EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Solid-organ transplant is the treatment of choice for all patients with end-stage diseases. Long-term graft function and survival rely on suitable immunosuppressive treatment to prevent rejection. Besides this desired effect, a reduced immunocompetence in the transplant recipient increases the risk of developing infectious diseases and malignancies. An ideal biomarker should be sensitive, allow early visibility, be accessible in peripheral blood, and be associated with a known mechanism. Torque teno virus or transfusion transmitted virus is a virus that has gained attention as a possible marker of immune function. This virus rarely causes disease in healthy individuals, but torque teno viral load in immunosuppressed patients is shown to be higher than in healthy controls. Replication of torque teno virus is inversely correlated with number and especially functions of T lymphocytes. Torque teno virus could join the current list of predictive biomarkers in transplantation to detect whether the patient is over- or under-immunosuppressed. More studies are needed to confirm or disprove this possibility.

Key words : Biomarker, Immunosuppression, Transfusion transmitted virus

Solid-organ transplant is the treatment of choice for all patients with end-stage diseases of heart, lung, liver, and kidney. Donors, both living and deceased, often have different human leukocyte antigens (HLA), resulting in most recipients needing immunosuppressive drugs for life. Long-term graft function and survival rely on suitable immunosuppressive treatment to prevent rejection. Along with this desired effect, transplant recipients have reduced immunocompetence and thus increased risk of developing infectious diseases, which may lead to patient death.¹ An ideal biomarker should be sensitive, have early visibility, be accessible in peripheral blood, and be associated with a known mechanism.²

The torque teno virus or transfusion-transmitted virus (TTV) is a virus that has gained attention as a possible marker of immune function because it is found in water, air, soil, and also in human body samples.³

In organ transplant, it has been understood that, although patients may develop infections as a result of overimmunosuppression, infections with TTV do not often occur. In the general population, this virus may be present but rarely causes disease in healthy individuals. However, in immunosuppressed patients, TTV viral load is higher than in healthy individuals. Replication of TTV is inversely correlated with number and especially functions of T lymphocytes.⁴

In kidney transplant recipients, it has been questioned whether the viral load of TTV over time actually relates to the risk of severe infection and rejection episodes. If the answer is yes, then does adjusting the immunosuppressive medication doses in kidney transplant patients according to TTV viral load decrease the risk of both infection and rejection episodes? In this work, we investigated the different opinions on TTV level as a biomarker to assess the immunocompetence of the host and intensity of immunosuppression in transplant recipients. If the viral load of TTV over time actually relates to the risk of severe infection and rejection episodes, we investigated the possibility of whether adjusting immunosuppressive doses in kidney transplant patients according to TTV viral load would decrease the risk of both infection and rejection episodes.

What Is Torque Teno Virus?
Torque teno virus is a nonenveloped, circular, single-strand DNA virus. It was first discovered in Japan in 1997 in 5 patients with acute posttransfusion non-A to G hepatitis.⁵ The name is derived from the Latin words torque, meaning “necklace,” and tenuis, meaning “thin.” It is also known as N22 and was assigned to the family of Anelloviridae.⁵

The virus is 30 to 50 nm in diameter, and the genome is 3.8?kb in length and contains 3739 base pairs expressing 3 messenger RNAs. It is estimated that >3.8 × 10¹⁰ copies/mL of TTV virions are generated in the healthy human body per day and that >90% of these are cleared by the immune system. Consequently, in cases with detectable viral load, TTV viral load remains about 10² to 10⁸ copies/mL.⁶

Hsiao and colleagues⁵ found the prevalence of TTV in the general population may reach up to 95% in healthy Taiwanese individuals. In Egypt, however, prevalence was 60% in patients with thalassemia and 57% in healthy individuals.⁷

Detection of Torque Teno Virus
Various sets of primers were designed on distinct regions of the TTV genome to evaluate their capacity for detection of TTV DNA by polymerase chain reaction (PCR). Furthermore, the nucleotide sequences of amplification products were determined and compared among TTV isolates of different genotypes. The results obtained highlighted a deep influence of primer use for PCR for the detection of TTV DNA due to an extremely wide range of sequence divergence among TTV of distinct genotypes.⁸

Torque teno virus is transmitted by parenteral, transplacental, breast milk, respiratory, and fecal-oral routes.⁹ This virus can be found in most tissues and cells except red blood cells and platelets.¹⁰

Torque teno virus can be detected by quantitative or qualitative PCR techniques in different clinical specimens.^11-13 Furthermore, immunoassays, including enzyme immunoassay and enzyme-linked immunosorbent assay, can be used to detect antibodies against ORF1 (anti-N22 polypeptide) or ORF2 proteins.^14,15

Up to now, there are no widely standardized methods for TTV diagnosis. One method used is PCR, which is an accurate method; however, the application of different primers results in variable diagnostic sensitivity.¹⁶

The viral load of TTV increases under compromised immune response conditions.¹⁷ Replication of TTV is inversely correlated with number and especially function of T lymphocytes.¹⁸

Relationship Between Torque Teno Virus and BK Nephropathy
Since the early 2000s, an increase in the prevalence of nephropathy caused by human BK polyomavirus (BKPyV) in renal transplant recipients has resulted in a renewed interest in this pathogen. Infections with BKPyV may induce progressive disease, mainly during the first 3 to 6 months after transplant, over 3 successive stages: viruria, viraemia, and, if viral replication persists, nephropathy. However, it is difficult to predict patients after transplant who could replicate BKPyV.¹⁹

