Polymorphic Forms of Human Cytomegalovirus Glycoprotein O Protect against Neutralization of Fibroblast Entry by Antibodies Targeting Epitopes Defined by Glycoproteins H and L

Abstract

Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection. Which cell types and neutralizing activities are important for protective efficacy in vivo remain unclear. Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural infection.

Keywords: cytomegalovirus; glycoprotein O; hyperimmune globulin; immune evasion; neutralizing antibody; polymorphism.

Figure 1

(A) Structure of the CMV trimer illustrating relevant immunogenic sites. The CMV trimer, as determined by cryoelectron microscopy [35], indicates the locations of gO (blue), gL (red), and gH (green) with the C-terminal transmembrane domain of gH oriented at the bottom. Approximate locations of immunogenic sites and the mAbs that target each site were inferred from mAb-gH/gL or mAb-trimer complexes characterized by negative staining electron microscopy [34] and cryoelectron microscopy [35]. (B) Schematic illustration of chimeric viruses. UL74 sequences encoding TB40/E gO in the parental virus GT1c were fully or partially replaced by UL74 sequences from the indicated CMV strains. All other sequences, including UL75 (encoding gH), are of the background strain TB40/E.

Figure 2

Strain Towne gO impairs the fibroblast entry neutralizing activities of mAbs targeting epitopes in gH/gL. Viruses GT1c (encoding TB40/E gO) and GT4 (encoding Towne gO) were incubated for 1 h at 37 °C in medium (Ø) or medium containing 100 µg/mL of mAbs 223.4, 124.4, or TRL345, then added to MRC-5 fibroblast or ARPE-19 epithelial cell monolayers in 96-well plates. Following incubation at 37 °C for 48 h, infected cells were detected by immunofluorescent staining for the CMV IE1/2 proteins. Representative images are shown.

Figure 3

Towne gO impairs fibroblast entry neutralization by mAbs targeting three independent gH/gL epitopes but does not impact net neutralizing activities of anti-CMV antibodies induced by natural infection. Viruses GT1c or GT4 were incubated for 1 h at 37 °C with serial dilutions of mAbs, HIG, or human sera, then added in triplicate to 96-well cultures of MRC-5 fibroblasts (A,C) or ARPE-19 epithelial cells (B). After incubation at 37 °C for 48 h, luciferase activity in each well was measured and the % of maximal luciferase activity (means of triplicate wells) was plotted vs. log of antibody concentration or dilution.

Figure 4

Polymorphisms of gO amino acid sequences encoded by diverse CMV strains. Predicted gO amino acid sequences from CMV strains TB40/E, AD169, Merlin, Han16, BE/29/2011, and Towne were aligned using clustalW. Numbers refer to amino acid positions in Merlin gO; the red box indicates the most highly polymorphic region.

Figure 5

Other gO types influence fibroblast entry neutralization by gH/gL-specific mAbs in an epitope-specific manner. Four additional TB40/E-background chimeric viruses encoding gOs from strains AD169, Merlin, Han16, or BE/29/2011 were used in fibroblast (A) or epithelial cell (B) entry neutralizing assays as described in Figure 3.

Figure 6

Inhibitory activity maps to the polymorphic N-terminal region of gO. (A) Predicted gO amino acid sequences from CMV strains TB40/E (red), Towne (black), and Han16 (blue) were aligned using clustalW to illustrate the respective sequences in intrastrain gO chimeras GT1c/3 and GT1c/4. Intrastrain gO chimeric viruses GT1c/3 and GT1c/4 were used in fibroblast (B) or epithelial cell (C) entry neutralizing assays as described in Figure 3. Data from chimeras encoding full-length gOs that comprise parts of each intrastrain gO chimera are included for comparison.