polynucleotide encoding the gB protein (or immunogenic fragment thereof) described herein is stably integrated into the genome of the CHO cell.
Different CHO cell lines are also available from the European Collection of Cell Cultures (ECACC), or the American Type Culture Collection (ATCC), for example CHO cell lines hCBE11 (ATCC® PTA-3357-), E77. 4 (ATCC® PTA-3765® *), hLT-B: R-hG1 CHO # 14 (ATCC® CRL-11965 "), MOR-CHO-MORAb-003-RCB (ATCC® PTA-7552"), AQ. C2 clone 11B (ATCC® PTA-3274 ™), AQ.C2 clone 11B (ATCC® PTA-3274 ™), hsAQC2 in CHO-DG44 (ATCC® PTA-3356 ™), xrs5 (ATCC® CRL-2348 ™) , CHO-K1 (ATCC® CCL-61 ™), Lecl [initially Pro-5WgaRI3C] (ATCC® CRL-1735 "), Pro-5 (ATCC® CRL-1781 ™), ACY1-E (ATCC® 65421") , ACY1-E (ATCC® 65420 "), pgsE-606 (ATCC® CRL-2246 ™), CHO-CD36 (ATCC® CRL-2092"), pgsC-605 (ATCC® CRL-2245 ™), MC2 / 3 (ATCC® CRL-2143 ™), CHO-ICAM-1 (ATCC® CRL-2093 ™), and pgsB-618 (ATCC® CRL-2241 ™). Any of these CHO cell lines can be used. Other commercially available CHO cell lines include, for example, CHO-S Freestyle ™ cells and the Life Technologies CHO Flp-In ™ cell line.
Methods of expressing recombinant proteins in CHO cells have generally been described. See, for example, U.S. Patent Nos. 4,816,567 and 5,981,214.
In some embodiments, the recombinant nucleic acids are codon nucleic acids optimized for the expression of a selected prokaryotic or eukaryotic host cell.
To facilitate replication and expression, the nucleic acids may be incorporated into a vector, such as a prokaryotic or eukaryotic expression vector. Examples of vectors include plasmids that are capable of autonomous replication or replication in a host cell. Typical expression vectors contain suitable promoters, enhancers, and terminators that are useful for regulating the expression of the coding sequence (s) in the expression construct. The vector may also include selectable markers to provide a phenotypic trait for selection of transformed host cells (e.g. confer antibiotic resistance such as ampicillin or neomycin).
Examples of procedures sufficient to guide those skilled in the art of producing recombinant CMV gB nucleic acids can be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements to 2003); and Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley &amp; Sounds, 1999. The invention also relates to a cell culture comprising the host cell described herein. The cell culture may be on a large scale, for example, at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least approximately 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 150 L, at least about 200 L, at least about 250 L, at least about 300 L, at least about 400 L L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1000 L, at least about 2000 L, at least about 3 L 000 L, at least about 4,000 L, at least about 5,000 L, at least about 6,000 L, at least about 10,000 L, at least about 15,000 L, at least about 20,000 L, at least about 25,000 L, at least about 30,000 L, at least about 35,000 L, at least about 40,000 L, at least about 45,000 L, at least about 50,000 L, at least about 55,000 L, at least about 60,000 L , at least e approximately 65,000 L, at least approximately 70,000 L, at least approximately 75,000 L, at least approximately 80,000 L, at least approximately 85,000 L, at least approximately 90,000 L, at least approximately 950,000 L, at least approximately 100,000 L, etc.
In some embodiments, the yield of the gB protein (or an immunogenic fragment thereof of the cell culture is at least about 0.01 g / L, at least about 0.02 g / L, at least about 0.03 g / L, at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.15 g / L, at least about 0.20 g / L, at least about 0.25 g / L, at least about 0.3 g / L, at least about 0.35 g / L, at least about about 0.4 g / L, at least about 0.45 g / L, at least about 0.5 g / L, at least about 0.55 g / L, at least about 0 , 6 g / L, of at least about 0.65 g / L, of at least about 0.7 g / L, of at least about 0.75 g / L, of at least about 0.8 g / L, at least about 0.85 g / L, at least about 0.9 g / L, at least about 0.95 g / L, or at least about 1.0 g / L.
The present invention further relates to a method of producing the cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, comprising: (i) culturing the host cell described herein in a condition suitable, thereby expressing said gB protein, or an immunogenic fragment thereof; and (ii) harvesting said gB protein, or an immunogenic fragment thereof, from the culture.
In some embodiments, the gB protein (or an immunogenic fragment thereof) described herein is purified. The gB protein (or an immunogenic fragment thereof) can be purified using suitable methods, such as HPLC, different types of chromatography (e.g. hydrophobic interaction, ion exchange, affinity, chelation and steric exclusion), electrophoresis, density gradient centrifugation, solvent extraction, or the like. As adapted, the gB protein (or an immunogenic fragment thereof) may further be purified, if necessary, to substantially remove any proteins that are also secreted in the medium or that result from lysis of the host cells, to provide a product that is at least substantially free of host debris, e.g., proteins, lipids, and polysaccharides. See, for example, those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocol Handbook Humana Press, NJ, Harris & Angal (1990) Protein Purification Applications: A Practical
Approach IRL Press at Oxford, Oxford, U.K .; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High
Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ. If desired, the gB protein (or an immunogenic fragment thereof) may include a "tag" that facilitates purification, as described above.
For example, methods of purifying CMV gB protein by immunoaffinity chromatography have been described. Ruiz-Arguello et al., J. Gen. Virol., 85: 3677-3687 (2004).
5. PHARMACEUTICAL COMPOSITIONS AND METHODS OF
TREATMENT The invention relates to pharmaceutical compositions and methods of treatment using the cytomegalovirus (CMV) gB protein (or immunogenic fragments thereof) described herein, or a nucleic acid encoding such a gB protein (or immunogenic fragments thereof) described herein. For example, the immunogenic proteins or fragments may be administered directly as components of an immunogenic composition, or nucleic acids that encode the gB proteins or immunogenic fragments may be administered to produce the CMV protein or immunogenic fragment in vivo. Certain preferred embodiments, such as protein formulations, recombinant nucleic acids (e.g., self-replicating RNA), and alphavirus replicon (VRP) particles that contain sequences encoding gB proteins or Immunogenic fragments are further described herein. A. Protein Compositions
In one aspect, the invention relates to an immunogenic composition comprising the CMV recombinant gB protein (or an immunogenic fragment thereof) described herein.
The immunogenic composition may include additional CMV proteins, such as gO, gH, gL, p1L128, pUL130, pUL131, an immunogenic fragment thereof, or a combination thereof. For example, gB (or an immunogenic fragment thereof) may be combined with a CMV pentameric complex comprising: gH or a pentamer forming moiety thereof, or a pentamer forming moiety thereof, pUL128 or a pentamer-forming fragment thereof, pUL130 or a pentamer-forming moiety thereof, and pUL131 or a pentamer-forming moiety thereof. The gB (or an immunogenic fragment thereof) can also be combined with a CMV trimer complex comprising: gH or a trimer-forming fragment thereof, gL or a trimer-forming fragment thereof, and gO or a trimer-forming moiety thereof.
The immunogenic composition may comprise an adjuvant. Examples of adjuvants for improving the effectiveness of the composition include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc .; ; (2) oil-in-water emulsion formulations (with or without other specific adjuvants such as mullam peptides (see below) or bacterial cell wall components), such as, for example, ( a) MF59 (PCT publication No. WO 90/14837), containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing different amounts of MTP-PE (see below), although this is not necessary) formulated in submicron particles using a microfluidizer such as model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% squalane, 0.4% Tween 80, 5% pluronic-blocked L121 polymer, and thr-MDP (see below) either microfluidized to a submicron emulsion or vortexed to generate a more particle size emulsion. large, and (c) the adjuvant system Ribi "(RAS), (Ribi Immunochem,
Hamilton, Mont.) Containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components of the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); (3) saponin adjuvants, such as Stimulon ™ (Cambridge Bioscience, Worcester, Mass.) May be used or particles generated therefrom such as ISCOM (immunostimulatory complex); (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc. ; and (6) other substances that act as adjuvants to improve the effectiveness of the composition.
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine (MTP-PE), etc.
In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the adjuvant is an oil-in-water emulsion, such as MF59.
