WO2017153954A1 - Polypeptide gb de cytomégalovirus humain - Google Patents

Polypeptide gb de cytomégalovirus humain Download PDF

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Publication number
WO2017153954A1
WO2017153954A1 PCT/IB2017/051401 IB2017051401W WO2017153954A1 WO 2017153954 A1 WO2017153954 A1 WO 2017153954A1 IB 2017051401 W IB2017051401 W IB 2017051401W WO 2017153954 A1 WO2017153954 A1 WO 2017153954A1
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Prior art keywords
polypeptide
seq
amino acid
acid sequence
cmv
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PCT/IB2017/051401
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English (en)
Inventor
Xiaoyuan Sherry CHI
Xinzhen Yang
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Pfizer Inc.
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Priority to EP17713783.3A priority Critical patent/EP3426676A1/fr
Priority to CA3016867A priority patent/CA3016867A1/fr
Priority to US16/082,177 priority patent/US10611800B2/en
Publication of WO2017153954A1 publication Critical patent/WO2017153954A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16171—Demonstrated in vivo effect

Definitions

  • the present invention relates to human cytomegalovirus (HCMV)
  • HCMV Human cytomegalovirus
  • HCMV Human cytomegalovirus
  • HCMV Human cytomegalovirus
  • HCMV is a double stranded DNA virus of the ⁇ - herpesvirus family.
  • HCMV is the leading cause of congenital and neonatal hearing loss resulting from vertical virus transmission following infection or reactivation of latent virus in pregnant women.
  • HCMV is a common opportunistic pathogen affecting immunosuppressed transplant patients, such as solid organ / stem cell transplant patients, AIDS patients, etc.
  • development of a vaccine against HCMV has been listed as a top priority by the Institute of Medicine, none has been licensed to date.
  • the HCMV genome encodes several envelope glycoproteins, one of which is glycoprotein B (gB).
  • Glycoprotein B is an important surface target for neutralizing antibody (nAb) response in natural infection and is required for virus entry into cells by functioning as a fusogen.
  • HCMV subunit vaccines incorporating gB have been under development. Studies have suggested that gB subunit vaccines were safe and immunogenic, though further improvements in potency and durability of protection were desirable.
  • the present invention relates to a polypeptide that includes at least one mutation in the fusion loop 1 (FL1) region of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes at least one mutation in the fusion loop 2 (FL2) region of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes at least one mutation in the fusion loop 1 (FL1) region and the fusion loop 2 (FL2) region of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes at least one mutation in the furin-like cleavage site of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes at least two mutations in the fusion loop 2 (FL2) region of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes at least two mutations in the fusion loop 1 (FL1) region and the fusion loop 2 (FL2) region of an
  • the invention relates to a polypeptide that includes at least two mutations in the furin-like cleavage site of an HCMV gB polypeptide.
  • the invention relates to a polypeptide that includes a mutation at position Y155, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions Y155 and 1156, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions Y155, 1156, and H 157, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions 1156 and H157, as compared to SEQ ID NO: 6. In one aspect, the invention relates to a polypeptide that includes a mutation at positions Y155, 1156, H157, and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions Y155 and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions Y155, H 157, and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes a mutation at positions Y155 and H157, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutation Y155G, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G and I 156H, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G, I 156H, and H157R, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations I 156H and H157R, as compared to SEQ I D NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G, I 156H, H157R, and W240A, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G and W240A, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G, H157R, and W240A, as compared to SEQ ID NO: 6.
  • the invention relates to a polypeptide that includes the mutations Y155G and H157R, as compared to SEQ ID NO: 6. In one aspect, the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 1.
  • the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 2.
  • the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 3.
  • the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 5.
  • the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 7.
  • the invention relates to a polypeptide that includes an amino acid sequence that is at least about 60% identical to SEQ ID NO: 8.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 1.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 3.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 5.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 7.
  • the invention relates to a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 8.
  • the polypeptide does not include a mutation at any one of the following positions: (i) R236, (ii) G237, (iii) T158; (iv) Y242.
  • the polypeptide does not include any one of the following mutations: (i) R236N, (ii) G237N, (iii) T158N; (iv) Y242T; (v) Y242S; (vi) Y242C.
  • the amino acid sequence SEQ ID NO: 9 is a part of the polypeptide sequence.
  • the amino acid sequence SEQ ID NO: 10 is a part of the polypeptide sequence. In one embodiment, the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10 is a part of the polypeptide sequence. In one embodiment, the polypeptide does not include a protease cleavage site. In one embodiment, the polypeptide does not include a wild-type CMV protease cleavage site. In one embodiment, the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type CMV protease cleavage site.
  • the polypeptide does not include an N- glycosylation site that includes N-X-S/T/C motif, wherein X is any amino acid residue.
  • the polypeptide does not include a modified amino acid sequence that introduces an O- linked glycosylation site.
  • the polypeptide does not include a deletion or substitution of any one of the amino acid residues selected from the group consisting of 154, 158, 159, 160, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 241 , and 242, according to the numbering of SEQ ID NO: 6.
  • the polypeptide does not include a mutation of any one of the amino acid residues: Y160, R236, S238, T239, and Y242, according to the numbering of SEQ ID NO: 6. In one embodiment, the polypeptide does not include the
  • the polypeptide does not contain an insect cell pattern of glycosylation. In one embodiment, the polypeptide is contacted with
  • EDTA ethylenediaminetetraacetic acid
  • the invention relates to a composition that includes the polypeptide described herein, and a diluent.
  • the invention relates to a composition that includes the polypeptide described herein, and an adjuvant.
  • the composition is immunogenic.
  • the composition is for use in inducing an immune response against cytomegalovirus.
  • the invention relates to a recombinant nucleic acid molecule encoding the polypeptide described herein, wherein the polypeptide undergoes a structural conformation change in response to a pH change.
  • said recombinant nucleic acid (a) is not a self-replicating RNA molecule; (b) is not an alphavirus replicon; (c) does not encode any alphavirus nonstructural proteins, such as NSP1 , NSP2, NSP3 and NSP4; (d) does not contain: an Internal Ribosomal Entry Site (IRES), such as EMCV or EV71 ; and/or (e) does not contain a viral 2A site, such as FMDV.
  • IRS Internal Ribosomal Entry Site
  • the invention relates to a method for raising antibodies using the polypeptide described herein.
  • said antibody is for use in a diagnostic assay.
  • said antibody is labelled directly or indirectly.
  • said antibody is for use in therapy.
  • the invention relates to a method of eliciting an immune response in a mammal, the method that includes administering to the mammal an effective amount of the polypeptide described herein.
  • SS represents a signal sequence;
  • D1 represents the Domain I;
  • FL represents a fusion loop;
  • RxxR mutation represents knock-out mutations of the furin-like cleavage site;
  • 6h represents an optional 6xHis tag.
  • FIG. 2 - sequence of HCMV gB705 (SEQ ID NO: 2).
  • FIG. 3 - The gB705 protein was transiently expressed in Expi293 and 293T cells after transfection respectively.
  • the target protein was secreted into culture medium, precipitated by an affinity tag, and resolved on SDS-PAGE under reducing condition.
