A broadly neutralizing human monoclonal antibody generated from transgenic mice immunized with HCMV particles limits virus infection and proliferation

Abstract

Human cytomegalovirus (HCMV) is a β-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases.

Importance: Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.

Keywords: combinatorial therapeutic; gH/gL complexes; human cytomegalovirus; neutralizing antibody; synergy.

Fig 1

Screening and selection of hybridoma clones that neutralize HCMV elicited from VelocImmune mice. (A) Hybridoma clones (1,440) generated from a VelocImmune mouse immunized with TB40/E and VHL/E were screened using a neutralization assay for AD169R (MOI = 0.2) in ARPE-19 epithelial cells. Percent infection was calculated relative to virus alone (set at 100%) and plotted as a heat map for clones assessed across 10 96-well plates. Clones selected for further study and characterization are outlined in white. (B) Hybridoma clones 7H6, 1C10, 1H1, 2D4, 4F9, 7G07, 8A5, and 9H1 were assessed for neutralization of AD169R (MOI = 0.2) in NHDF and ARPE-19 epithelial cells. Controls included untreated virus and virus incubated with CytoGam, anti-gH mAb 5C3, and anti-influenza HA mAb M2E10. Relative infection (%) was calculated based on virus alone as 100%. (C) The gH/gL complexes expressed in U373-gH/gL cell lysates were recovered by immunoprecipitation (IP) with mAbs 5C3, 1C10, 4F9, 8A5, 2D4, and 7H6. The U373-gH/gL total cell lysates (lane 1) and IPs of protein A beads alone (lane 3), W6/32 antibody (anti-MHC-I, lane 4), anti-HCMV mAbs (lanes 5–9), and isotype control clone 7H6 (lane 10) were resolved on a non-reducing SDS-polyacrylamide gel and then subjected to anti-gL immunoblot. Lane 2 was blank. The relative molecular weight markers and recovered polypeptides are indicated.

Fig 2

Evaluation of anti-HCMV mAbs to neutralize infection of HCMV strains in epithelial cells. mAbs 1C10 (A), 2D4 (B), 4F9 (C), and 8A5 (D) and HCMV hyperimmune globulin (CytoGam, E) (0–50 µg/mL) were evaluated for the neutralization of TB40/E, AD169R, and TR (MOI = 0.2) in ARPE-19 epithelial cells. Relative infection (%) was calculated based on virus alone as 100%. Error bars represent standard deviation from the mean. Statistical significance is denoted as ^*P < 0.05; ^***P < 0.001; ^****P < 0.0001 based on the untreated sample, respectively.

Fig 3

Evaluation of anti-HCMV mAbs to neutralize infection of HCMV strains in fibroblasts. mAbs 1C10 (A), 2D4 (B), 4F9 (C), and 8A5 (D) and HCMV hyperimmune globulin (CytoGam, E) (0–50 mg/mL) were evaluated for the neutralization of TB40/E, AD169R, TR, and AD169-IE2-YFP (MOI = 0.2) in NHDF cells. Relative infection (%) was calculated based on virus alone as 100%. Error bars represent standard deviation from the mean. Statistical significance is denoted as ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 based on untreated sample, respectively.

Fig 4

Anti-HCMV mAb 1C10 neutralizes HCMV infection of placenta- and monocyte-derived cells. mAb 1C10 (A and C) and HCMV hyperimmune globulin (CytoGam, B and D) (0–50 mg/mL) were analyzed for neutralization of TB40/E and AD169R (MOI = 0.2) in HTR-8/SVneo placenta-derived trophoblast cells (A and B) and THP-1 monocyte-derived cells (C and D). Relative infection (%) was calculated based on virus alone as 100%. Error bars represent standard deviation from the mean. Statistical significance is denoted as ^*P < 0.05; ^***P < 0.001; ^****P < 0.0001 based on the untreated sample, respectively.