In some embodiments, the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod.
In some embodiments, the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate.
Adjuvants described herein may be used alone or in combination, such as an alum / TLR7 combination. B. Alphavirus replicon particles
In some embodiments, the CMV gB proteins (or immunogenic fragments thereof) described herein are administered using alphavirus replicon particles (VRP). As used herein, the term "alphavirus" has its conventional meaning in the art and includes various species such as Venezuelan equine encephalitis virus (VEE), for example, Trinidad donkey, TC83CR, etc.), Semliki Forest Virus (SFV), Sindbis virus, Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SA AR86 virus, Everglades virus, Mucambo virus, Barmah forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.
An "alphavirus replicon particle" (VRP) or "replicon particle" is an alphavirus replicon packaged in structural alphavirus proteins.
An "alphavirus replicon" (or "replicon") is an RNA molecule that can direct its own amplification in vivo in a target cell. The replicon encodes the polymerase (s) that catalyze the amplification of RNA (nsP1, nsP2, nsP3, nsP4) and contains the cis RNA sequences necessary for replication that are recognized and used by the encoded polymerase (s). An alphavirus replicon typically contains the following elements, in the order: 5 'viral sequences required in cis for replication, sequences that encode biologically active alphavirus non-structural proteins (nsP1, nsP2, nsP3, nsP4), 3 'viral sequences required in cis for replication, and a polyadenylated tail. An alphavirus replicon may also contain one or more promoters of the viral subgenomic "junction region", directing the expression of heterologous nucleotide sequences, which may, in some embodiments, be modified to increase or decrease transcription of the subgenomic fragment and the heterologous sequence (s) to be expressed. Other control elements may be used, such as IRES or 2A sequences. C. Nucleic acid delivery systems
The recombinant nucleic acid molecule that encodes the CMV gB proteins or immunogenic fragments described herein can be administered to induce production of the encoded CMV gB proteins or immunogenic fragments and an immune response thereto. The recombinant nucleic acid may be DNA (e.g., plasmid or viral DNA) or RNA, preferably self-replicating RNA, and may be monocistronic or polycistronic. Any suitable DNA or RNA can be used as the vector nucleic acid that carries the open reading frames that encode the CMV gB proteins or immunogenic fragments thereof. Suitable nucleic acid vectors have the ability to carry and direct expression of one or more CMV gB proteins or immunogenic fragments. Such nucleic acid vectors are well known in the art and include, for example, plasmids, DNA DNA viruses such as vaccinia virus vectors (e.g., NYVAC, see OS 5,494 807), and poxvirus vectors (e.g., ALVAC canarypox vector, Sanofi Pasteur), and RNA obtained from suitable RNA viruses, such as an alphavirus. If desired, the recombinant nucleic acid molecule may be modified, for example, contain nucleobases and / or modified linkages as described below.
The self-replicating RNA molecules of the invention are based on genomic RNA virus RNA, but do not possess the genes encoding one or more structural proteins. Self-replicating RNA molecules can be translated to produce nonstructural proteins of RNA viruses and CMV gB proteins encoded by self-replicating RNA. Self-replicating RNA usually contains. at least one or more genes selected from the group consisting of viral replicase, viral proteases, viral helicases and other non-structural viral proteins, and further comprises cis-active replication sequences at the 5 'ends and 3 ', and a heterologous sequence that encodes one or more desired CMV gB proteins. A subgenomic promoter that directs the expression of the heterologous sequence (s) may be included in the self-replicative RNA. If desired, a heterologous sequence can be. merged in phase with other coding regions in self-replicating RNA and / or may be under the control of an internal ribosome entry site (IRES).
The self-replicating RNA molecules of the invention may be designed such that the self-replicating RNA molecule can not induce production of the infectious virus particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary for the production of viral particles in self-replicative RNA. For example, when the self-replicating RNA molecule is based on an alphavirus, such as Sinbis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE), or one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, may be omitted. If desired, the self-replicating RNA molecules of the invention can be designed to induce the production of infectious viral particles that are attenuated or virulent, or to produce virus particles that are capable of a single subsequent infection cycle .
A self-replicating RNA molecule can, when administered to a vertebrate cell even without protein, lead to the production of multiple RNA girls by transcription from itself (or from a copy antisense of itself). The self-replicating RNA can be directly translated after the administration to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces transcripts from the administered RNA.
Thus, the administered RNA leads to the production of multiple daughter RNAs. These transcripts are antisense to the administered RNA and can be translated themselves to provide in situ expression of the encoded CMV protein, or can be transcribed to provide more transcripts in the same sense as the administered RNA that are translated to provide in situ expression of the coded CMV protein (s).
A preferred self-replicating RNA molecule thus encodes (i) an RNA-dependent RNA polymerase that transcribes RNA from the self-replicating RNA molecule and (ii) one or more CMV gB proteins or immunogenic fragments of them. The polymerase may be an alphavirus replicase, for example, comprising nsP1-nsP4 alphavirus nonstructural proteins.
The self-replicating RNA molecules of the invention may contain one or more modified nucleotides and therefore have improved stability and resistance to degradation and clearance in vivo, and other advantages. There are more than 96 naturally occurring nucleoside modifications found on mammalian RNA. See, for example, Limbach et al., Nucleic Acids Research, 22 (12): 2183-2196 (1994). The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, for example, US Pat. Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, and 5,132. 418, 5,153,319, 5,262,530, all of which are incorporated herein by reference, and a large number of modified nucleosides and modified nucleotides are available commercially. If desired, the self-replicating RNA molecule may contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. The self-replicating RNA described herein is suitable for administration in a variety of modalities, for example, the administration of naked RNA or in combination with lipids, polymers or other compounds that facilitate entry into the cells. Self-replicating RNA molecules can be introduced into target cells or subjects using any suitable technique, by. for example, direct injection, microinjection, electroporation, lipofection, biolistics, and the like. The self-replicating RNA molecule can also be introduced into cells by means of receptor-mediated endocytosis. See, for example, U.S. Patent No. 6,090,619; Wu and Wu, J. Biol. Chem., 263: 14621 (1988); and Curiel et al., Proc. Natl. Acad. Sci. USA, 88: 8850 (1991). For example, U.S. Patent No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by combining the nucleic acid with a polycationic moiety (e.g., a poly-L-lysine having 3 to 100 amino acid lysine), which is itself coupled to an integrin receptor binding moiety (e.g., a cyclic peptide having the Arg-Gly-Asp sequence).
Self-replicating RNA molecules can be administered into cells via amphiphiles. See, for example, U.S. Patent No. 6,071,890.
Typically, a nucleic acid molecule can form a complex with the cationic amphiphile. Mammalian cells brought into contact with the complex can rapidly absorb. The self-replicative RNA can be administered in the form of a naked RNA (for example, simply in the form of an aqueous solution of RNA) but, to improve its entry into cells and subsequent intracellular effects, Self-replicating RNA is preferably administered in combination with a delivery system, such as a particulate or emulsion delivery system. A large number of delivery systems are well known to those skilled in the art. Three particularly useful delivery systems are (i) liposomes, (ii) biodegradable and nontoxic polymeric microparticles, and (iii) cationic oil-in-water emulsions, less than one micron in size. The invention also relates to an immunogenic composition comprising the nucleic acids (e.g., self-replicating RNA) described herein. The immunogenic composition may comprise an adjuvant as described above. Preferred adjuvants include, for example, an aluminum salt or an oil-in-water emulsion (such as MF59). D. Pharmaceutical formulations
