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.

Keywords: HCMV; gH/gL/pUL128–131; glycoprotein; neutralizing epitope; pentameric complex; protein complex; protein purification; vaccine; virus entry.

FIGURE 1.

Purification of gH/gL and gH/gL/pUL128–131 pentameric complex (PC). A, UV trace of Strep Tactin chromatogram. B, UV trace of CEX chromatogram to purify pentameric complex. C, reinjection of gH/gL and pentameric complex on the HPLC CEX column. D, reducing SDS-PAGE of pentameric complex purification fractions. E, nonreducing SDS-PAGE of pentameric complex purification fractions. F, reducing SDS-PAGE of deglycosylated gH/gL and pentameric complex by PNGase and α(2–3,6,8,9)-neuraminidase (NM). Protein identification was confirmed by in-gel digestion followed by nano LC-MS/MS. For SDS-PAGE, samples were loaded at 10 μg/lane. IMAC, product from immobilized metal affinity chromatography; StrepFT, flowthrough from Strep Tactin column. Strep, product from Strep Tactin purification.

FIGURE 2.

Binding activity of purification samples to gH/gL specific mAb 124.4 and pentameric complex specific mAb 57.4. A, dose-response curve of purification samples binding to mAb 124.4. B, dose-response curve of purification samples binding to mAb 57.4. In the Sandwich ELISA assay, either mAb 124.4 or 57.4 was immobilized on the plates at a fix concentration of 2 μg/ml, and a serial dilution of samples were tested for binding. C, relative binding activities compared with sample IMAC. Pentameric complex is enriched in the CEX by 6-fold. Low binding activity of CEXFT sample toward mAb 57.4 suggests a low level of pentameric complex is remaining in the CEXFT.

FIGURE 3.

Binding activities of seven elite neutralizing antibodies to the soluble gH/gL and pentameric complex. The elite neutralizing mAbs were isolated from a rabbit vaccinated with the pentameric complex restored AD169 (15). Among the seven elite neutralizers, two bind to the pentameric complex only (A and C) consistent with their binding to the AD169 revertant virus (category 1), and five bind to both gH/gL and the pentameric complex (B and C) consistent with their binding to both AD169 parental and revertant virus (category 2).

FIGURE 4.

Biophysical characterization of the soluble gH/gL and gH/gL/pUL128–131 PC. A, charge heterogeneity of gH/gL and PC by imaged capillary isoelectric focusing (NanoPro Technology). Electropherograms show that gH/gL is detected by mAb 124.4 only and PC is detected by both mAb 124.4 and mAb 57.4. B, size exclusion chromatogram of gH/gL (solid line) and PC (dashed line) measured at UV 280 nm. The calculated molecular mass is shown as dots above each peak. The gH/gL complex has a total molecular mass of 250 kDa, and the PC has a total molecular mass of 180 kDa. The dotted line shows a PC purchased from a reagent vendor. It reveals co-purified (gH/gL)₂ homodimer present in the PC preparation. C, two-dimensional class average calculated using particles selected from the EM images of gH/gL overlaid with the coordinates of the HSV-2 gH/gL structure (Protein Data Bank code 3M1C) is on the left, and the two-dimensional class average of PC is on the right.

FIGURE 5.

A, RP-HPLC separation of gH/gL and gH/gL/pUL128–131 PC under reducing and nonreducing conditions. Samples were prepared with 1% SDS for nonreducing condition and 1% SDS and 100 mm DTT for reducing condition. Samples of 30 μg of gH/gL and PC were separated on Applied Biosystems Poros R2 column at 75 °C. The chromatogram shows baseline separation among pUL131, pUL128, pUL130, gL, and gH peaks. B, peak identification by SDS-PAGE and nano LC-MS/MS. Samples R1–R5 were collected from PC under reducing condition. Samples N1–N3 were collected from PC under nonreducing condition.

FIGURE 6.

Evaluation of viral entry inhibition by soluble gH/gL and gH/gL/pUL128–131 PC. Pentameric complex restored AD169 viruses were preincubated with soluble gH/gL, soluble gH/gL/pUL128–131 PC, CMV-HIG, and a negative control IgG, and the mixtures were used to infect ARPE-19 cells. Viral entry was quantitated at 20 h postinfection by IE2 protein expression.

FIGURE 7.

Evaluation of CMV-HIG depleted by soluble gH/gL and gH/gL/pUL128–131 PC. A, the efficiency of depletion as evaluated by the binding activities of depleted CMV-HIG toward PC. The purified PC was immobilized on the plate for direct ELISA. B, neutralization (Neut) of HCMV in infecting epithelial ARPE-19 cells by depleted CMV-HIG. Infectivity was quantitated at 21 h postinfection by IE1 protein expression. C, reduction of binding and neutralization activities of gH/gL and PC depleted CMV-HIG compared with mock control. Conc., concentration.