Fig 5

mAb 1C10 competes with broadly neutralizing anti-gH mAb 15G11. Competition assays were performed with U373-gH/gL cells incubated with AF647-labeled 1C10 (1C10^AF647, 2 µg/mL) with increasing concentrations (0.4, 2, 4, and 20 µg/mL) of unlabeled anti-gH antibodies 1C10, 5C3, or 15G11. The competition of 1C10^AF647 with the irrelevant influenza antibody PY102 (20 µg/mL) or AF647-labeled mouse IgG (20 µg/mL) was used to define the relative mean fluorescent intensity as 100%. Binding of 1C10^AF647 to cell surfaces was measured via flow cytometry. All conditions were performed over at least n = 2 experimental replicates.

Fig 6

Anti-gH/gL mAbs limit virus infection of fibroblast and epithelial cells in a post-attachment step. mAbs 1C10, 2D4, 4F9, 7H6, 8A5, and CytoGam (5 µg/mL) were incubated with TB40/E pre-attachment to cells or added following virus addition as a post-attachment treatment. NHDF (A) or ARPE-19 epithelial (B) cells were evaluated for virus infection upon treatment of antibody with virus alone set to 100%. (C) Schematic depiction of the time-of-addition study to evaluate mAbs’ kinetics of inhibition. Antibodies 1C10, 2D4, 4F9, 7H6, 8A5, and CytoGam were added to NHDF (D) or ARPE-19 epithelial (E) cells at the indicated timepoints relative to infection of TB40/E (MOI = 0.2) and evaluated for infection (24 hpi) with virus alone set to 100%. Error bars represent the standard deviation from the mean.

Fig 7

Anti-gH/gL mAbs limit HCMV proliferation. mAbs 1C10, 2D4, 4F9, 7H6, and 8A5 (5 µg/mL) were examined in a TB40/E (MOI = 0.01) proliferation assay in ARPE-19 epithelial cells. Relative virus infection (%) (A) and number of viral foci (>5,000 µm²) (B) were quantified at 9 dpi. CytoGam, ganciclovir, and no treatment (−) were utilized as controls. Relative infection (%) was calculated based on virus alone set as 100%. mAbs 1C10, 4F9, 8A5, 2D4, and 7H6 (5 or 10 µg/mL) were evaluated in a virus proliferation assay by measuring relative HCMV genome levels in ARPE-19 epithelial (C) or NHDF (D) cells infected with TB40/E (MOI = 0.01) at 9 dpi. CytoGam, ganciclovir, and no treatment (−) were utilized as controls. mAbs 1C10 and 15G11 (5 or 20 µg/mL) were evaluated for relative HCMV genome levels in ARPE-19 cells infected with a ganciclovir-resistant clinical HCMV isolate CH7 (E). Isotype mAb 7H6 (20 µg/mL) and letermovir (20 nM) were used as controls. HCMV genome levels were quantified relative to mock (uninfected) cells after normalizing to housekeeping gene RPS11 expression. Error bars represent the standard deviation from the mean. Statistical significance is denoted as ^*P < 0.05; ^***P < 0.001; ^****P < 0.0001 based on the untreated sample, respectively.

Fig 8

Anti-gH and anti-gB mAb combinations effectively limit HCMV proliferation. Combinations of increasing concentrations of mAbs 1C10 and 8F9 were evaluated for limiting TB40/E (MOI = 0.05) proliferation in ARPE-19 epithelial cells. Relative infection (%) was calculated at 9 dpi based on virus alone as 100%, (A) and these values were used to determine the Loewe synergy scores (B). Error bars represent the standard deviation from the mean. Statistical significance is denoted as ^****P < 0.0001 based on the untreated sample.

Fig 9

Anti-gH mAb/ganciclovir combinations synergistically inhibit HCMV proliferation. Combinations of increasing concentrations of mAb 1C10 and ganciclovir were evaluated for limiting TB40/E (MOI = 0.05) proliferation in ARPE-19 epithelial (A and C) and NHDF (B and D) cells at 9 dpi. Relative infection (%) was calculated based on virus alone as 100% (A and B), and these values were used to determine the Loewe synergy scores (C and D). Error bars represent the standard deviation from the mean. Statistical significance is denoted as ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 based on the untreated sample, respectively.