Each of the immunogenic compositions described herein may be used alone or in combination with one or more antigens, the one or more antigens from either the same viral pathogen or another or other viral pathogenic source. These pharmaceutical formulations may be prophylactic (to prevent infection) or therapeutic (to treat the disease after infection).
These pharmaceutical formulations comprise an immunogenic composition, generally in combination with "pharmaceutically acceptable carriers", which include any carrier which does not itself induce the production of harmful antibodies to the individual receiving the composition. Suitable vehicles are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets, or liposomes), and inactive viral particles. These vehicles are well known to those skilled in the art. In addition, these vehicles can function as adjuvants. In addition, the antigen may be conjugated to a bacterial toxoid, such as diphtheria, tetanus, cholera, H. pylori, etc. pathogens
The pharmaceutical formulations may include an adjuvant as described above.
The pharmaceutical formulations (e.g., the immunogenic composition, the pharmaceutically acceptable carrier, and the adjuvant) typically contain diluents, such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in these vehicles.
Typically, the pharmaceutical formulations are prepared in the form of injectables, in the form of liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for improved adjuvant effect, as discussed above, in the pharmaceutically acceptable vehicles section.
The pharmaceutical formulations comprise an immunologically effective amount of the antigenic polypeptides, as well as any of the above-mentioned components, as needed. "Immunologically effective amount" means that the administration of that amount to an individual, in a single dose or as part of a series of doses, is effective for the treatment or prevention of a condition, condition or condition. an infection or a disease. This quantity varies according to the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (for example, a non-human primate, a primate, etc.), the capacity of the system the individual's immunity to synthesize the antibodies, the desired degree of protection, the vaccine formulation, the medical assessment of the attending physician, and other relevant factors. It is expected that a therapeutically effective amount will fall within a relatively broad range that can be determined by routine testing.
The pharmaceutical formulations are typically administered parenterally, for example by injection, subcutaneous or intramuscular injection. Additional formulations of other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. The treatment may be a single dose regimen or a multiple dose regimen. The immunogenic composition may be administered together with other immunoregulatory agents.
5. PROCESSING PROCESS
In another aspect, the invention relates to a method of inducing an immune response against cytomegalovirus (CMV), comprising administering to a subject in need thereof an immunologically effective amount of the immunogenic composition described herein. which comprises proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), or VRPs, as described above.
In some embodiments, the immune responses include the production of neutralizing antibodies against CMV. In some embodiments, the neutralizing antibodies are complement-independent.
The immune response can include a humoral immune response, a cell-mediated immune response, or both. In some embodiments, an immune response is induced against each administered CMV protein. A cell-mediated immune response includes a helper T cell response (Th), a CD8 + cytotoxic T cell (CTL) response, or both. In some embodiments, the immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral cell infection. CMV infects epithelial cells and fibroblasts. In some embodiments, the immune response reduces or prevents infection of both cell types. Neutralizing antibody responses may be complement-dependent or complement-independent. In some embodiments, the neutralizing antibody response is complement-independent. In some embodiments, the neutralizing antibody response results in cross-neutralization; in other words, an antibody generated against the administered composition neutralizes a CMV virus of a strain other than the strain used in the composition.
A useful measure of power in the art is the "50% neutralization title". To determine the 50% neutralization titer, the serum of immunized animals is diluted to assess how well the serum can be diluted while retaining the ability to block entry of 50% of the viruses into the cells. For example, a titer of 700 means that the serum has retained the ability to neutralize 50% of the virus after being diluted 700 times. Thus, higher titers indicate stronger responses of neutralizing antibodies. In some embodiments, this title is in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, of about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6 000, about 6,500, or about 7,000. The 50% neutralization titre range may have an upper limit of about 400, about 600, about 800, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 120,00 about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000. For example, the 50% neutralization title can be from about 3,000 to about 6,500. "About" means plus or minus 10% of the proposed value. The neutralization titre can be measured as described in the specific examples below.
An immune response can be stimulated by administering proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), or VRP to an individual, typically a mammal, including a human being . In some embodiments, the immune response induced is a protective immune response, in other words, the response reduces the risk or severity of a CMV infection. Stimulation of a protective immune response is particularly desirable in certain populations particularly at risk for CMV infection and the disease it involves. For example, at-risk populations include patients with solid organ transplantation (SOT), bone marrow transplant patients, and patients with hematopoietic stem cell transplantation (HSCT). VRP can be administered to a transplant donor prior to transplantation, or to a transplant recipient before and / or after transplantation. Because mother-to-child vertical transmission is a common source of newborn infec- tion, VRP administration to a woman who is or may become pregnant is particularly useful.
Any suitable route of administration may be used. For example, a composition can be administered intramuscularly, intraperitoneally, subcutaneously, or transdermally. Some embodiments will be administered by an intramucosal route, for example, intra-orally, intranasally, intravaginally, and intrarectally. The compositions may be administered according to any suitable scheme. The invention also relates to a method of inhibiting the entry of cytomegalovirus (CMV) into a cell, comprising contacting the cell with the immunogenic composition described herein. The invention is further illustrated by the following examples, which should not be construed as being restrictive.
EXAMPLES EXAMPLE 1 - Generation of a soluble gB construct and resolution of the crystal structure of the CMV H glycoprotein B bound to a human neutralizing antibody Fab fragment The CMV gB ectodomain, residues 1 to 698, with a 6-His tag (SEQ ID NO: 8) at the C-terminus was expressed in 293GnTI cells (Figure 1). The sequence of the ectodomain WT could not be expressed as a secreted protein. To increase protein secretion, we mutated three hydrophobic residues in the fusion loops with the corresponding amino acids of HSV-1 gb (I157H, H158R and W240R), which are more hydrophilic. We also mutated the canonical furin cleavage site to reduce the heterogeneity of the protein caused by incomplete treatment during expression (R457S / R460S), as well as Cys246 in Ser to prevent the formation of parasitic disulfide bonds (Figure 1). In spite of these modifications, diffusion-exclusion chromatography (SEC, Figure 2A) and negative-staining electron microscopy (data not shown) revealed that the protein (gB-698) had formed dimeric trimers of the post-trimeric trimers. fusion with three characteristic lobes. Analysis of the electron microscopy images suggested that the dimerization was mediated by the base of the gB trimer, presumably because of the intrinsic hydrophobicity of its surface. Thus, we introduced a glycosylation site in fusion loop 2 (W240N, Y242T), which was predicted to be exposed to solvent in the trimer, to interfere with dimerization of trimers. MOE and SEC confirmed that this construct, gB-698glyc, did not dimerize (to form dimeric trimers) even at a high protein concentration (Figure 2A and data not shown).