  • the presence of the gB705 protein was visualized by a Western blot analysis using poly-clonal anti-gB serum. As shown, gB705 exhibited a single band with an apparent molecular weight about 130kDa, consistent with the design of the furin- cleavage site being knocked out by mutations.
  • FIG. 4A-B - FIG. 4A Binding of human anti-HCMV gB monoclonal antibodies to gB705 protein
  • FIG. 4B Binding of human anti-HCMV gB monoclonal antibodies to Sino gB protein (gB from Sino Biologicals, Inc.) (ELN# 00710043-0177).
  • FIG. 5 HCMV gB705 protein (SEQ ID NO: 2) was analyzed on SDS-PAGE and exhibited a single band under a fully reduced condition and showed one additional high molecular weight band under partially reduced conditions, representing a trimer.
  • FIG. 6 Sedimentation Velocity analysis of gB705 (ELN# 00708337-0110).
  • FIG. 7 Velocity sedimentation analysis of 10 mM EDTA-treated gB705 (ELN# 00708337-01 10).
  • FIG. 8A-C Sedimentation velocity analysis of EDTA-treated gB705 at pH 5.2 (FIG. 8A) and pH 8.7 (FIG. 8B); Cryo electron microscopy analysis of EDTA-treated gB705 proteins pH 5.2 (left) or pH 8.7 (right) (FIG. 8C)
  • FIG. 9A-B - FIG. 9A Far UV CD analysis;
  • FIG. 9B intrinsic fluorescence
  • FIG. 10 Near UV CD spectroscopy analysis of gB705 at pH 5.2 and pH 8.7.
  • Buffer 1 50 mM citrate, 100 mM phosphate; Buffer 2, 50 mM NaOAc; Buffer 3, 100 mM Na carbonate/bicarbonate; Buffer 4, 50 mM Na borate (ELN# 00708337-0115, 01 17).
  • FIG. 13 illustration of RhCMV gB674 (SEQ ID NO: 7).
  • SS represents a signal sequence;
  • FL represents a fusion loop;
  • 6h represents a 6xHis tag
  • FIG. 14 Graph showing anti-gB binding titers from six vaccines on week 9 after three doses of vaccination
  • FIG. 15A-C Six RhCMV(-) monkeys were immunized with placebo (A), recombinant RhCMV pentamer (B), or pentamer + RhCMV gB674 (C) intramuscularly in QS-21 adjuvant at week 0, 4 & 8. The animals were then challenged with live RhCMV (UCDE52 strain) orally for five times. Virus shedding in saliva samples were measured using qPCR (low limit of quantitation (LLOQ) is 125 DNA copy per ml) after virus challenge. Week 9 was one week after the third vaccination and one week prior to the first oral challenge. Week 15 was first sample at one week post the fifth virus challenge.
  • LLOQ low limit of quantitation
  • FIG. 16 - TEM image of negative staining of gB705 (62000X magnification) (ELN# 00702423-0124)
  • SEQ ID NO: 1 sets forth the amino acid sequence for HCMV VR1814 gB705.
  • SEQ ID NO: 2 sets forth the amino acid sequence for HCMV VR1814 gB705, without an initial methionine.
  • SEQ ID NO: 2 is identical to SEQ ID NO: 1 but for the absence of the initial methionine.
  • SEQ ID NO: 3 sets forth the amino acid sequence for a fragment of HCMV VR1814 gB705 (residues 25-705 OF SEQ ID NO: 1).
  • SEQ ID NO: 4 sets forth the amino acid sequence for the signal sequence of an HCMV VR1814 gB.
  • SEQ ID NO: 5 sets forth the amino acid sequence for HCMV VR1814 gB705 with linker
  • SEQ ID NO: 6 sets forth the amino acid sequence for GenBank Accession #
  • SEQ ID NO: 7 sets forth the amino acid sequence for an analogous construct of HCMV gB705 construct, named as RhCMV gB674, which was designed and tested in RhCMV(-) monkeys
  • SEQ ID NO: 8 sets forth the amino acid sequence for an analogous construct of HCMV gB705 construct, named as RhCMV gB674, which was designed and tested in RhCMV(-) monkeys, without an initial methionine.
  • SEQ ID NO: 8 is identical to SEQ ID NO: 7 but for the absence of the initial methionine.
  • SEQ ID NO: 9 sets forth the amino acid sequence for VVDPLPP 705 , a part of the membrane proximal region (MPR) of HCMV gB.
  • SEQ ID NO: 10 sets forth the amino acid sequence for RA 457 TKA 459 S.
  • SEQ ID NO: 11 sets forth the amino acid sequence for GenBank Accession # GU552457.1 for macacine herpesvirus 3 isolate 21252 glycoprotein B (RhUL55).
  • SEQ ID NO: 12 sets forth the amino acid sequence for the predicted fusion loop 1 region of the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], from GenBank Accession # ACZ79977.1.
  • SEQ ID NO: 13 sets forth the amino acid sequence for the predicted fusion loop 2 region of the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • SEQ ID NO: 14 sets forth the amino acid sequence for the furin-like cleavage site of the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • SEQ ID NO: 15 sets forth the amino acid sequence for the protease cleavage site of the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • CMV cytomegalovirus
  • the modified gB significantly improve the efficiency of production of a gB polypeptide; maintain and/or increase antigenicity of a gB polypeptide, as compared to the wild-type gB polypeptide; facilitate a focused immune response to gB; and reduce and/or eliminate steric occlusion of neutralizing epitopes of gB.
  • polypeptide also surprisingly demonstrated strong structural integrity and a potential to undergo structural conformational changes in tertiary structure in response to significant pH changes.
  • the structural changes were strictly pH-dependent.
  • the potential to undergo structural conformational changes in response to significant pH changes may be a newly recognized property of the HCMV gB ectodomain.
  • gB is an envelope glycoprotein B having numerous roles, one of which is the involvement in the fusion of the cytomegalovirus with host cells. It is encoded by the UL55 gene of HCMV genome.
  • the invention relates to a modified HCMV gB that is based on the VR1814 gB sequence (GenBank# ACZ79977.1 ; SEQ ID NO: 6).
  • the present invention is applicable to gB proteins originating from any CMV strain.
  • references to the numbering of amino acid residue positions of a CMV gB polypeptide as used herein are in relation to the amino acid sequence of the gB protein of SEQ ID NO: 6, from the clinical isolate VR1814 strain. Comparable amino acid positions in a gB protein of any other CMV strains can be determined by those of ordinary skill in the art. Accordingly, the term "CMV gB protein" or "HCMV gB protein” as used herein is to be understood as a HCMV gB protein from any human HCMV strain (not limited to the VR1814 strain). The actual residue position number may need to be adjusted for gB proteins from other human CMV strains depending on the actual sequence alignment.
  • the modified HCMV gB polypeptide includes amino acids
  • the modified HCMV gB polypeptide includes at least one mutation in the fusion loop 1 region of an HCMV gB
  • fusion loop 1 region of a wild-type VR1814 gB polypeptide The precise boundaries of the fusion loop 1 region of a wild-type VR1814 gB polypeptide are currently not fully defined, however, the predicted sequence of the fusion loop 1 region of a wild-type VR1814 gB polypeptide is Y 153 AYIHT 158 (SEQ ID NO: 12).
  • the modified HCMV gB polypeptide includes at least one mutation in the fusion loop 2 region of an HCMV gB polypeptide.
