title: Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.
abstract: Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: John W
authors.initials: JW
authors.lastname: Loughney
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.affiliation: Vaccine Research, and.
authors.forename: Dai
authors.initials: D
authors.lastname: Wang
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Troutman
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Dick
authors.affiliation: Biologics Bioprocess Development, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: Guanghua
authors.initials: G
authors.lastname: Li
authors.affiliation: Biologics Bioprocess Development, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Vaccine Research, and.
authors.forename: Fengsheng
authors.initials: F
authors.lastname: Li
authors.affiliation: Vaccine Research, and.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Freed
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Colleen E
authors.initials: CE
authors.lastname: Price
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Van M
authors.initials: VM
authors.lastname: Hoang
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Culp
authors.affiliation: From the Vaccine Bioprocess Research & Development.
authors.forename: Pete A
authors.initials: PA
authors.lastname: DePhillips
authors.affiliation: Vaccine Research, and.
authors.forename: Tong-Ming
authors.initials: TM
authors.lastname: Fu
authors.affiliation: From the Vaccine Bioprocess Research & Development, sha_ha@merck.com.
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000821:virology*; D004847:Epithelial Cells / Q000276:immunology / Q000821:virology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D053586:Virus Internalization
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D008562:Membrane Glycoproteins; C532797:UL128 protein, human cytomegalovirus; C502890:UL130 protein, human cytomegalovirus; D014759:Viral Envelope Proteins; C071440:glycoprotein H, Cytomegalovirus
keywords: HCMV; gH/gL/pUL128–131; glycoprotein; neutralizing epitope; pentameric complex; protein complex; protein purification; vaccine; virus entry