Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex

Abstract

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC(90)] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.

Figures

FIG. 1.

Microneutralization assays on human fibroblasts and epithelial cells reveal two types of HCMV human monoclonal neutralizing antibodies. Human IgG memory B cells from HCMV-immune donors were immortalized with EBV and B-cell clones producing MAbs capable of neutralizing HCMV infection of fibroblasts (MRC-9) or retinal epithelial cells (ARPE-19) were isolated. (A) An appropriate dilution of the HCMV clinical isolate VR1814 was incubated for 2 h in the absence or presence of MAbs at 1 μg/ml before addition to target cells. Infected cells were detected by staining with anti-pp72 mouse MAb 48 h after infection. Representative staining of antibodies belonging to the two groups is shown (one field of acquisition of nine fields analyzed). (B) Microneutralization assays with different concentrations of three representative antibodies performed as for panel A. The mean of measurements of duplicate wells ± standard error from one experiment out of three performed was plotted, and nonlinear sigmoidal dose-response fit curves for each MAb were calculated. IC90 values for all antibodies and target cells are shown in Table 1.

FIG. 2.

Staining of HEK293T cells expressing HCMV antigens determines the antigenic specificity of human MAbs. HCMV genes UL128, UL130, UL131A, gH, and gL were cloned and transfected in HEK293T cells in different combinations. The cells were fixed, permeabilized, and stained with the neutralizing antibodies. Ectopic expression of antigens was confirmed by reactivity with mouse antibodies (not shown). Shown are representative profiles for MAbs 15D8, 1F11, 2C12, and 8I21.

FIG. 3.

15D8 binds to a conserved region of UL128. HEK293T cells expressing wild-type or pan-mutant UL128 were stained with different concentrations of human MAb 15D8 and a noncompeting mouse anti-UL128 MAb. Shown are the dot plots of the two-color staining.

FIG. 4.

Antigenic map of gH/gL/UL128-131A complex. Based on minimal antigen requirement for binding and on cross-competition experiments, human monoclonal neutralizing antibodies define nine distinct antigenic sites on gH/gL/UL128-131A. Gray ovals represent sites defined by group 1 antibodies specific for gH, whereas white ovals represent sites recognized by group 2 antibodies. Dashed ovals represent sites defined on the basis of lack of competition by antibodies 7I13 and 10P3 when tested at 100-fold excess.