  • the precise boundaries of the fusion loop 2 region of a wild-type VR1814 gB polypeptide are currently not fully defined, however, the predicted sequence of the fusion loop 2 region of a wild-type VR1814 gB polypeptide is G 237 STWLYRE 244 (SEQ ID NO: 13).
  • the modified HCMV gB polypeptide includes at least one mutation in the fusion loop 1 region and in the fusion loop 2 region of an HCMV gB polypeptide. In another preferred embodiment, the modified HCMV gB polypeptide includes a total of at most four mutations in the fusion loop 1 region and in the fusion loop 2 region of an HCMV gB polypeptide.
  • the modified HCMV gB polypeptide includes at least one mutation in the furin-like cleavage site of an HCMV gB polypeptide. In a preferred embodiment, the modified HCMV gB polypeptide includes at most two mutations in the furin-like cleavage site of an HCMV gB polypeptide.
  • the modified HCMV gB polypeptide includes at least one mutation in the fusion loop 1 region, at least one mutation in the fusion loop 2 region, and at least one mutation in the furin-like cleavage site of an HCMV gB polypeptide.
  • the sequence of the furin-like cleavage site of a wild-type VR1814 gB polypeptide is R 456 TKR 459 (SEQ ID NO: 14).
  • the modified HCMV gB polypeptide includes at least two mutations in the fusion loop 1 region, at least one mutation in the fusion loop 2 region, and at least one mutation in the furin-like cleavage site of an HCMV gB polypeptide.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at position Y155, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155 and 1156, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155, 1156, and H157, as compared to SEQ ID NO: 6. In one aspect, the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions 1156 and H157, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155, 1156, H157, and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155 and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155, H157, and W240, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having a mutation at positions Y155 and H157, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutation Y155G, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G and I 156H, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G, I 156H, and H157R, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations I 156H and H157R, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G, I156H, H 157R, and W240A, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G and W240A, as compared to SEQ ID NO: 6. In one aspect, the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G, H157R, and W240A, as compared to SEQ ID NO: 6.
  • the invention relates to a CMV gB polypeptide or immunogenic fragment thereof having the mutations Y155G and H157R, as compared to SEQ ID NO: 6.
  • the gB polypeptide does not include a mutation at any one of the following positions, individually or in combination: (i) R236, (ii) G237, (iii) T158; (iv) Y242.
  • the gB polypeptide does not include any one of the following mutations, individually or in combination: (i) R236N, (ii) G237N, (iii) T158N; (iv) Y242T; (v) Y242S; (vi) Y242C.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 includes the amino acid sequence VVDPLPP 705 (SEQ ID NO: 9) as a part of the polypeptide sequence.
  • sequence set forth in SEQ ID NO: 9 may be highly hydrophobic and structurally stiff, which facilitates immunogenicity of the polypeptide. Accordingly, in one embodiment, the sequence set forth in SEQ ID NO: 9 is hydrophobic.
  • polypeptide having at least about 60%, 65%,
  • SEQ ID NO: 1 includes the amino acid sequence RA 457 TKA 459 S (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the sequence set forth in SEQ ID NO: 10 may prevent cleavage of the gB polypeptide by cellular proteases.
  • the polypeptide of the invention does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site, e.g., RTKR 459 (SEQ ID NO: 15) for the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • the polypeptide does not include a non- naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • the polypeptide does not include a protease cleavage site at or encompassing residue R 459 at position 459 of a human CMV gB, according to the numbering of SEQ ID NO: 6, wherein the protease cleavage site includes at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, or 3 amino acid residues in length.
  • the polypeptide does not include a protease cleavage site encompassing residue R 459 at position 459 of a human CMV gB, according to the numbering of SEQ I D NO: 6, wherein the protease cleavage site includes at most 6, more preferably at most 5, and most preferably at most 4 amino acid residues in length.
  • polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 includes both the amino acid sequence VVDPLPP 705 (SEQ ID NO: 9) and the amino acid sequence RA 457 TKA 459 S (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.
  • polypeptide having at least about 60%, 65%, 70%,
  • SEQ ID NO: 2 includes the amino acid sequence VVDPLPP 705 (SEQ ID NO: 9) as a part of the polypeptide sequence.
  • polypeptide having at least about 60%, 65%,
  • SEQ ID NO: 2 includes the amino acid sequence RA 457 TKA 459 S (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the polypeptide does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site, e.g., RTKR 459 (SEQ ID NO: 15) for the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • the polypeptide does not include a non-naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • polypeptide having at least about 60%, 65%, 70%,
  • SEQ ID NO: 3 includes the amino acid sequence VVDPLPP 705 (SEQ ID NO: 9) as a part of the polypeptide sequence.
  • polypeptide having at least about 60%, 65%,
  • SEQ ID NO: 3 includes the amino acid sequence RA 457 TKA 459 S (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the polypeptide does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site, e.g., RTKR 459 (SEQ ID NO: 15) for the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • the polypeptide does not include a non-naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.
  • polypeptide having at least about 60%, 65%, 70%,
  • SEQ ID NO: 5 includes the amino acid sequence VVDPLPP 705 (SEQ ID NO: 9) as a part of the polypeptide sequence.
  • polypeptide having at least about 60%, 65%,
  • sequence identity to SEQ ID NO: 5, includes the amino acid sequence RA 457 TKA 459 S (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the polypeptide does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site, e.g., RTKR 459 (SEQ ID NO: 15) for the envelope glycoprotein B of strain VR1814 [Human beta-herpesvirus 5], based on GenBank Accession # ACZ79977.1.
  • the polypeptide does not include a non-naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the polypeptide does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • the invention relates to a polypeptide having the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • polypeptide having at least about 60%, 65%, 70%,
  • SEQ ID NO: 8 includes the amino acid sequence VVDPLPP (SEQ ID NO: 9) as a part of the polypeptide sequence.
  • polypeptide having at least about 60%, 65%,
  • sequence identity to SEQ ID NO: 8 includes the amino acid sequence RATKAS (SEQ ID NO: 10) as a part of the polypeptide sequence.
  • the polypeptide does not include a protease cleavage site.
  • the polypeptide does not include a wild-type, natural CMV protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site.
  • the polypeptide does not include a non-naturally occurring protease cleavage site that replaces the wild-type, natural CMV protease cleavage site.
  • CMV gB may be referred to as a glycoprotein, this nomenclature should not be taken to mean that the polypeptides described herein must be glycosylated when used with the invention. In some embodiments of the invention, the modified gB polypeptide is not glycosylated.
  • the gB polypeptide does not include a glycosylation site in the fusion loop regions of the gB polypeptide. That is, the gB polypeptide does not include a glycan moiety attached to the gB polypeptide within the fusion loop 1 region or within the fusion loop 2 region.
  • the fusion loop 1 region of the VR1814 (SEQ ID NO: 6) is located at residues 153-158.
  • the fusion loop 2 region of the VR1814 is located at residues 237-244.
  • the gB polypeptide does not include a modified amino acid sequence that introduces an N-linked glycosylation site.
  • the modified gB polypeptide does not include an N- glycosylation site comprising N- X-S/T/C motif, wherein X is any amino acid residue.
  • the modified gB polypeptide does not include a modified amino acid sequence that introduces an O-linked glycosylation site.