Initial attempts to crystallize gB-698glyc by itself or in a complex with a neutralizing antibody Fab fragment have not been successful. We therefore deglycosylated the protein with endoglycosidase H (endo H) and performed limited in situ proteolysis with subtilisin E to remove the flexible regions that could interfere with crystallization. This treatment resulted in crystals which, however, only diffracted to a resolution of 4.3 Å. To improve diffraction, we have deleted 63 N-terminal residues of gaectodomaine (ANgB, devoid of residues 25-86) shown to be flexible in HSV-1 gB (Heldwein et al., Science, 313 ( 5784): 217-220, 2006). The deglycosylated ANgB-1G2 Fab complex crystallized easily without the need for protease treatment. After screening several crystals, a dataset of resolution of 3.6 Å was obtained and the structure was determined by molecular replacement (Figure 3).
Example 2 - Mutant Constructs of CMV gB
Based on the crystal structure of gB, we have designed mutations in the hydrophobic surface (covering both fusion loops and residues in the vicinity of the two fusion loops) to explore mutants that allow expression of 1 '. ectodomain of gB (monomeric trimer). The mutants were prepared and tested in an expression experiment. Figure 4 shows the Western blot of cell culture supernatant with anti-His antibody. All constructs with the exception of the ectodomain of gB and R236E / S238E had detectable secreted expression under boiling and reduction conditions (left panel).
The following constructions were prepared and tested:
1. WT
2. R236N
3. G237N
4. T158N / Y160T
5. Y160E
6. R236E
7. R236E / S238E 8. NGT inserted before W240 9. I156H / H157R / W240N / Y242T (B-698glyc)
All constructs contained the following additional mutations: R457S / R460S (furin cleavage site mutations) and C246S. T158N / Y160T (lane 4) shows a monomer size band under boiling / non-reducing conditions, similar to gB-698glyc (lane 9). The rest (with the exception of lanes 1 and 6) seems to form oligomeric structures of larger order, only visible after decomposition with heat and DTT. These results indicate that the insertion of a glycosylation site just after the fusion loop 1 is probably sufficient to allow the soluble expression of an ectodomain of gB even if residues of the wild-type fusion loop are present. Having a glycosylation site just outside of the fusion loop 1 is sufficient to allow secretion without the necessity of mutating the hydrophobic residues within FL1. A glycosylation site outside FL1 provided comparable results to the mutation of residues within the fusion loops to add a glycosylation site.
The various elements and embodiments of the present invention, to which reference is made in the individual sections apply, as appropriate, to the other sections, mutatis mutandis. Accordingly, the elements specified in one section may be combined with other elements specified in other sections, as appropriate.
The specification will be more fully understood in light of the teachings of the references cited in the specification. Embodiments in the specification provide an illustration of the embodiments of the invention and should not be understood as limiting the scope. Those skilled in the art will readily recognize that a large number of other embodiments are covered by the invention. All publications, patents, and sequences of GenBank cited in this specification are incorporated herein by reference in their entirety. If the documents incorporated by reference contradicted this application or were inconsistent with this application, this application would prevail over these documents. The fact that references of any kind are cited herein is not tantamount to admitting that they constitute the prior art of the present invention.
Those skilled in the art will recognize, or will be able to verify without resorting to experiments other than routine experiments, a large number of equivalents to the specific embodiments of. the invention described herein. These equivalents are intended to be included in the following embodiments. A recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation that results in a glycosylation site within a hydrophobic surface 1. 2. The recombinant gB protein according to embodiment 1 wherein said glycosylation site is an N-glycosylation site comprising an NXS / T motif, wherein X is any amino acid residue except proline. 3. The recombinant gB protein according to embodiment 1 or 2, wherein said mutation is selected from the group consisting of (i) R236N, (ii) G237N, (iii) T158N, (iv) W240N and Y242S, (v) ) W240N and Y242T, and a combination thereof. 4. The recombinant gB protein according to any one of embodiments 1 to 3, wherein said mutation comprises an insertion of the sequence N-X-S / T, wherein X is any amino acid residue except proline. 5. The recombinant gB protein according to embodiment 4, wherein said mutation comprises an insertion of the NXS / T sequence, in which X is any amino acid residue except proline, in the fusion loop 1 ( FL1), the merging loop 2 (FL2), or both. 6. The recombinant gB protein according to embodiment 5, wherein said mutation comprises an insertion of the NXS / T sequence, wherein X is any amino acid residue except proline, without mutation of other residues in FL1 and FL2. 7. The recombinant gB protein according to any one of embodiments 4 to 6, wherein said mutation comprises the mutation of 236RGSTW (SEQ ID NO: 12) to 236RGSTNGTW (SEQ ID NO: 13); 240WLYR (SEQ ID NO: 14) in 240WLYNGTR (SEQ ID NO: 15), or a combination thereof. 8. The recombinant gB protein according to any one of embodiments 1 to 7, further comprising a mutation which results in a reduction of the overall hydrophobicity index of said hydrophobic surface 1. The recombinant gB protein according to Embodiment 8, wherein (i) said mutation comprises replacing a hydrophobic amino acid residue with an amino acid residue which comprises a charged side chain or a polar side chain; or (ii) said mutation is at residue 1156 (e.g., I156H), H157 (e.g., H157R), or a combination thereof. 10. The recombinant gB protein according to embodiment 9, wherein said hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. recombinant gB protein according to embodiment 9 or 10, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 12. The recombinant gB protein according to the mode embodiment 9 or 10, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q, and Y. 13. The recombinant gB protein according to embodiment 8, wherein said mutation comprises deleting a hydrophobic amino acid residue within hydrophobic surface 1. The recombinant gB protein according to embodiment 13, wherein said hydrophobic amino acid residue is selected from the group consisting of of: A, V, L, I, P, M, F, G, and W. The recombinant gB protein according to embodiment 8, wherein said mutation comprises insertion of an amino acid residue which comprises a charged side chain or a polar side chain in the hydrophobic surface 1. The recombinant gB protein according to embodiment 15, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 17. The recombinant gB protein according to embodiment 15, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q and Y. 18. The recombinant gB protein according to any one of embodiments 1 to 17, comprising a mutation which replaces Y160 with an amino acid residue which comprises a charged side chain or a polar side chain. 19. The recombinant gB protein according to embodiment 18, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. The recombinant gB protein according to US Pat. Embodiment 18 or 19, comprising a Y160E mutation. 21. The recombinant gB protein according to embodiment 18, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, and Q. 22. The recombinant gB protein according to Embodiment 18 or 21, comprising a Y160T mutation. 23. The recombinant gB protein according to any one of embodiments 1 to 18 and 21 to 22, comprising a mutation α1 ty160 en15aNTT160. 24. The recombinant gB protein according to any one of embodiments 1 to 23, comprising a mutation which replaces S238 with an amino acid residue which comprises a charged side chain. 25. The recombinant gB protein according to embodiment 24, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 26. The recombinant gB protein according to US Pat. Embodiment 25, comprising an S238E mutation. 27. The recombinant gB protein according to any one of embodiments 1 to 26, comprising a mutation which replaces T239 with an amino acid residue which comprises a charged side chain. 28. The recombinant gB protein according to embodiment 27, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. The recombinant gB protein according to Embodiment 28, comprising a T239E mutation. 30. The recombinant gB protein according to any one of embodiments 1 to 29, comprising an S238E mutation and a T239E mutation. 31. The recombinant gB protein according to any one of embodiments 1 to 30, comprising a R236E mutation or a R236D mutation. 32. The recombinant gB protein according to embodiment 31, wherein said mutation is a R236E mutation. 33. The recombinant gB protein according to any one of embodiments 1 to 32, comprising mutations selected from the group consisting of: (i) R236E and S238E; (ii) R236E and T239E; and (iii) R236E, S238E, and T239E. 34. A recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane (TM) domain; and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation that results in a glycosylation site, wherein said glycosylation site is (1) within the hydrophobic surface 2; or (2) at a residue that is at most 20 angstroms from the melting loop 1 (FL1) or the melting loop 2 (FL2). 