Broad-spectrum antiviral brincidofovir inhibits Epstein-Barr virus and related gammaherpesvirus in human and nonhuman primate cells

Abstract

Epstein-Barr virus (EBV) is of growing interest for its potential role in neurodegenerative diseases such as multiple sclerosis (MS) and its possible utility as a therapeutic target in herpesvirus-associated chronic diseases. The effects of brincidofovir (BCV) on EBV reactivation were evaluated in vitro using EBV-infected spontaneous lymphoblastoid cell lines (SLCLs) and peripheral blood mononuclear cells (PBMCs) derived from patients with MS and healthy controls. In addition, a B lymphoblastoid cell line and PBMCs from common marmosets (Callithrix jacchus) naturally infected with an EBV-related gammaherpesvirus (Callitrichine herpesvirus 3, CalHV-3) were used to measure BCV efficacy in a nonhuman primate model. BCV significantly inhibited gammaherpesvirus reactivation, with decreased lytic and latent viral transcript expression. These results suggest that BCV may be a useful antiviral for inhibiting EBV activity in patients with MS. Additionally, this work further validates the utility of CalHV-3 in marmosets as a translational model for the investigation of successful EBV-targeting therapeutics.

Keywords: Multiple sclerosis; Neuroscience; Virology.

Figure 1. Brincidofovir decreases EBV DNA in a human EBV-infected cell line and reduces viral mRNA following reactivation.

(A) Viability of one EBV-infected SLCL, SMS2 (red triangle), and the Ramos EBV-negative cell line (black square) after 7 days in culture with BCV. (B and C) Average EBV DNA copy number ± SEM per 1 × 10⁶ cells of the SMS2 SLCL detected by ddPCR for EBV BamHI DNA following 5 days in culture (B) and 7 days in culture (C) with BCV. (D–F) Average of EBV mRNA expression ± SEM in HC (open circles, n = 4) and MS (black circles, n = 6) SLCLs cultured with BCV and with (dark blue bars, to right of arrow) or without (light blue bars, under arrow) NaB+TPA to induce viral reactivation (arrow). (D) EBNA1 expression; (E) BZLF1 expression; (F) BFRF3 expression. Data analyzed using 1-way ANOVA with Tukey’s multiple comparisons (A–C) and the Kruskal-Wallis test with Dunn’s test for pairwise comparisons (D–F), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. BCV, Brincidofovir; EBV, Epstein-Barr virus; HC, healthy control; SMS, stable multiple sclerosis patient.

Figure 2. Brincidofovir reduces EBV mRNA expression in PBMCs from an individual with active EBV infection following viral reactivation.

(A–C) Representative ddPCR plots of EBNA1 detection from HC6 PBMCs cultured (A) without NaB+TPA and no BCV, (B) with NaB+TPA and no BCV, and (C) with NaB+TPA and 125 nM BCV. Y axis represents the FAM-MGBNFQ fluorescent amplitude, corresponding to EBNA1 detection in this plot (blue dots + orange dots, upper quadrants). X axis represents the VIC-MGBNFQ fluorescent amplitude, which corresponds to the HPRT housekeeping gene (green dots, bottom right quadrant). Lower left quadrant represents droplets negative for both EBNA1 and HPRT (gray dots). (D–F) Quantification of EBV mRNA expression in HC6 PBMCs cultured with BCV and with or without NaB+TPA to induce viral reactivation (arrow). Treatment with BCV and no NaB+TPA (light green bars, under arrow); treatment with BCV plus NaB+TPA (dark green bars, right of arrow). (D) EBNA1 expression; (E) BZLF1 expression; (F) BFRF3 expression. Open circle intersecting x-axis indicates mRNA expression undetected by ddPCR. BCV, Brincidofovir; EBV, Epstein-Barr virus; HC, healthy control.

Figure 3. Brincidofovir reduces EBV mRNA expression in PBMCs from an individual with stable multiple sclerosis (SMS) following viral reactivation.