  • the modified gB polypeptide does not include a carbohydrate moiety linked to the hydroxyl oxygen of serine and threonine.
  • the modified gB polypeptide does not include an O- linked glycosylation at tyrosine, 5-hydroxylysine, or 4-hydroxyproline.
  • the modified gB polypeptide does not include a deletion or substitution of any one of the amino acid residues selected from the group consisting of 154, 158, 159, 160, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 241 , and 242, according to the numbering of SEQ ID NO: 6, or any combination thereof.
  • the gB polypeptide does not include a mutation of any one of the amino acid residues selected from the group consisting of Y160, R236, S238, T239, and Y242, according to the numbering of SEQ ID NO: 6, or any combination thereof.
  • cytoplasmic tail of HCMV gB may help to focus the mammal's immune response to the ectodomains of gB. Additionally, deletion of the cytoplasmic tail may reduce steric occlusion and facilitate access of antibodies to important neutralizing epitopes on gB.
  • the native C-terminal cytoplasmic domain of the gB polypeptide (e.g., 100% of the amino acids of the cytoplasmic domain) is deleted.
  • the native C-terminal cytoplasmic domain is not deleted to a varying extent. That is, no less than 100% of the native C-terminal cytoplasmic domain is deleted.
  • the cytoplasmic tail of HCMV gB refers the amino acid sequence located at positions 771-905 of SEQ ID NO: 6.
  • the invention relates to nucleic acids that encode a gB polypeptide of the invention.
  • the recombinant nucleic acid molecule (a) is not a self- replicating RNA molecule; (b) is not an alphavirus replicon; (c) does not encode any alphavirus nonstructural proteins, such as NSP 1 , NSP2, NSP3 and NSP4; (d) does not contain: an Internal Ribosomal Entry Site (IRES), such as EIV1CV or EV71 ; and/or (e) does not contain a viral 2A site, such as FMDV.
  • IRS Internal Ribosomal Entry Site
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the invention relates to a nucleic acid sequence that encodes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • the invention also provides a host cell comprising the nucleic acids described herein.
  • the nucleic acids can express a gB polypeptide.
  • said gB polypeptide forms a
  • the gB polypeptide of the invention does not form a dimeric association of two trimers.
  • the gB polypeptide of the invention does not form a higher order association of trimers.
  • the monodispersed trimer can be secreted from the host cell.
  • the host cells are mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster).
  • mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vera cells, HeLa cells, PERC.6 cells
  • ECACC deposit number 96022940 Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160), Madin-Darby bovine kidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cells, such as BHK21 -F, HKCC cells, and the like.
  • MDCK Madin-Darby bovine kidney
  • MDCK Madin-Darby canine kidney
  • BHK baby hamster kidney
  • the host cell is EXPI293FTM (ThermoFisher Scientific) human cells, derived from the 293 cell line.
  • the host cell is a CHO cell.
  • the polynucleotide encoding the gB polypeptide described herein is stably integrated into the genome of the CHO cell.
  • CHO cell lines are also available from European Collection of Cell Cultures (ECACC), or American Type Culture Collection (ATCC), such as CHO cell lines hCBE1 1 (ATCC® PTA-3357TM), E77.4 (ATCC® PTA-3765TM), hLT-B: R-hG1 CHO #14 (ATCC® CRL-1 1965TM), MOR-CHO- MORAb-003-RCB (ATCC® PTA- 7552TM), AQ.C2 clone 1 1 B (ATCC® PTA-3274TM), AQ.C2 clone 1 1 B (ATCC® PTA-3274TM), hsAQC2 in CHO-DG44 (ATCC® PTA-3356TM), xrs5 (ATCC® CRL- 2348TM), CHO-K1 (ATCC® CCL-61TM), Led [originally named Pro-5WgaRI3C] (ATCC® CRL-1735TM), Pro-5 (ATCC® CRL-1781TM), A
  • CHO-ICAM-1 ATCC® CRL-2093TM
  • pgsB-618 ATCC® CRL-2241TM
  • Any one of these CHO cell lines may be used.
  • CHO cell lines include, e.g., FREESTYLETM CHO-S Cells and Flp-lnTM-CHO Cell Line from Life Technologies.
  • Alternative suitable host cells may also 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, non- human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster)), avian cells (e.g., chicken, duck, and geese), bacteria (e.g., E. coli, Bacillus subtilis, and
  • Streptococcus spp. yeast cells (e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica), Tetrahymena cells (e.g., Tetrahymena thermophila) or combinations thereof.
  • yeast cells e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica
  • Tetrahymena cells e.g., Tetrahymena thermophila
  • Suitable insect cell expression systems such as baculovirus systems, are known to those of skill in the art. Materials and methods for baculo virus/insect cell expression systems are commercially available. Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (Invitrogen)).
  • the host cell is a mammalian cell, not an insect cell. Accordingly in a preferred embodiment, the polypeptide does not have an insect cell pattern of glycosylation.
  • the recombinant nucleic acids are codon optimized for expression in a selected prokaryotic or eukaryotic host cell.
  • the nucleic acids can be incorporated into a vector, such as a prokaryotic or a eukaryotic expression vector.
  • exemplary vectors include plasmids that are able to replicate autonomously or to be replicated in a host cell.
  • Typical expression vectors contain suitable promoters, enhancers, and terminators that are useful for regulation of the expression of the coding sequence(s) in the expression construct.
  • the vectors may also comprise selection markers to provide a phenotypic trait for selection of transformed host cells (such as conferring resistance to antibiotics such as ampicillin or neomycin).
  • CMV cytomegalovirus
  • the gB polypeptide described herein is purified.
  • the gB polypeptide can be purified using any suitable methods, such as HPLC, various types of chromatography (such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion), electrophoresis, density gradient centrifugation, solvent extraction, or the like.
  • the gB polypeptide may be further purified, as required, so as to remove substantially any polypeptides which are also secreted in the medium or result from lysis of host cells, so as to provide a product which is at least substantially free of host debris, e.g., polypeptides, lipids and polysaccharides.
  • a purified protein or polypeptide is a protein or polypeptide which is recombinantly or synthetically produced, or produced by its natural host, and has been isolated from other components of the recombinant or synthetic production system or natural host such that the amount of the protein relative to other macromolecular components present in a composition is substantially higher than that present in a crude preparation.
  • a purified protein will be at least about 50% homogeneous and more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogeneous.
  • the process of purifying the modified gB polypeptide of the invention allows for production of the polypeptide at a purity of >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94% or >95% of total protein by mass, as determined by gel electrophoresis.
  • These high levels of purity make the modified gB polypeptide suitable for use as an immunogen in diagnostic applications or as an antigen in immunogenic compositions.
  • the immunogenic polypeptides prepared as described above may be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g. , mouse, rabbit, goat, guinea pig, horse, etc.) is immunized with an immunogenic polypeptide bearing a CMV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a CMV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
  • Monoclonal antibodies directed against CMV epitopes can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against CMV epitopes can be screened for various properties; i.e., for isotype, epitope affinity, etc.
  • Antibodies both monoclonal and polyclonal, which are directed against CMV epitopes are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Both the recombinant polypeptides which react immunologically with serum containing CMV antibodies, and the antibodies raised against these recombinant polypeptides, are useful in immunoassays to detect the presence of CMV antibodies, or the presence of the virus, in biological samples, including for example, blood or serum samples.
  • Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art.
  • the immunoassay may utilize the polypeptide having the sequence set forth in SEQ ID NO: 2.
  • the immunoassay may use a combination of viral antigens derived from the gB polypeptides described herein.
  • Protocols may be based, for example, upon competition, or direct reaction, or may be sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known;
  • assays which utilize biotin and avidin examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
  • Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the recombinant polypeptides of the invention containing CMV epitopes or antibodies directed against epitopes in suitable containers, along with the remaining reagents and materials required for the conduct of the assay, as well as a suitable set of assay instructions.
  • the polynucleotide probes can also be packaged into diagnostic kits.
  • Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.
  • the invention relates to compositions and methods of treatment using the cytomegalovirus gB polypeptide described herein, or a nucleic acid encoding such gB polypeptide described herein.
  • the polypeptide of the invention can be delivered directly as a component of an immunogenic composition.
  • nucleic acids that encode the gB polypeptide of the invention can be administered to produce the CMV protein or immunogenic fragment in vivo.
  • Certain preferred embodiments such as protein formulations, recombinant nucleic acids (e.g., DNA, RNA, self-replicating RNA, or any variation thereof) and viral vectors (e.g., live, single-round, non-replicative assembled virions, or otherwise virus-like particles, or alphavirus VRP) that contain sequences encoding gB polypeptides are further described herein and may be included in the composition.
  • recombinant nucleic acids e.g., DNA, RNA, self-replicating RNA, or any variation thereof
  • viral vectors e.g., live, single-round, non-replicative assembled virions, or otherwise virus-like particles, or alphavirus VRP
  • the invention provides an immunogenic composition
  • immunogenic composition can include additional CMV proteins, such as gO, gH, gl_, pUL128, pUL130, pUL131 , an immunogenic fragment thereof, or a combination thereof.
  • the gB polypeptide can be combined with CMV pentameric complex comprising: gH or a pentamer-forming fragment thereof, gl_ or a pentamer- forming fragment thereof, pUL128 or a pentamer-forming fragment thereof, pUL130 or a pentamer-forming fragment thereof, and pUL131 or a pentamer-forming fragment thereof.
  • the gB polypeptide of the invention can also be combined with CMV trimeric complex comprising: gH or a trimer-forming fragment thereof, gl_ or a trimer-forming fragment thereof, and gO or a trimer- forming fragment thereof.
  • the immunogenic composition may include an adjuvant.
  • adjuvants to enhance 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 muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No.
  • WO 90/14837 containing 5% Squalene, 0.5% TWEEN 80, and 0.5% Span 85 formulated into submicron particles using a microfluidizer
  • SAF containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121 , and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion
  • RAS RIBITM adjuvant system
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as QS-21 , STIMULONTM (Cambridge Bioscience, Worcester, Mass.)
  • Each of the immunogenic compositions discussed herein may be used alone or in combination with one or more other antigens, the latter either from the same viral pathogen or from another pathogenic source or sources. These compositions may be used for prophylactic (to prevent infection) or therapeutic (to treat disease after infection) purposes.
  • the composition may include a "pharmaceutically acceptable carrier,” which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as adjuvants.
  • the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and etc. pathogens.
  • the composition includes a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • a diluent such as water, saline, glycerol, ethanol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • compositions described herein may include an immunologically effective amount of the polypeptide, as well as any other of the above-mentioned
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for eliciting an immune response.
  • the immune response elicited may be sufficient, for example, for treatment and/or prevention and/or reduction in incidence of illness, infection or disease.
  • This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the composition may be administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the immunogenic composition may be administered in conjunction with other immunoregulatory agents.
  • the invention provides a method of eliciting an immune response against cytomegalovirus (CMV), comprising administering to a subject in need thereof an immunologically effective amount of a modified CMV gB polypeptide and/or an immunogenic composition described herein, which comprises the proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), or VRPs as described above.
  • CMV cytomegalovirus
  • the immune response comprises the production of neutralizing antibodies against CMV.
  • the immune response can comprise a humoral immune response, a cell- mediated immune response, or both.
  • an immune response is induced against each delivered CMV protein.
  • a cell-mediated immune response can comprise a Helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both.
  • the immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies.
  • Neutralizing antibodies block viral infection of cells. CMV infects epithelial cells and also fibroblast cells. In some embodiments the immune response reduces or prevents infection of both cell types. Neutralizing antibody responses can be complement-dependent or complement- independent. In some embodiments the neutralizing antibody response is complement- independent. In some embodiments the neutralizing antibody response is cross-neutralizing; i.e., an antibody generated against an administered composition neutralizes a CMV virus of a strain other than the strain used in the composition.
  • the modified CMV gB polypeptide and/or immunogenic composition described herein may also elicit an effective immune response to reduce the likelihood of a CMV infection of a non-infected mammal, or to reduce symptoms in an infected mammal, e.g., reduce the number of outbreaks, CMV shedding, and risk of spreading the virus to other mammals.
  • the modified CMV gB polypeptide and/or immunogenic composition described herein reduces viral shedding in a mammal.
  • viral shedding is used herein according to its plain ordinary meaning in medicine and virology and refers to the production and release of virus from an infected cell.
  • the virus is released from a cell of a mammal.
  • virus is released into the environment from an infected mammal.
  • the virus is released from a cell within a mammal.
  • the invention relates to a method for reducing CMV viral shedding in a mammal.
  • the method includes administering the modified CMV gB polypeptide and/or immunogenic composition described herein to the mammal that is infected with or is at risk of a CMV infection.
  • the reduction in CMV viral shedding in a mammal is as compared to the viral shedding in mammals that were not administered the modified CMV gB.
  • the reduction in CMV viral shedding in a mammal is as compared to the viral shedding following an administration of a CMV pentamer alone or following an administration of a CMV pentamer in the absence of the modified CMV gB.
  • the challenge cytomegalovirus strain is a human CMV strain. In one embodiment, the challenge cytomegalovirus strain is homologous to the CMV strain from which the modified CMV gB polypeptide is derived. In another embodiment, the challenge cytomegalovirus strain is homologous to the VR1814 CMV strain.
  • the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the CMV strain from which the modified CMV gB polypeptide is derived.
  • the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the VR1814 CMV strain.
  • the challenge cytomegalovirus strain is the VR1814 CMV strain.
  • the challenge cytomegalovirus strain is a rhesus CMV strain homologous to the macacine herpesvirus 3 isolate 21252 CMV strain.
  • the challenge cytomegalovirus strain is the macacine herpesvirus 3 isolate 21252 CMV strain.
  • a useful measure of antibody potency in the art is "50% neutralization titer.”
  • Another useful measure of antibody potency is any one of the following: a "60% neutralization titer”; a “70% neutralization titer”; a “80% neutralization titer”; and a “90% neutralization titer.”
  • serum from immunized animals is diluted to assess how dilute serum can be yet retain the ability to block entry of 50% of infectious viruses into cells.
  • a titer of 700 means that serum retained the ability to neutralize 50% of infectious virus after being diluted 700-fold.
  • higher titers indicate more potent neutralizing antibody responses.
  • this titer is in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000.