35. The recombinant gB protein according to embodiment 34, wherein said glycosylation site is an N-glycosylation site comprising an N-X-S / T motif, wherein X is any amino acid residue except proline. 36. The recombinant gB protein according to embodiment 34 or 35, wherein said mutation comprises an insertion of the N-X-S / T sequence, wherein X is any amino acid residue except proline. 37. The recombinant gB protein according to any one of embodiments 34 to 36, wherein said glycosylation site is at a residue that is at most 10 angstroms from the fusion loop 1 (FL1) or of the merging loop 2 (FL2). 38. The recombinant gB protein according to any one of embodiments 34 to 37, wherein said mutation is between residues 696-698. 39. The recombinant gB protein according to any one of embodiments 34 to 38, further comprising a mutation which results in a reduction of the overall hydrophobicity index of said hydrophobic surface 2. 40. The recombinant gB protein according to any of embodiments 34 to 39, further comprising a mutation which results in a reduction in the overall hydrophobicity index of the hydrophobic surface 1. 41. The recombinant gB protein according to embodiment 39 or 40 wherein (i) said mutation comprises replacing a hydrophobic amino acid residue with an amino acid residue which comprises a charged side chain or a polar side chain; or (ii) said mutation is at residue 1156 (eg, I156H), H157 (eg, H157R), or a combination thereof. 42. The recombinant gB protein according to embodiment 41, wherein said hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. 43. recombinant gB protein according to embodiment 41 or 42, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 44. The recombinant gB protein according to the mode embodiment 41 or 42, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q and Y. 45. The recombinant gB protein according to embodiment 39 or 40 wherein said mutation comprises deleting a hydrophobic amino acid residue. 46. The recombinant gB protein according to embodiment 45, wherein said hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. Recombinant gB protein according to embodiment 39 or 40, wherein said mutation comprises inserting an amino acid residue which comprises a charged side chain or a polar side chain. 48. The recombinant gB protein according to embodiment 47, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 49. The recombinant gB protein according to US Pat. embodiment 47, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q and Y. 50. A recombinant cytomegalovirus (CMV) gB protein, or a fragment an immunogen thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation in a hydrophobic surface 1, wherein said mutation results in a reduction in the overall hydrophobicity index of said hydrophobic surface 1; wherein said mutation is not a deletion or substitution of an amino acid in the fusion loop 1 (FL1) and in the fusion loop 2 (FL2). 51. The recombinant gB protein according to embodiment 50, wherein said mutation comprises replacing a hydrophobic amino acid residue with an amino acid residue that comprises a charged side chain or a polar side chain. 52. The recombinant gB protein according to embodiment 51, wherein said hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. 53. recombinant gB protein according to embodiment 51 or 52, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 54. Recombinant gB protein according to the mode embodiment 51 or 52, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q and Y. 55. The recombinant gB protein according to embodiment 50, in which wherein said mutation comprises deleting a hydrophobic amino acid residue. 56. The recombinant gB protein according to embodiment 55, wherein said hydrophobic amino acid residue is selected from the group consisting of: A, V, L, I, P, M, F, G, and W. 57. Recombinant gB protein according to embodiment 50, wherein said mutation comprises inserting an amino acid residue which comprises a charged side chain or a polar side chain. 58. The recombinant gB protein according to embodiment 57, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of D, E, K, R, and H. 59. The recombinant gB protein according to US Pat. Embodiment 57, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, Q and Y. 60. The recombinant gB protein according to any of the embodiments 50-59, comprising a mutation that replaces Y160 with an amino acid residue that includes a charged side chain or a polar side chain. 61. The recombinant gB protein according to embodiment 60, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of R, K, D, and E. 62. The recombinant gB protein according to the method of Embodiment 61, comprising a Y160E mutation. 63. The recombinant gB protein according to embodiment 60, wherein said amino acid residue comprising a polar side chain is selected from the group consisting of S, T, C, N, and Q. 64. The recombinant gB protein according to US Pat. Embodiment 61, comprising a Y160T mutation. 65. The recombinant gB protein according to any one of embodiments 50 to 60 and 63 to 64, comprising a mutation ι ^ βχ ^ γιβο in ise ^ TT160. 66. The recombinant gB protein according to any one of embodiments 50 to 65, comprising a mutation which replaces S238 with an amino acid residue which comprises a charged side chain. 67. The recombinant gB protein according to embodiment 66, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of R, K, D, and E. 68. The recombinant gB protein according to the method of Embodiment 67, comprising an S238E mutation. 69. The recombinant gB protein of any one of embodiments 50 to 68, comprising a mutation that replaces T239 with an amino acid residue that comprises a charged side chain. 70. The recombinant gB protein according to embodiment 69, wherein said amino acid residue comprising a charged side chain is selected from the group consisting of R, K, D, and E. 71. The recombinant gB protein according to the method of embodiment 70, comprising a T239E mutation. 72. The recombinant gB protein according to any one of embodiments 50 to 71, comprising an S238E mutation and a T239E mutation. 73. The recombinant gB protein according to any one of embodiments 50 to 72, comprising a R236E mutation or a R236D mutation. 74. The recombinant gB protein according to embodiment 73, wherein said mutation is a R236E mutation. 75. The recombinant gB protein according to any one of embodiments 50 to 72, comprising mutations selected from the group consisting of: (i) R236E and S238E; (ii) R236E and T239E; and (iii) R236E, S238E, and T239E. 76. The recombinant gB protein according to any one of embodiments 1 to 75, comprising a heterologous sequence of at least 12 residues at the C-terminus. 77. The recombinant CMV gB protein according to embodiment 76, wherein said heterologous sequence is at least 20 residues in length. 78. The recombinant CMV gB protein according to embodiment 76 or 77, wherein said heterologous sequence may comprise an amphipathic peptide. 79. A recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); (ii) said gB protein, or said immunogenic fragment thereof, comprises an ectodomain; and (iii) said gB protein, or said immunogenic fragment thereof, comprises a heterologous sequence of at least 12 residues at the C-terminus. 80. The CMV recombinant gB protein according to embodiment 79, wherein said heterologous sequence is at least 20 residues in length. 81. The recombinant CMV gB protein according to embodiment 79 or 80, wherein said heterologous sequence may comprise an amphipathic peptide. 82. A CMV complex comprising the recombinant gB protein according to any one of embodiments 1 to 81. 83. The CMV complex according to embodiment 82, wherein said complex is a monomeric trimer consisting of three subunits. units of the protein gB. 84. An immunogenic composition comprising the CMV recombinant gB protein according to any one of embodiments 1 to 81, or the complex of embodiment 82 or 83. 85. The immunogenic composition according to embodiment 84, comprising in addition to a CMV protein selected from the group consisting of gH, gL, pUL128, pUL130, pUL131, gO, an immunogenic fragment thereof, and a combination thereof. 86. The immunogenic composition according to embodiment 84 or 85, further comprising the CMV pentameric complex comprising: gH or a pentamer forming moiety thereof, or a pentamer forming moiety thereof, pUL128 or a pentamer-forming fragment thereof, pUL130 or a pentamer-forming moiety thereof, and pUL131 or a pentamer-forming moiety thereof. 87. The immunogenic composition of any one of embodiments 84 to 86, further comprising an adjuvant. 88. The immunogenic composition according to embodiment 86, wherein said adjuvant comprises an aluminum salt, a TLR7 agonist, or an oil-in-water emulsion. 89. The immunogenic composition according to embodiment 88, wherein said oil-in-water emulsion is MF59. 90. An isolated nucleic acid comprising a polynucleotide sequence encoding the recombinant CMV gB protein according to any one of embodiments 1 to 81. 