Evaluation of Torquetenovirus (TTV) Particle Integrity Utilizing PMAxx™

Abstract

Torquetenovirus (TTV) is a ubiquitous, non-pathogenic DNA virus that has been suggested as a biomarker of immune competence, with the viral load correlating with the level of immunosuppression. However, by detecting non-intact viral particles, standard PCR-based quantification may overestimate the TTV viremia. To improve the clinical relevance of TTV quantification, in this study, we investigated the use of PMAxx™, a virion viability dye that selectively blocks the amplification of compromised virions. Serum samples from 10 Hepatitis C Virus-positive (HCV+) individuals, 81 liver transplant recipients (LTRs), and 40 people with HIV (PWH) were treated with PMAxx™ and analyzed for TTV DNA loads by digital droplet PCR (ddPCR). Furthermore, anti-SARS-CoV-2 IgG levels and neutralizing antibody (nAbs) titers were measured post-COVID-19 vaccination. Using ddPCR, the PMAxx™ treatment significantly reduced the TTV DNA levels in all the groups (mean reduction: 0.66 Log copies/mL), indicating the abundant presence of non-intact, circulating viral genomes. However, correlations between TTV DNA and SARS-CoV-2 IgG or nAbs were weak or absent in both PMAxx™-treated and untreated samples. These findings suggest that while PMAxx™ enhanced the specificity of TTV quantification, it did not improve the predictive value of TTV viremia at assessing vaccine-induced humoral responses.

Keywords: COVID-19 vaccination; Torquetenovirus; genome quantification; propidium monoazide (PMA).

Figure 1

TTV results of testing samples by ddPCR before and after the PMAxx™ treatment. (A) Fresh samples (n. 10, in blue) untreated (NT) and treated with PMAxx™. (B) Frozen PWH samples (n. 40, in green), untreated (NT) and treated with PMAxx™. (C) Frozen LTR samples (n. 81, in orange), untreated (NT) and treated with PMAxx™. (D) Δ TTV DNA stratified by study population (HCV+, LTR, and PWH). Black lines indicate the mean and standard error of the mean (SEM); asterisks indicate statistical significance levels (** p < 0.01; **** p < 0.0001; ns = not significant).

Figure 2

Correlation of the TTV levels in untreated samples with Δ TTV DNA between treated and untreated samples. Each point represents an individual sample. Fresh samples are depicted in blue, while frozen samples are depicted in green and orange, from PWH and LTRs, respectively. The regression line, confidence interval, p-value, and r value are shown.

Figure 3

Correlations of the untreated and PMAxx™-treated TTV DNA levels with the anti-SARS-CoV-2 IgG levels and nAbs responses in the PWH. Correlations between the TTV DNA and the (A) SARS-CoV-2 anti-RBD IgG and (B) nAbs. Correlations between the PMAxx™-treated TTV DNA and the (C) SARS-CoV-2 anti-RBD IgG and (D) nAbs. Correlations between the Δ TTV DNA and the (E) SARS-CoV-2 anti-RBD IgG and (F) nAbs. Each point represents an individual sample. Regression lines, confidence intervals, p-values, and r values are shown.

Figure 4

Correlations of the untreated and PMAxx™-treated TTV DNA levels with the anti-SARS-CoV-2 IgG levels and nAbs responses in the LTR patients. Correlations between the TTV DNA and the (A) anti-SARS-CoV-2 RBD IgG levels and (B) nAbs titers. Correlations between the PMAxx™-treated TTV DNA and the (C) anti-SARS-CoV-2 RBD IgG levels and (D) nAbs titers. Correlations between the Δ TTV DNA and the (E) SARS-CoV-2 anti-RBD IgG and (F) nAbs. Each point represents an individual sample. Regression lines, confidence intervals, p-values, and r values are shown.

Figure 5

TTV DNA kinetics of untreated and PMAxx™-treated samples from 5 LTR patients. (A) TTV DNA levels within 15 days post-transplantation. (B) TTV DNA levels until 90 days post-transplantation. Each point represents the mean Log copies/mL from five patients; error bars indicate the standard error of the mean (SEM). NT, untreated samples.