(A–C) Representative ddPCR plots of EBNA1 detection from SMS6 PBMCs cultured (A) without NaB+TPA and no BCV, (B) with NaB+TPA and no BCV, and (C) with NaB+TPA and 125nM BCV. Y axis represents the FAM-MGBNFQ fluorescent amplitude, corresponding to EBNA1 detection in this plot (blue dots + orange dots, upper quadrants). X axis represents the VIC-MGBNFQ fluorescent amplitude, which corresponds to the HPRT housekeeping gene (green dots, bottom right quadrant). Lower left quadrant represents droplets negative for both EBNA1 and HPRT (gray dots). (D–F) Quantification of EBV mRNA expression in SMS6 PBMCs cultured with BCV and with or without NaB+TPA to induce viral reactivation (arrow). (D) EBNA1 expression; (E) BZLF1 expression; (F) BFRF3 expression. Open circle intersecting x-axis, mRNA expression undetected by ddPCR; BCV, Brincidofovir; EBV, Epstein-Barr virus; SMS, stable multiple sclerosis patient; Control, cells treated with only media.

Figure 4. Treatment of EBV-infected human PBMCs with brincidofovir attenuates viral reactivation.

(A–C) Average of EBV mRNA expression ± SEM in human PBMCs cultured with BCV and with or without NaB+TPA to induce viral reactivation (arrow). HC PBMCs, open circles (n = 4). MS PBMCs, black circles (n = 5). (A) EBNA1 expression; (B) BZLF1 expression; (C) BFRF3 expression. Data analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons, ****P ≤ 0.0001. H, healthy control; MS, multiple sclerosis patient.

Figure 5. CalHV-3–infected marmoset cells treated with brincidofovir show decreases in CalHV-3 DNA and mRNA following viral reactivation assay.

(A–C) Quantification of Callitrichine herpesvirus 3 (CalHV-3) DNA and mRNA expression in CalHV-3 infected cell line, CJ0149, with (dark blue bars, right) or without (light blue bar, left) NaB+TPA to induce viral reactivation. (A) CalHV-3 DNA expression; (B) ORF39 mRNA expression; (C) ORF45 mRNA expression. (D–F) Representative ddPCR plots of ORF39 mRNA expression in marmoset #3 PBMCs cultured (D) without NaB+TPA and no BCV, (E) with NaB+TPA and no BCV, and (F) with NaB+TPA and 250nM BCV. Y axis represents FAM-MGBNFQ fluorescent amplitude, which corresponds to ORF39 mRNA detection (blue dots + orange dots, upper quadrants). X axis represents the VIC-MGBNFQ fluorescent amplitude, which corresponds to the CJ-TBP housekeeping gene (green dots, bottom right quadrant). Lower left quadrant represents droplets negative for both ORF39 and CJ-TBP (gray dots). (G–I) Mean detection ± SEM of CalHV-3 gene expression in marmoset PBMCs (n = 4, circles) cultured with BCV and with (dark blue bars, right) or without (light blue bar, left) NaB+TPA to induce viral reactivation. (G) CalHV-3 DNA expression; (H) ORF39 mRNA expression; (I) ORF45 mRNA expression. Data analyzed using (G) Kruskal-Wallis test with Dunn’s comparisons, (H) a Welch’s ANOVA, and (I) 1-way ANOVA with Tukey’s multiple comparisons, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Control, cells treated with only media; BCV, brincidofovir.

Figure 6. Ability to reactivate EBV in human PBMCs correlates with ability to generate a spontaneous lymphoblastoid cell line.

(A) Table demonstrating correlation between successful reactivation of EBV and ability to generate an SLCL for 23 individuals (Fisher’s exact test, P < 0.0001). (B) Representation of number of individuals in which EBV was successfully reactivated (shaded area) compared with the total number of individuals tested. Total PBMCs reactivated: 9/23, 39%; HC PBMCs reactivated: 4/12, 33%; MS PBMCs reactivated: 5/11, 45%; SMS PBMCs reactivated: 3/7, 43%; AMS PBMCs reactivated: 2/4, 50%. HC, healthy control; MS, multiple sclerosis patient; SMS, stable multiple sclerosis patient; AMS, active multiple sclerosis patient; SLCL, spontaneous lymphoblastoid cell line.