91. The isolated nucleic acid according to embodiment 90, wherein said nucleic acid isolated is an RNA, preferably a self-replicating RNA. 92. The isolated nucleic acid according to embodiment 91, wherein said self-replicating RNA is an alphavirus replicon. 93. An alphavirus replication particle (VRP) comprising the alphavirus replicon according to embodiment 92. 94. An immunogenic composition comprising the nucleic acid according to any one of embodiments 90 to 92. 95. An immunogenic composition comprising the VRP of Embodiment 93. 96. The immunogenic composition of Embodiment 94 or 95, further comprising an adjuvant. 97. The immunogenic composition according to embodiment 96, wherein said adjuvant comprises an aluminum salt. 98. The immunogenic composition of embodiment 96, wherein said adjuvant comprises an oil-in-water emulsion. 99. The immunogenic composition of embodiment 98, wherein said oil-in-water emulsion is MF59. A host cell comprising the nucleic acid of any of embodiments 90 to 92. 101. The host cell of embodiment 100, wherein said nucleic acid is a DNA. 102. The host cell according to embodiment 101, wherein said cell is a mammalian cell. 103. The host cell according to embodiment 102, wherein said mammalian cell is a CHO cell or a HEK-293 cell. 104. The host cell of any one of embodiments 101 to 103, wherein said DNA encoding the CMV gB protein or the immunogenic fragment thereof is stably integrated into the genome of said host cell. 105. The host cell of any one of embodiments 101 to 104, wherein when cultured under appropriate conditions, said nucleic acid expresses a gB protein that forms a monomeric trimer. 106. The host cell according to embodiment 105, wherein said trimer is secreted by the host cell. A cell culture comprising the host cell according to embodiments 101 to 106, wherein said culture is at least 20 liters in size. 108. A cell culture comprising the host cell according to embodiments 101 to 107, wherein said culture is at least 100 liters in size. 109. A cell culture comprising the host cell according to embodiments 101 to 106, wherein said culture, wherein the yield of the gB protein is at least 0.05 g / L. 110. A cell culture comprising the host cell according to embodiments 101 to 106, wherein said culture, wherein the yield of the gB protein is at least 0.1 g / L. A method of producing a recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, comprising: (i) culturing the host cell according to any one of the embodiments of the invention; at 106 in an appropriate condition, thereby expressing said gB protein, or an immunogenic fragment thereof; and (ii) harvesting said gB protein, or said immunogenic fragment thereof, from the culture. The method of embodiment 111, further comprising purifying said recombinant gB protein or immunogenic fragment thereof. 113. A recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, produced by the method according to embodiment 111 or 112. 114. A method of inducing an immune response against cytomegalovirus ( CMV), comprising administering to a subject in need thereof an immunologically effective amount of the immunogenic composition of any one of embodiments 84 to 89 and 94 to 99. 115. The method according to embodiment 114, wherein the immune responses include the production of neutralizing antibodies against CMV. 116. The method according to embodiment 115, wherein the neutralizing antibodies are complement-independent. A method of inhibiting the entry of cytomegalovirus (CMV) into a cell, comprising contacting the cell with the immunogenic composition of any one of embodiments 84 to 89 and 94 to 99. The immunogenic composition of any of embodiments 84 to 89 and 94 to 99 for use in inducing an immune response against cytomegalovirus (CMV). 118. The immunogenic composition of any one of embodiments 84 to 89 and 94 to 99 for use in inducing an immune response against cytomegalovirus (CMV). 119. Use of the immunogenic composition according to any one of embodiments 84 to 89 and 94 to 99 in the manufacture of a medicament for inducing an immune response against cytomegalovirus (CMV). Amino acid sequence of human Merlin cytomegakwkus gB protein (SEQ ID No: 1) 10 20 30 40 50 60
MESRIWCLW CVtCCIVCLG AAVSSSSTRG TSATHSHHSS H7TSAAHSRS GSVSORVTSS 70 8 £ 90 100 110 120
Q7V5HGVNET IYKTTIKYGD W3VKTTKY YRVCSMf.QGT DLIRFERNiV CTSKKPIN3D
130 140 150 160 170 ISO EOEGIMVVYK RM1VAHKKV KVYQKOL'I'KR KSYAYiHiIIl.lGSK'KYVP PPMMEiHHiIi 190 200 210 220 230 240
SHSOCYSSYS RVIAGTVFVA YHRDSYEHXf MQLKPDDYEÎÎ TliSTRYVTVK DC'WÜSRGSTW 250 260 270 280 290 300
LYRETCNLÏ4C HVTITTAFSK YPYHFFAT5T GDWDISPFY MGTSRNRSYF GENA3KFFIF 310 320 330 340 350 360
PNYTIVSDFG RPKSAIETHR I.VAFERADS VISWDIQDSK NVTCQ2TFWE ASERTTRSSA 370 330 390 400 410 420
ED3YHFSSAK MTATFESKKQ EVKKSDSALD CVRDEAtNKË QölFKTSYNÖ TYEKYÜNVSV 430 440 450 460 470 430
FETTGGLWF WQGIKQKSLV ELERLANRSS LNLTHNRTKR SGDGKMATHL SMMEGVHNLV 490 500 510 520 530 540 YAQlÎFTYirr lrgyinrala oiaeawcvdq RRTÎEVFKSL skimpsails aiynkpiaar 550 560 570 580 590 600
FMÜDViCLAS CVT1NQTSVK VLRDMNVKII PGRCYSRPW IFKE'AMSSYV QYGQLGHDNE 610 620 630 640 650 660
ILLGNHRTEÊC0LPS1KIFÏ AGKSAYEYVD YLFKRMIDLS SÏSTVDSM'A LDIDP1ENTD 670 680 69 £ 700 710 720
FRVLELYSCK ELRS5MVFDL EEIKREFNSY KORVKYVEEK VVDPLPPYLK GLDDLKSGLG 730 74 £ 750 760 770 780 aagkavgvaT gavggavasv vegvatflkn pfgaftiilv aiawhtyi. tytrqrr; .ct 790 800 810 820 830 840
ÜPtOKLFPYL VSADGîrvrs "STKDTKUÎA PPSYEESVYR SCRWÎPGPPS SÛASTAAPPY 850 860 870 880 890 900
TNEQAYQMLL ALARLDAEQR AOQMGTDSLD GRTGTQDKGQ KPHLLDSLRÏÏ RKNGYRH3KD 907
SDEEENV Amino Acid Sequence of B protein gB of human cytomegalovirus strain AD169 (SB) ID NO: 21 10 20 30 <0 60 60
MEASURING A CVKLCIVCLG AAVSSSSTSTSTSSTJ1NGSÏÏSRSRTTSAQTR SVYSQHVT3S 70 80 90 100 1¾ 120
EAVSHRANET IYKTT1KYGD WGWTTK1 ' YFVCSKAQGT DLIRFERNII CTSKKPINSD 130 180 160 160 Π0 180 LOEGIMVVYK RNIVAHI'FKŸ RVYQKVLTFK RSYAY1ŸTTY LLÜSK'YEYVÂ PPMWE1KH1ÏÏ 190 200 210 220 230 240
KFAQCYSSYS RVIGGTVFVA YliRDSYENXf MQLIPDDYSN THSYP.YVTVK DQWHSRGSTO 260 260 270 280 2M 300. MZ.TITTAR5K YPYHFFATST GDWYISPFY KGTSRNASYF GEKADKFFI 310 320 330 340 350 360
PNYTTVSDF5 RPKAAPCTHR I.VAFI.F.RAI3S VISWDIQDEK RVTCOirFWE A5F.RTTRS. ~ A 370 380 390 400 410 420
EDSYHFSSAK MTATF1.SKXQ EVNKSDSALD CVRDEAINXI QQIPNTSYNÔ TYEKICKVSV 430 440 450 460 470 480
FETSGGLWF WQGIKQKSLV ELERLANR5S LNITHRTRRS TSDMNYTHLS SMESVHNLVY 490 500 510 520 530 540
AQLQFTYDTL RGYIKRALAQ IAEAWCVDQR RTLEVFKELS KIKPSAILSA IYNKPIAARF 550 560 570 580 590 600
mcovlglasc vnî) Q'.rsvKv lrdkkvxesF grcyskpvvT fnfanssyvo ygoegeomeT 610 620 630 640 660 660
LiGUHRTEEC C1PSLKIFIÄ GNSAYEYVDŸ LFKRMIDLSS ISTVDSMIAL DIDPLENTD 670 690 690 700 730 720
RVLELYSQKE LRPSKVFDLÊ EIMREFMSYK QRVKYVEDKV VDPLPPYLKG LDDLMSGLGA 730 74 £ 750 760 770 780
AGKAVGVMG AVGGAVASVV EGVATFI.KN FGAFTTXI.VA IAWT1TYM YTRÖRR1..CTQ 790 800 810 820 830 840 PLQMLFPYLV SAÜGrmSG STKDTSLQAP PSYKESVYNH ÜRKCPGPPSS DASTAAPPYÏ 850 860 870 880 890 900
HEQAYQMLLA LARIOAEQRA QQKGTDSLDG QTGTQDKGQK PNLL3RLRHR KNGYRULKDS 906
DEEEKV
SEQUENCE LISTING <110> GLAXOSMITHKLINE BIOLOGICALS sa
<120> ANTIGENS OF CYTOMEGALOVIRUS
<130> PAT056317-EP-EPT <140> <141> <160> 21 <170> Patentln version 3.5
<210> 1 <211> 907 <212> PRT <213> Human cytomegalovirus <400> 1
Met Glu Ser Arg Ile Trp Cys Leu Val Val Cys Val Asn Leu Cys Ile 15 10 15
Val Cys Leu Gly Ala Ser Val Ser Ser Ser Ser Ser Arg Arg Gly Thr Ser 20 25 30
Ala Thr His Ser His Ser Ser Ser His Thr Ser Ser Ala Ala His Ser 35 40 45
Arg Ser Gly Ser Val Ser Gin Arg Val Thr Ser Ser Gin Thr Val Ser 50 55 60
His Gly Val Asn Glu Thr Tire Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80
Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met 85 90 95
Ala Gin Gly Thr Asp Leu Ile Arg Phe Glu Arg Asn Ile Val Cys Thr 100 105 110
Ser Met Lys Pro Ile Asn Asp Asp Glu Asp Glu Gly Ile Met Val Val 115 120 125
Tyr Lys Arg Asn Val Ala Island His Thr Phe Lys Val Arg Val Tyr Gin 130 135 140
Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr Island His Thr Thr Tyr 145 150 155 160
Leu Leu Gly Ser Asn Thr Glu Tire Val Pro Ala Pro Met Trp Glu Ile 165 170 175
His His Island Asn Ser His Ser Gin Cys Tire Ser Ser Ser Ser Ser Arg Val 180 185 190
Ala Gly Thr Island Val Phe Val Ala Tyr His Arg Asp Ser Tire Glu Asn 195 200 205
Lys Thr Met Gin Leu Met Asp Asp Asp Ser Ser Thr Thr Thr Ser Ser 210 215 220
Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser Thr Trp 225 230 235 240
Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Thr Thr Thr Thr 245 250 255
Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Ser Thr Thr Gly Asp 260 265 270
Val Val Asp Asp Ser Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285
Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe Phe Island Pro Asn Tyr Thr 290 295 300
Val Ser Island Asp Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg 305 310 315 320
Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330 335
Gin Asp Glu Lys Asn Thr Thr Cys Gin Leu Thr Phe Thr Trp Glu Ala Ser 340 345 350
Glu Arg Thr Isle Arg Ser Glu Ala Asp Glu Ser Tyr His Ser Phe Ser 355 360 365
Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Glu Glin Val Asn Met 370 375 380
Ser Asp Ser Ala Leu Asp Asp Val Cys Asp Glu Ala Ile Asn Lys Leu 385 390 395 400