  • the 50%, 60%, 70%, 80%, or 90% neutralization titer range can have an upper limit of about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 1 1000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000.
  • the 50% neutralization titer can be about 3000 to about 6500.
  • "About" means plus or minus 10% of the recited value. Neutralization titer 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 VRPs to an individual, typically a mammal, including a human.
  • the immune response induced is a protective immune response, i.e., the response reduces the risk or severity of or clinical consequences of a CMV infection.
  • Stimulating a protective immune response is particularly desirable in some populations particularly at risk from CMV infection and disease.
  • at-risk populations include solid organ transplant (SOT) patients, bone marrow transplant patients, and hematopoietic stem cell transplant (HSCT) patients.
  • VRPs can be administered to a transplant donor pre-transplant, or a transplant recipient pre- and/or post-transplant. Because vertical transmission from mother to child is a common source of infecting infants, administering VRPs to a woman who is pregnant or can become pregnant is particularly useful.
  • compositions can be administered intramuscularly, intraperitoneally, subcutaneously, or transdermally.
  • Some embodiments will be administered through an intra-mucosal route such as intra-orally, intra- nasally, intra-vaginally, and intra-rectally.
  • compositions can be administered according to any suitable schedule.
  • CMV cytomegalovirus
  • Pfizer's expression construct of HCMV gB705 (SEQ ID NO: 1) is based on the
  • FIG. 1 shows the sequence of HCMV gB705 (SEQ ID NO: 2)
  • the initial assessment of gB705 expression was carried out by transfecting the construct DNA into HEK293T or Expi293F cells using Effectene Transfection Reagent (Qiagen) or Expifetamin 293 Transfection kit (Gibco), respectively.
  • the culture supernatants were harvested -48 hours after transfection.
  • the gB705 protein was pulled down from the culture supernatant of transfected cells using a
  • MAGNEHISTM Protein Purification System Promega
  • NUPAGE® NOVEX® 4-12% Bis-Tris protein gel
  • Presence of the target proteins was detected by Western blot using anti-HCMV polyclonal antibody (CytoGam).
  • CytoGam anti-HCMV polyclonal antibody
  • the result showed that recombinant gB705 existed as a single protein band with molecular weight about 120 kDa (FIG. 3). This apparent molecular weight is higher than that predicted from the peptide sequence of residues 25-705 [of SEQ ID NO: 1] from the mature gB705 polypeptide, indicating that the gB705 protein is heavily glycosylated.
  • the gB705 protein is expressed efficiently as a
  • EXAMPLE 3 Recombinant gB705 contains intact neutralizing epitopes recognized by human anti-HCMV gB monoclonal antibodies (mAbs)
  • gB705 SEQ ID NO: 2. Briefly, the culture supernatants from gB705 transfected Expi293F cells were added on a pre-blocked HISGRABTM Nickel Coated Plate (Pierce) and incubated for 1 hour at room temperature. After wash, serial diluted antibody solutions were added to the plate and incubated for 1 hour at room temperature, followed by addition of HRP-conjugated anti-human IgG secondary antibody.
  • Recombinant gB705 binds to three anti-AD5 mAbs including 2B11 , 5F1 and 4H9 (each described in Table 1 of US patent publication no. 20160280770 and Table 1 of WlPO publication no. WO2010007533), whereas recombinant gB proteins from a commercial source (Sino gB, from Sino Biologicals, Inc.) did not (FIG. 4B).
  • the 2B1 1 mAb has a sequence set forth in SEQ ID NO: 367 for the heavy chain and SEQ ID NO: 368 for the light chain; 5F1 mAb has a sequence set forth in SEQ ID NO: 290 for the heavy chain and SEQ ID NO: 291 for the light chain; and 4H9 mAb has a sequence set forth in SEQ ID NO: 308 for the heavy chain and SEQ ID NO: 309 for the light chain, as disclosed in US patent publication no.
  • FIG. 4A Binding of human anti-HCMV gB monoclonal antibodies to gB705 protein. Culture supernatants from gB705 transfected Expi293F cells were captured on a pre-blocked HISGRABTM Nickel Coated Plate (Pierce) and incubated serially diluted monoclonal antibodies followed by detection with HRP conjugated anti- human Ig secondary antibody (ELN# 00710043-0177).
  • FIG. 4B Binding of human anti-HCMV gB monoclonal antibodies to Sino gB protein (gB from Sino Biologicals, Inc.) (ELN# 00710043-0177).
  • the gB705 construct was transiently transfected into 1.8 liters of Expi293F cells and about 95 mg of protein was purified from the conditioned media collected on day 4 through a series of processes of diafiltration, Ni-sepharose and size exclusion chromatography.
  • the protein was analyzed on SDS-PAGE and exhibited a single band under reducing condition, as detected by Coomassie blue staining (FIG. 5). Under non-reducing conditions, there were higher molecular weight bands that may be corresponding to a trimeric form of the protein.
  • FIG. 5 shows an SDS-PAGE of gB705 under reducing and non-reducing conditions (ELN#00709755-0086).
  • EXAMPLE 5 Structural characterization of recombinant gB705 expressed in Expi293F cells: Oligomer formation and stability
  • Postfusion gB protein is in a trimer form. To characterize whether the gB705 forms higher order of oligomers, a series of experiments were carried out as summarized below.
  • Recombinant gB705 protein was subjected to electron microscopy analysis after negative staining.
  • the image shows that the protein forms a singular trimer as well as larger oligomeric complexes (that is, associations between trimers).
  • the singular trimer of gB705 exhibited strong similarity in general morphology with the postfusion gB structure as published in Burke et al., PLoS Pathog. 2015 Oct; 11 (10).
  • gB706 a modified gB protein, termed "gB706,” which does not include the inventive combination of fusion loop mutations, forms a dimer and trimer of trimers, as discussed in in Sharma et al., Virology.
  • gB706 protein of Sharma et al. is described as encoding the mature ectodomain of HCMV (strain AD169) gB, residues 25-706, with a signal sequence.
  • Sharma et al. further describe gB706 as lacking the hydrophobic membrane- proximal region, the transmembrane region, and the cytoplasmic domain of gB.
  • the purified gB705 protein (SEQ ID NO: 2) was also subjected to velocity analytical ultracentrifugation analysis and the result showed that the protein formed multiple peaks corresponding to the sizes of singular trimer or larger / high-order complexes (FIG. 6).
  • FIG. 6 velocity analytical ultracentrifugation analysis of gB705 (ELN# 00708337-01 10). Transforming recombinant gB705 to homogeneous singular trimer by treatment of ethylenediaminetetraacetic acid (EDTA)
  • EDTA ethylenediaminetetraacetic acid
  • FIG. 7 shows Velocity sedimentation analysis of 10 mM EDTA-treated gB705 ( ELN# 00708337-01 10).
  • oligomer formation of gB705 trimer may be a result of the existence of the 6xHis affinity tag, rather than due to some unknown intrinsic properties of gB705 trimer.
  • EXAMPLE 6 Structural characterization of gB705 trimer at different pH conditions:
  • EDTA-treated gB705 protein was dialyzed into buffers at pH 5.2 and pH 8.7 and subjected to a series of biophysical characterization experiments to determine the changes, if any, in the secondary, tertiary and quaternary structure or hydrodynamic properties.
