Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

Abstract

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

Fig. 1. VUN100bv binds and inhibits US28 signaling.

a ELISA binding of monovalent VUN100 and bivalent VUN100bv to membrane extracts of US28-expressing HEK293T cells. Representative figure of three independent experiments. b Displacement of ¹²⁵I-CX3CL1 from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and VUN100bv. Representative figure of three independent experiments. c Effect of nanobodies on US28-mediated NFAT (nuclear factor of activated T cells) activation. HEK293T cells expressing either NFAT-luciferase reporter only (Mock) or NFAT-luciferase reporter together with US28 wildtype receptor (WT), US28 Y16F mutant (Y16F), US28 ΔN (2–22) mutant (ΔN (2–22)) or US28 R129A mutant (R129A). Cells were untreated (untr) or treated with a non-targeting nanobody (NT Nb), VUN100, or VUN100bv for 24 h prior to luminescence measurement. Data were normalized to the untreated WT samples. Representative figure of three independent experiments. d Immunofluorescence microscopy of nanobody binding to US28-expressing THP-1 cells. US28 was detected using a polyclonal rabbit-anti-US28 antibody (US28 mAb). Cells were incubated without nanobody (No Nb), an NT Nb, VUN100, or VUN100bv. Bound nanobody was detected using the Myc-tag present on the nanobodies and an anti-Myc antibody (Nb). Representative figure of three independent experiments. e Western blot detection for total IFI16 levels of lysates of untreated THP-1 mock transduced cells (THP-1 Mock) or US28-expressing THP-1 cells (THP-1 US28 WT). THP-1 US28 WT cells were untreated (Untr) or treated with NT Nb, VUN100, or VUN100bv for 48 h. IFI16 protein levels were determined and normalized to actin protein levels. Relative IFI16 protein levels were normalized to untreated THP-1 mock cell lysates. n = 3 independent experiments from three independent biological replicates. All data are plotted as mean ± S.D. For all data, except for Fig. 1c, statistical analyses were performed using an unpaired two-tailed t test. For Fig. 1c, statistical significance was determined using the Holm–Sidak method (two-sided with alpha = 0.05). Source data are provided as a Source Data file.

Fig. 2. VUN100bv induces immediate-early expression but no full viral reactivation.

a CD14⁺ monocytes were isolated, infected with HCMV IE2-eYFP, and were left untreated or treated with a non-targeting nanobody, VUN100 or VUN100bv. Two days post infection, cells were fixed and stained for immediate-early (IE) expression. b CD14⁺ monocytes were isolated, infected with HCMV IE2-eYFP, and were left untreated (Untr) or treated with a non-targeting nanobody (NT Nb), VUN100, or VUN100bv. As a positive control, CD14⁺ monocytes were pre-treated with 20 ng/ml PMA before infection (PMA). IE-positive nuclei were counted 6 days post infection. c Six days post infection, untreated (untr), nanobody-treated monocytes (NT Nb, VUN100, and VUN100bv) or monocytes pre-treated with 20 ng/ml PMA before infection (PMA) were co-cultured with Hff1 fibroblasts. IE-positive infectious foci formation was quantified after 4 days of co-culturing. d Six days post infection, HCMV-infected CD14+ cells were left untreated (Untr) or were treated with NT Nb, VUN100, VUN100bv, or 20 ng/ml PMA (PMA). IE-positive nuclei were counted 8 days post infection. e Eight days post infection, untreated (Untr), nanobody-treated (NT Nb, VUN100, and VUN100bv) or PMA-treated monocytes were co-cultured with Hff1 fibroblasts. IE-positive infectious foci were quantified after 8 days of co-culturing. Representative figures, showing technical replicates, from two (Fig. 2d, e) or four (Fig. 2a b) biological replicates are shown. All data are plotted as mean ± S.D. For all figures, statistical analyses were performed using unpaired two-tailed t test. ns, p > 0.05. Source data are provided as a Source Data file.

Fig. 3. VUN100bv increases immediate-early, early and late gene but no true late gene expression.

CD14⁺ monocytes were left uninfected (Uninf) or infected with HCMV IE2-eYFP. Infected monocytes were left untreated (Untr), treated with a non-targeting nanobody (NT Nb), VUN100, VUN100bv, or pre-treated with 20 ng/ml PMA before infection (PMA). Six days post infection, RNA was isolated and IE72 (a), UL44 (b), UL32 (c), and US11 (d) gene expression was measured by RT-qPCR. Representative figures, showing technical replicates, from two biological replicates are shown. All data are plotted as mean ± S.D. For all figures, statistical analyses were performed using unpaired one-way ANOVA with Tukey’s multiple comparison test. ns, p > 0.05. Source data are provided as a Source Data file.

Fig. 4. HCMV-infected CD14⁺ monocytes are targets for HCMV-specific T cells upon VUN100bv treatment.

a Counting of IE-positive CD14⁺ monocytes before co-culture with CD4/CD8⁺ T cells or T-cell-depleted PBMCs. CD14⁺ monocytes were treated with a non-targeting nanobody (NT Nb), VUN100bv for 6 days post infection or pre-treated with 20 ng/ml PMA before infection. b Counting of IE-positive CD14⁺ monocytes after co-culture of T-cell-depleted PBMCs (depleted PBMC) or T cells (CD4/CD8) and differentiation of CD14⁺ monocytes to mature dendritic cells. c Counting of IE-positive CD14⁺ monocytes before co-culture with CD4/CD8⁺ T cells or T-cell-depleted PBMCs. Six days post infection, CD14⁺ monocytes were treated with NT Nb, VUN100bv, or 20 ng/ml PMA for 1 day. d Counting of IE-positive CD14⁺ monocytes after co-culture of T-cell-depleted PBMCs (depleted PBMC) or T cells (CD4/CD8) and differentiation of CD14⁺ monocytes to mature dendritic cells. Representative figures, showing technical replicates, from two biological replicates are shown. All data are plotted as mean ± S.D. For all figures, statistical analyses were performed using unpaired two-tailed t test. ns, p > 0.05. Source data are provided as a Source Data file.

Fig. 5. VUN100bv treatment drives a T-cell-mediated reduction of HCMV reactivation without driving viral DNA replication in latently infected cells.

a Quantification of HCMV gDNA copy numbers of uninfected (Uninf) or HCMV-US2-6 infected CD14⁺ monocytes before co-culture with CD4/CD8⁺ T cells. Infected monocytes were untreated (Untr), treated with a non-targeting nanobody (NT Nb), VUN100bv, or 20 ng/ml PMA (PMA) for 6 days. Total genomic DNA was harvested and HCMV gDNA copy numbers were determined by quantifying GAPDH and UL44 gene numbers via qPCR. HCMV gDNA copy numbers were normalized to the untreated infected wells. b Quantification of HCMV gDNA copy numbers of untreated NT Nb or VUN100bv treated HCMV-US2-6 infected CD14⁺ monocytes after co-culturing with CD4/CD8⁺ T cells. HCMV gDNA copy numbers were determined prior to (Pre) and after (Post) differentiation of CD14⁺ monocytes to mature dendritic cells and co-culturing with fibroblasts for 8 days. Total genomic DNA was harvested and HCMV gDNA copy numbers were determined by quantifying GAPDH and UL44 gene numbers via qPCR. HCMV gDNA copy numbers were normalized to the untreated wells prior to differentiation to mature dendritic cells. Representative figures, showing technical replicates, from three biological replicates are shown. All data are plotted as mean ± S.D. For all figures, statistical analyses were performed using unpaired two-tailed t test. ns, p > 0.05. Source data are provided as a Source Data file.