Gin Gin Island Phe Asn Thr Ser Tire Asn Gin Thr Tyr Glu Lys Tyr Gly 405 410 415
Asn Val Ser Val Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gin 420 425 430
Gly Ile Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 '445
Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Asp Asp Gly 450 455 460
Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val His Asn Leu Val 465 470 475 480
Tyr Ala Gin Leu Gin Phe Thr Asp Asp Asp Thr Leu Arg Gly Tire Asn Island 485 490 495
Arg Ala Leu Ala Gin Ala Gla Ala Island Trp Cys Val Asp Gin Arg Arg 500 505 510
Thr Leu Glu Val Phe Lys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile 515 520 525
Leu Ser Ala Island Tyr Asn Lys Pro Ala Ala Island Arg Phe Met Gly Asp 530 535 540
Val Leu Gly Leu Ala Ser Cys Thr Val Asn Island Gin Thr Ser Val Lys 545 550 555 560
Val Leu Arg Asp Met Asn Val Lys Glu Pro Ser Gly Arg Cys Tyr Ser 565 570 575
Arg Pro Val Val Ile Phe Asn Phe Ala Asn Ser Ser Tyr Val Gin Tyr 580 585 590
Gly Gin Leu Gly Glu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr 595 600 605
Glu Glu Cys Gin Leu Pro Ser Leu Lys Ile Phe Ile Ala Gly Asn Ser 610 615 620
Ala Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met Ile Asp Leu Ser 625 630 635 640
Ser Ser Ser Ser Asp Val Asp Ser Met Ile Ala Leu Asp Asp Asp Asp Leu 645 650 655
Glu Asn Asp Asp Phe Arg Val Leu Glu Leu Tyr Ser Ser Gin Lys Glu Leu 660 665 670
Arg Ser Ser Asn Val Phe Asp Leu Glu Glu Met Island Arg Glu Phe Asn 675 680 685
Ser Tyr Lys Gin Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro 690 695 700
Leu Pro Pro Leuk Tyr Lys Gly Leu Asp Leu Asp Met Ser Gly Leu Gly 705 710 715 720
Ala Ala Gly Lys Ala Val Gly Val Ala Ally Gly Ala Val Gly Ala 725 730 735
Val Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe 740 745 750
Gly Ala Phe Thr Island Ile Leu Val Ala Ile Ala Val Val Island Ile Thr 755 760 765
Tyr Leu Tyr Island Arg Arg Arg Arg Arg Leu Cys Thr Gin Leu Gin Pro 770 775 780
Asn Leu Phe Pro Tire Leu Val Ser Ala Asp Gly Thr Ser Thr Thr Val 785 790 795 800
Gly Ser Thr Lys Asp Ser Ser Ser Gin Ala Pro Pro Ser Tyr Glu Glu 805 810 815
Ser Val Tyr Asn Ser Gly Arg Lys Pro Gly Pro Pro Ser Ser Asp 820 825 830
Ala Ser Thr Ala Pro Ala Pro Tyr Asn Thru Glin Gin Ala Tyr Gin Met 835 840 845
Leu Leu Ala Leu Ala Arg Leu Asp Ala Glu Gin Arg Ala Gin Gin Asn 850 855 860
Gly Thr Asp Ser Asp Asp Gly Arg Thr Gly Thr Asp Aspen Gly Gin 865 870 875 880
Lys Pro Asn Leu Leu Asp Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg 885 890 895
His Leu Lys Asp Asp Asp Asp Glu Glu Glu Asn Val 900 905
<210> 2 <211> 906 <212> PRT <213> Human cytomegalovirus <400> 2
Met Glu Ser Arg Ile Trp Cys Leu Val Val Cys Val Asn Leu Cys Ile 15 10 15
Val Cys Leu Gly Ala Ala Ser Ser Ser Ser Ser Ser Ser Ser His Ala Thr 20 25 30
Ser Ser Thr His Asn Gly Ser His Thr Ser Arg Thr Ser Ser Ala Gin 35 40 45
Thr Arg Ser Val Ser Ser Gin His Ser Thr Val Ser Ser Glu Ala Val Ser 50 55 60
His Arg Ala Asn Glu Thr Tire Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80
Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met 85 90 95
Ala Gin Gly Thr Asp Leu Ile Arg Phe Glu Arg Asn Island Ile Cys Thr 100 105 110
Ser Met Lys Pro Ile Asn Glu Asp Leu Asp Glu Gly Ile Met Val Val 115 120 · 125
Tyr Lys Arg Asn Val Ala Island His Thr Phe Lys Val Arg Val Tyr Gin 130 135 140
Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr Island Tyr Thr Thr Tyr 145 150 155 160
Leu Leu Gly Ser Asn Thr Glu Tire Val Pro Ala Pro Met Trp Glu Ile 165 170 175
His His Island Asn Lys Phe Ala Gin Cys Tire Ser Ser Ser Ser Ser Ara Val 180 185 190
Gly Gly Thr Island Val Phe Valley Val Ala Tyr His Arg Asp Ser Tire Glu Asn 195 200 205
Lys Thr Met Gin Leu Pro Island Asp Asp Tyr Ser Asn Thr His Ser Thr 210 215 220
Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser Thr Trp 225 230 235 240
Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Leu Thr Thr Thr 245 250 255
Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Ser Thr Thr Gly Asp 260 265 270
Val Val Tyr Ser Island Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285
Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe Phe Island Pro Asn Tyr Thr 290 295 300
Val Ser Island Asp Phe Gly Arg Pro Asn Ala Ala Pro Glu Thr His Arg 305 310 315 320
Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330 335
Gin Asp Glu Lys Asn Thr Thr Cys Gin Leu Thr Phe Thr Trp Glu Ala Ser 340 345 350
Glu Arg Thr Isle Arg Ser Glu Ala Asp Glu Ser Tyr His Ser Phe Ser 355 360 365
Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Glu Glin Val Asn Met 370 375 380
Ser Asp Ser Ala Leu Asp Asp Val Cys Asp Glu Ala Ile Asn Lys Leu 385 390 395 400
Gin Gin Island Phe Asn Thr Ser Tire Asn Gin Thr Tyr Glu Lys Tyr Gly 405 410 415
Asn Val Ser Val Phe Glu Ser Ser Gly Gly Leu Val Val Phe Trp Gin 420 425 430
Gly Ile Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445
Ser Ser Leu Asn Thr Thr His Arg Thr Arg Arg Ser Ser Ser Asp Asn 450 455 460
Asn Thr Thrn His Ser Ser Ser Glu Ser Ser Val His Asn Leu Val Tyr 465 470 475 480
Ala Gin Leu Gin Phe Thr Tyr Asp Aspl Leu Arg Gly Tyr Asn Arg Island 485 490 495
Ala Leu Ala Gin Ala Gla Ala Trp Cys Val Asp Gin Arg Arg Thr 500 505 510
Leu Glu Val Phe Lys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile Leu 515 520 525
Ser Ala Island Tyr Asn Lys Pro Ala Ala Island Arg Phe Met Gly Asp Val 530 535 540
Leu Gly Leu Ala Ser Cys Thr Val Asn Island Gin Thr Ser Val Lys Val 545 550 555 560
Leu Arg Asp Met Asn Val Lys Glu Pro Ser Gly Arg Cys Tyr Ser Arg 565 570 575
Pro Val Val Ile Phe Asn Phe Ala Asn Ser Ser Tyr Val Gin Tyr Gly 580 585 590
Gin Leu Gly Glu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr Glu 595 600 605
Glu Cys Gin Leu Pro Ser Leu Lys Island Phe Island Ala Gly Asn Ser Ala 610 615 620
Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met Ile Asp Leu Ser Ser 625 630 635 640
Ser Ser Thr Val Asp Ser Met Ala Leu Asp Asp Asp Asp Leu Glu 645 650 655
Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gin Lys Glu Leu Arg 660 665 670
Ser Ser Asn Val Phe Asp Leu Glu Glu Met Island Arg Glu Phe Asn Ser 675 680 685
Tyr Lys Gin Arg Val Lys Tyr Val Glu Val Asp Lys Val Val Asp Pro Leu 690 695 700
Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly Ala 705 710 715 720
Ala Gly Lys Ala Val Gly Val Ala Gly Ala Val Gly Ala Val 725 730 735
Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly 740 745 750
Ala Phe Thr Island Ile Leu Val Ala Ile Ala Val Val Ile Ile Thr Tyr 755 760 765
Leu Tyr Island Arg Arg Arg Arg Arg Leu Cys Thr Gin Leu Gin Asn Pro 770 775 780
Leu Phe Pro Tire Leu Val Ser Ala Asp Thr Thr Thr Thr Thr Ser Gly 785 790 795 800
Ser Thr Lys Asp Ser Ser Ser Gin Ala Pro Ser Pro Ser Glu Glu Ser 805 810 815
Val Tyr Asn Ser Gly Arg Lys Pro Gly Pro Gly Pro Ser Ser Asp Ala 820 825 830
Ser Thr Ala Pro Ala Pro Tyr Asn Glu Gin Ala Tyr Gin Met Leu 835 840 845
Leu Ala Leu Ala Arg Al Asp Ala Glu Gin Ala Gin Gin Arg Asin Gly 850 855 860
Thr Asp Ser Leu Asp Gly Gin Gly Thr Asp Gin Asp Lys Gly Gin Lily 865 870 875 880
Pro Asn Leu Leu Arg Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg His 885 890 895
Leu Ser Asp Asp Asp Glu Glu Glu Asn Val 900 905
<210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 3
Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 15 10 15
Ala Phe Leu Ala 20
<210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 4
Glu Leu Leu Glu Lys Trp Lily Glu Ala Leu Ala Ala Leu Ala Glu Lily 15 10 15
Leu Lys
<210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 5
Phe Trp Leu Lys Ala Phe Tyr Asp Lily Val Ala Glu Lily Leu Lys Glu 1 5 10 15
Ala Phe
<210> 6 <211> 29 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 6
Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 15 10 15
Ala Phe Arg Leu Thr Arg Lys Arg Gly Leu Lys Leu Ala 20 25
<210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 7
Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 15 10 15
Ala Phe
<210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic 6xHis tag" <4 0 0> 8
His His His His His
<210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 9
Asp Asp Asp Asp Asp Asp Asp Lys 1 5
<210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 10
Ala .Trp Arg His Pro Gin Phe Gly Gly 1 5 <210> 11
<211> 8 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note = "Description of Artificial Sequence: Synthetic Peptide" <400> 11
Trp Ser His Pro Gin Phe Glu Lys 1 5
<210> 12 <211> 5 <212> PRT <213> Human cytomegalovirus <400> 12
Arg Gly Ser Thr Trp 1 5
<210> 13 <211> 8 <212> PRT <213> Human cytomegalovirus <400> 13
Arg Gly Ser Thr Asn Gly Thr Trp 1 5
<210> 14 <211> 4 <212> PRT <213> Human cytomegalovirus <400> 14 Trp Leu Tyr Arg 1 <210> 15 <211> 7
<212> PRT <213> Human cytomegalovirus <400> 15 ·
Trp Leu Tyr Asn Gly Thr Arg 1 5
<210> 16 <211> 5 <212> PRT <213> Human cytomegalovirus <400> 16
Asn Gly Ser Thr Trp 1 5
<210> 17 <211> 5 <212> PRT <213> Human cytomegalovirus <400> 17
Arg Asn Ser Thr Trp 1 5
<210> 18 <211> 5 <212> PRT <213> Human cytomegalovirus <400> 18 '
Glu Gly Glu Thr Trp 1 5
<210> 19 <211> 5 <212> PRT <213> Human cytomegalovirus <400> 19
Glu Gly Glu Glu Trp 1 5 '<210> 20,
<211> 4 <212> PRT <213> Human Cytomegalovirus <400> 20 Arg Thr Arg1 Lys <210> 21 <211> 4 <212> PRT '<213> Human Cytomegalovirus <400> 21 Arg Gin Arg Arg 1