  • FIG. 8A-B Sedimentation velocity analysis of EDTA-treated gB705 at pH 5.2 (A) and pH 8.7 (B).
  • FIG. 9A Far UV CD
  • FIG. 9B intrinsic fluorescence spectroscopy
  • FIG. 11 A-B DSC analysis of gB705 at (FIG. 11 A) pH 5.2 and (FIG. 11 B) pH 8.7. At pH 8.7, the observed melting curve (in block line) was fit to the 4-state unfolding model and resolved to a three-phase transition pattern (in red lines) using the software provided by the DSC manufacturer. (ELN# 00708337-01 15, 0117).
  • ANS is a fluorescent dye that binds to hydrophobic surfaces of protein, resulting in increased fluorescence intensity and shift in the emission maximum towards lower wavelengths. It was observed that ANS fluorescence intensity of gB705 was increased and its maximal emission was shifted to shorter wavelength of UV at pH 5.2, relative to those at pH 8.7 or pH 7.4 (FIG. 12A). These data are consistent with that hydrophobic surfaces of gB705 trimer are more exposed under acidic pH than under neutral or basic pH conditions.
  • FIG. 12A-C (A) ANS binding of gB705 at pH 5.2, pH 7.4 and pH 8.7 (ELN#
  • HCMV gB705 protein was produced.
  • the protein contains the ectodomain of the VR1814 gB and mutations in the fusion loops and furin cleavage site. This protein may be in the postfusion conformation under neutral pH.
  • the protein forms various oligomers after purification but can be transformed into homogenous singular trimer upon EDTA treatment. No significant difference was observed in the secondary structure of EDTA-treated gB705 at pH 5.2 and pH 8.7, based on assays of far-UV CD spectroscopy, intrinsic fluorescence as well as sedimentation velocity analysis. However, significant differences in conformation of gB705 trimer are observed with various assays.
  • the gB705 protein exhibits a pH-dependent transition of conformation, which is a unique property and is biologically important.
  • a non-postfusion conformation of gB (for example, gB705 at pH 8.7) may be a presented in a specific chemical/physical condition or by stabilizing its conformation with treatment of crosslinking agents.
  • Such an immunogen may assume conformation state(s) that have a potential to elicit more robust nAb response than for example, recombinant gB protein in the postfusion conformation.
  • the pH-dependent transition of tertiary conformation of the gB705 trimer may be useful for improving solubility of the gB immunogen in an immunogenic composition, for improving stability of the
  • immunogen for improving binding of the immunogen to aluminum or other adjuvants, and/or for improving immunization of a vaccine comprising the
  • immunogen as compared, for example, to a recombinant gB protein that does not exhibit a pH-dependent transition of tertiary conformation, and/or as compared to a recombinant gB protein that does not include the following mutations Y155G, I 156H, H157R, and W240A.
  • MREFNSYKQRVKYVEDKVVDPLPP (SEQ ID NO: 2) >HCMV VR1814 gB705 (residues 25-705 OF SEQ ID NO: 1 )
  • PLPPGSG (SEQ ID NO: 5) >ACZ79977.1 envelope glycoprotein B [Human betaherpes virus 5]
  • CMV is a species-specific virus. Just has humans are infected by HCMV, rhesus macaques are infected by rhCMV, which has a biology similar to HCMV. This provides an animal model system for testing CMV vaccine inventions and technologies in ways that may be impractical or unethical in humans, such as certain infectious challenge experiments. Therefore, rhCMV analogues of HCMV antigens may be made to allow experimental investigation. RhCMV has a gB with a structure and function very similar to those of HCMV gB. A satisfactory correlation exists between effects in rhesus macaques relating to CMV and effects ultimately observed in humans.
  • RhCMV gB674 an analogous construct of HCMV gB705 construct, named as RhCMV gB674, was designed and tested in RhCMV(-) monkeys.
  • RhCMV gB674 Design Following the same design of HCMV gB705, Pfizer's expression construct of RhCMV gB674 (SEQ ID NO: 7) was based on gB sequence of RhCMV strain UCD52, which contains amino acids 1-674 of the ectodomains and a 6xHis affinity tag at the C- terminus.
  • the gB674 protein contains three mutations (F128G, I 129H, W213A) in the predicted fusion loops, because a fourth change in HCMV gB705 is pre-existing in the natural UCD52 sequence.
  • mutations R429A/R430T/R432A were introduced to totally inactivate the furin-like cleavage site, as illustrated.
  • the gB674 protein was transiently expressed in Expi293 cells and purified via the C-terminal 6xHis tag. On SDS-PAGE, the protein exhibited a single band under reducing (R) conditions, demonstrating the lack of protease cleavage as designed and >98% purity. Some oligomeric forms were shown under a non-reducing condition. See FIG. 17A.
  • the gB674 protein was treated with EDTA after purification and analyzed on Superose-6 column. As observed with HCMV gB705, gB674 protein was eluted as a major peak, consistent with a homogenous individual trimer. See FIG. 17B.
  • RhCMV(-) rhesus monkeys were screened and proven as seronegative for anti- gB by an ELISA test (data not shown).
  • Six animals were immunized intramuscularly with 100 ug of gB674 per dose and 50 ⁇ g of RhCMV pentamer in 50 ug of QS-21 adjuvant at week 0, 4 and 8.
  • a group of six animals were injected with a placebo regimen lacking only the gB protein, as controls.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigens.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigens.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigens.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigens.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigens.
  • RhCMV pentamer complex containing the gH, gL, pUL128, pUL130, and pUL131 RhCMV antigen
  • Virus infection was monitored by the presence of viremia and by virus shedding in saliva and urine compartments. The presence of virus in these target samples was quantified by a qPCR assay with a limit of quantitation of 125 DNA copies/ml.
  • Fig 15 panel A five oral challenges led to virus shedding in saliva in 5 of 6 animals in the control group. Week 9 and week 15 are one week prior to the first virus challenge and one week after the fifth virus challenge, respectively. The same five control animals had viremia and high level of virus shedding in urine (data not shown).
  • a polypeptide comprising the mutations Y155G, H157R, and W240A, as compared to SEQ ID NO: 6.
  • C24 A polypeptide comprising an amino acid sequence that is at least about 60% identical to SEQ ID NO: 1.
  • C25 A polypeptide comprising an amino acid sequence that is at least about 60% identical to SEQ ID NO: 2.
  • a polypeptide comprising an amino acid sequence that is at least about 60% identical to SEQ ID NO: 5.
  • polypeptide does not comprise a mutation at any one of the following positions: (i) R236, (ii) G237, (iii) T158; (iv) Y242.
  • polypeptide does not comprise any one of the following mutations: (i) R236N, (ii) G237N, (iii) T158N; (iv) Y242T; (v) Y242S; (vi) Y242C.
  • polypeptide does not comprise a protease cleavage site.
  • polypeptide does not comprise a wild-type CMV protease cleavage site.
  • polypeptide does not comprise a non-naturally occurring protease cleavage site that replaces the wild-type CMV protease cleavage site.
  • polypeptide does not comprise an N- glycosylation site comprising N-X-S/T/C motif, wherein X is any amino acid residue.
  • polypeptide does not comprise a modified amino acid sequence that introduces an O-linked glycosylation site.