权利要求:
Claims (11)
[1]
A recombinant cytomegalovirus (CMV) gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a hydrophobic surface region 1, which corresponds to amino acid residues 154-160 and 236-243 of the sequence SEQ.ID.NO: 1 and which comprises a mutation that results in a glycosylation site within said region.
[2]
The recombinant gB protein of claim 1, wherein said glycosylation site is an N-glycosylation site comprising an N-X-S / T motif, wherein X is any amino acid residue except proline.
[3]
The recombinant gB protein according to claim 1 or 2, wherein said mutation of the region which corresponds to amino acid residues 154-160 and 236-243 of the sequence SEQ.ID.NOrl and which is selected from the group consisting of i) R236N, (ii) G237N, (iii) T158N, (iv) W240N and Y242S, (v) W240N and Y242T, and a combination thereof.
[4]
The recombinant gB protein according to any one of claims 1 to 3, wherein said mutation comprises an insertion of the NXS / T sequence, wherein X is any amino acid residue except proline, in the fusion 1 (FL1) which corresponds to amino acid residues 155-157 of the sequence QEQ.ID.NO: 1, the fusion loop 2 (FL2) corresponding to amino acid residues 240-242 of the sequence SEQ.ID.NO : 1, or both.
[5]
The recombinant gB protein according to any one of claims 1 to 4, further comprising a mutation which results in a reduction in the overall hydrophobicity index of said hydrophobic surface 1.
[6]
The recombinant gB protein according to any one of claims 1 to 5, wherein a mutation in a region which corresponds to amino acid residues 154-160 and 236-243 of sequence SEQ.ID.NO: 1 in 1156, H157. , R236, S238, T239, W240, Y242, or a combination thereof.
[7]
7. Cytomegalovirus (CMV) recombinant gB protein, or an immunogenic fragment thereof, wherein (i) said gB protein, or said immunogenic fragment thereof, does not comprise a transmembrane domain (TM); and (ii) said gB protein, or said immunogenic fragment thereof, comprises a mutation which results in a glycosylation site, wherein said glycosylation site is (1) within the hydrophobic surface 2 which corresponds to the amino acid residues 145-167 and 230-252 of the sequence SEQ.ID.NOrl; or (2) a residue which is at most 20 angstroms from the fusion loop 1 (FL1) which corresponds to the amino acid residues 155-157 of the sequence SEQ.ID.NOrl or the fusion loop 2 (FL2) which corresponds to amino acid residues 240-242 of the sequence SEQ.ID.NOrl.
[8]
The recombinant gB protein of claim 7, wherein said glycosylation site is an N-glycosylation site comprising an N-X-S / T motif, wherein X is any amino acid residue except proline.
[9]
9. The recombinant gB protein according to claim 7 or claim 8, wherein said mutation is between residues which correspond to residues 696 to 698 of the sequence SEQ.ID.NOrl.
[10]
10. An immunogenic composition comprising the CMV recombinant gB protein according to any one of claims 1 to 9, and optionally an adjuvant.
[11]
Immunogenic composition according to claim 10 for use in inducing an immune response against cytomegalovirus (CMV).

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同族专利:

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公开号 | 公开日

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BE1024516A9|2018-04-17|

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BE1024515A9|2018-04-04|

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BE1024515A1|2018-03-21|

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BE1024516B1|2018-03-27|

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US20180265551A1|2018-09-20|

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BE1024515B1|2018-03-27|

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BE1023390A1|2017-03-01|

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US10364273B2|2019-07-30|

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BE1024516B9|2018-04-23|

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BE1024516A1|2018-03-21|

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EP3031822A1|2016-06-15|

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ES2765484T3|2020-06-09|

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EP3230305B1|2019-11-06|

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EP3230305A2|2017-10-18|

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WO2016092460A2|2016-06-16|

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WO2016092460A3|2016-11-10|

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法律状态:
2019-10-02| MM| Lapsed because of non-payment of the annual fee|Effective date: 20181231 |

优先权:

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申请号 | 申请日 | 专利标题

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EP14196854.5A|EP3031822A1|2014-12-08|2014-12-08|Cytomegalovirus antigens|

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EP14196854.5|2014-12-08|

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