  • polypeptide does not include a deletion or substitution of any one of the amino acid residues selected from the group consisting of 154, 158, 159, 160, 230, 231 ,
  • polypeptide does not include a mutation of any one of the amino acid residues: Y160, R236, S238, T239, and Y242, according to the numbering of SEQ ID NO: 6.
  • polypeptide does not include the cytoplasmic tail of HCMV gB.
  • polypeptide does not contain an insect cell pattern of glycosylation.
  • polypeptide is contacted with ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • polypeptide undergoes a structural conformation change in response to a pH change.
  • polypeptide according to clause C51 wherein the polypeptide exhibits improved solubility or stability, as compared to a recombinant gB protein that does not include the following mutations Y155G, I156H, H157R, and W240A.
  • composition comprising the polypeptide according to any one of clauses C1-C51 , and a diluent.
  • composition comprising the polypeptide according to any one of clauses C1-C51 , and an adjuvant.
  • C55 The composition according to any one of clauses C53-C54, wherein the composition is immunogenic.
  • I RES Internal Ribosomal Entry Site
  • C62 The antibody according to clause C59, wherein said antibody is for use in therapy.
  • C63 A method of eliciting an immune response in a mammal, the method
  • method comprising administering to the mammal an effective amount of the polypeptide according to any one of clauses C1-C51.

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Abstract

La présente invention concerne un polypeptide qui comprend au moins une mutation au niveau de la région de boucle de fusion 1 et/ou au niveau de la région de boucle de fusion 2 et/ou au niveau du site de clivage de type furine d'un polypeptide gB de cytomégalovirus humain. Dans un mode de réalisation, le polypeptide subit un changement de conformation structurale en réponse à un changement de pH.
PCT/IB2017/051401 2016-03-11 2017-03-09 Polypeptide gb de cytomégalovirus humain WO2017153954A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020121983A1 (fr) 2018-12-10 2020-06-18 Ｋｍバイオロジクス株式会社 Vaccin pour la prévention ou le traitement d'une infection congénitale par le cytomégalovirus
JPWO2021251384A1 (fr) * 2020-06-09 2021-12-16

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
WO2010007533A2 (fr) 2008-07-16 2010-01-21 Institute For Research In Biomedicine Anticorps neutralisant le cytomégalovirus humain et utilisation de ceux-ci
WO2012049317A2 (fr) * 2010-10-15 2012-04-19 Glaxosmithkline Biologicals S.A. Nouvel antigène
EP3031822A1 (fr) * 2014-12-08 2016-06-15 Novartis AG Antigènes du cytomégalovirus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
WO2010007533A2 (fr) 2008-07-16 2010-01-21 Institute For Research In Biomedicine Anticorps neutralisant le cytomégalovirus humain et utilisation de ceux-ci
US20160280770A1 (en) 2008-07-16 2016-09-29 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
WO2012049317A2 (fr) * 2010-10-15 2012-04-19 Glaxosmithkline Biologicals S.A. Nouvel antigène
EP3031822A1 (fr) * 2014-12-08 2016-06-15 Novartis AG Antigènes du cytomégalovirus

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BURKE ET AL., PLOS PATHOG., vol. 11, no. 10, October 2015 (2015-10-01)
DATABASE Geneseq [online] 7 June 2012 (2012-06-07), "Cytomegalovirus glycoprotein B (gB)-SLP12-Delta725 polypeptide, SEQ 10.", XP002769687, retrieved from EBI accession no. GSP:AZV29616 Database accession no. AZV29616 *
DATABASE UniProt [online] 15 June 2010 (2010-06-15), TARANTAL: "Envelope glycoprotein B, Macanine betaherpesvirus 3, Rhesus cytomegalovirus", XP002769689, Database accession no. D5KB35 *
DATABASE UniProt [online] 19 March 2014 (2014-03-19), DAVISON: "Envelope glycoprotein B, human cytomegalovirus", XP002769688, Database accession no. V9LN55 *
GALE SMITH ET AL: "Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats", PLOS ONE, vol. 7, no. 11, 30 November 2012 (2012-11-30), pages e50852, XP055060314, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0050852 *
SAPNA SHARMA ET AL: "HCMV gB shares structural and functional properties with gB proteins from other herpesviruses", VIROLOGY, vol. 435, no. 2, 22 October 2012 (2012-10-22), pages 239 - 249, XP055192519, ISSN: 0042-6822, DOI: 10.1016/j.virol.2012.09.024 *
SHARMA ET AL., VIROLOGY, vol. 435, no. 2, 20 January 2013 (2013-01-20), pages 239 - 49

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EP3895730A4 (fr) * 2018-12-10 2022-10-19 KM Biologics Co., Ltd. Vaccin pour la prévention ou le traitement d'une infection congénitale par le cytomégalovirus
AU2019397719B2 (en) * 2018-12-10 2023-11-09 Km Biologics Co., Ltd. Vaccine for preventing or treating congenital infection with cytomegalovirus
KR20210101274A (ko) 2018-12-10 2021-08-18 케이엠 바이올로직스 가부시키가이샤 사이토메갈로바이러스의 선천성 감염을 예방 또는 치료하기 위한 백신
JPWO2020121983A1 (ja) * 2018-12-10 2021-10-28 Ｋｍバイオロジクス株式会社 サイトメガロウイルスの先天性感染を予防又は治療するためのワクチン
JP7179872B2 (ja) 2018-12-10 2022-11-29 Ｋｍバイオロジクス株式会社 サイトメガロウイルスの先天性感染を予防又は治療するためのワクチン
KR102609106B1 (ko) 2018-12-10 2023-12-05 케이엠 바이올로직스 가부시키가이샤 사이토메갈로바이러스의 선천성 감염을 예방 또는 치료하기 위한 백신
CN113164585A (zh) * 2018-12-10 2021-07-23 Km生物医薬股份公司 用于预防或治疗巨细胞病毒的先天性感染的疫苗
WO2020121983A1 (fr) 2018-12-10 2020-06-18 Ｋｍバイオロジクス株式会社 Vaccin pour la prévention ou le traitement d'une infection congénitale par le cytomégalovirus
CN113164585B (zh) * 2018-12-10 2023-08-29 Km生物医薬股份公司 用于预防或治疗巨细胞病毒的先天性感染的疫苗
JPWO2021251384A1 (fr) * 2020-06-09 2021-12-16
KR20230022160A (ko) 2020-06-09 2023-02-14 케이엠 바이올로직스 가부시키가이샤 사이토메갈로바이러스의 gB와 펜타머의 융합 단백질 및 당해 융합 단백질을 포함하는 백신
AU2021287508B2 (en) * 2020-06-09 2023-11-09 Km Biologics Co., Ltd. Fusion protein of pentamer and gB of cytomegalovirus, and vaccine containing said fusion protein
JP7271794B2 (ja) 2020-06-09 2023-05-11 Ｋｍバイオロジクス株式会社 サイトメガロウイルスのｇＢとペンタマーとの融合タンパク質及び該融合タンパク質を含むワクチン
WO2021251384A1 (fr) 2020-06-09 2021-12-16 Ｋｍバイオロジクス株式会社 Protéine de fusion de pentamère et gb de cytomégalovirus, et vaccin contenant ladite protéine de fusion

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