Until now, no data have been available to address whether there is any correlation between TTV and BKPyV viral load after solid-organ transplant. Herrmann and colleagues²⁰ found that TTV viral load in serum correlated with BKPyV viral load in urine and observed a trend for the mean TTV viral load in serum to be weakly higher in patients without BK viruria compared with patients with BK viruria. In addition, the 2 patients with BK viremia in their study had no TTV DNA in urine, but both were positive for TTV in serum. Similar observations have been made by Beland and associates.²¹ It may also be possible that both BKPyV and TTV replicate in the urogenital tract in the same cells and use similar host factors or that BKPyV replication suppresses TTV replication. However, because of the lack of an in vitro cell culture system for TTV, it is difficult to follow-up on this hypothesis.²⁰

Handala and colleagues²⁴ retrospectively analyzed 116 recipients in which BKPyV loads were assessed monthly for the first 6 months and every 3 months thereafter. The authors found no differences in plasma TTV DNA levels throughout the first 3 months between patients with or without BKPyV replication and observed high variations in viral load levels with high variations between populations, with many factors seeming to influence the level of TTV replication, such as individual factors (for example, age and sex). However, many of these factors, factors related to the immunosuppressive regimen, and probably also factors specific to the virus remained unidentified. In addition, the authors did not demonstrate a clinically predictive TTV viral load threshold. This study found no correlation between TTV viral load and BKPyV replication after kidney transplant. However, this study had limitations, as it was a retrospective cohort study.²²

Studies on larger multicenter cohorts with higher rates of BKPyV-associated nephropathy should be encouraged. In addition, further efforts should be directed at standardizing the reporting units for TTV DNAemia across centers.²³

Torque Teno Virus Viral Load in Cases of Rejection
In kidney transplant recipients, the prevalence of those with detectable TTV DNA has been estimated to range between 10% and 100%.²⁴ The prevalence of various TTV genotypes in kidney transplant recipients differs in different geographical areas; for example, genotypes 2 and 5 are the most prevalent genotypes in Brazilian kidney transplant recipients,25 whereas genotype 1 is the most common genotype in Hungary.²⁶ The detectability of the virus in peripheral blood, stool, saliva, and pharyngeal mucus suggests several possible routes of transmission and a high probability of exposure.²⁷

A correlation between increasing TTV levels in peripheral blood and plasma and the immune status of patients after transplant has been suggested, which indicates the possibility of exploiting posttransplant quantitative TTV analyses in solid-organ transplant recipients, particularly as a surrogate marker for immune reconstitution.²⁸ The use of TTV is emerging as a promising marker to assess global functional immune competence and to predict posttransplant immune-related adverse events, which would eventually allow the customization of immunosuppression.

Several studies have evaluated TTV load as a biomarker for defining the net state of immunosuppression in transplant recipients. A comparison of TTV levels in transplant recipients who received either tacrolimus- or cyclosporine-based therapy revealed that cyclosporine-treated patients had significantly lower TTV DNA levels in serum at 1 month compared with tacrolimus-treated patients.²⁹ This study reported that TTV DNA load appears to reflect the function of the immune system after transplant, depending on the type of immunosuppressive treatment. However, there was no association between either TTV load and infectious events or acute rejections, which suggests a limited clinical applicability as a biomarker to predict short-term outcomes related to the net state of immunosuppression. On the other hand, plasma TTV levels <7.0 log 10 copies/mL posttransplant were shown to be significantly associated with a high risk for the development of chronic rejection in lung transplant recipients.³⁰ In concordance with these data, other investigators have found that anellovirus levels in heart and lung transplant recipients with acute rejection episodes were lower than levels in patients without rejection. It is tempting to speculate that a higher immunocompetence in TTV-negative patients at transplant could be responsible for the higher incidence of rejection episodes observed during the first year posttransplant.³¹

Antibody-mediated rejection (ABMR) represents one of the cardinal causes of late allograft loss after kidney transplant. Considerable increases in TTV DNA load have been shown, with a peak within the first 3 months, followed by a slow decrease over the following 2 years. In the BORTEJECT trial, 715 kidney allograft recipients were subjected to ABMR screening at a median of 6.3 years posttransplant. Forty-six of these patients (6%) were diagnosed with ABMR (44 patients with acute or chronic active ABMR, 2 with chronic inactive ABMR). Torque teno virus DNA was detected in 678 patients (95%) with a median of 2.3 × 10⁵ copies/mL2.3 × 105 copies/mL.³²

Torque teno viral load has been shown to be highest in patients screened 6 to 12 months after transplant, with a median of 3.5 × 10⁶ copies/mL. Antibody induction therapy and tacrolimus-based initial immunosuppression were associated with 2-fold higher TTV levels. In contrast, TTV levels were significantly lower in patients on cyclosporine- or mammalian target of rapamycin inhibitor-based treatment. A remarkable finding was that initial treatment with belatacept was associated with a 45-fold increase in viral load. When the investigators³² analyzed immunosuppressive therapy at the time of screening, they found that patients on triple immunosuppression had 2-fold higher TTV levels than patients with dual immunosuppression or single therapy; in addition, lower levels were detected in patients who were off steroids or antimetabolites. Again, tacrolimus was associated with higher and cyclosporine with lower levels of TTV, without any relationship to trough levels. Finally, TTV load in patients on belatacept at the time of screening was 35 times higher.³²

Torque teno viral load among ABMR-positive recipients was 4-fold lower than in patients who had no ABMR (6.6 ×10⁴ vs 2.6 × 10⁵ copies/mL; P = .003). A robust and linear inverse association between TTV load and ABMR was confirmed using score tests. Univariate logistic regression revealed a decrease in risk of ABMR of 0.91 per TTV log level (95% CI, 0.87-0.96; P = .001). Logistic regression models demonstrated that associations between TTV load and ABMR were independent of potential confounders. Similarly, a generalized linear model revealed an inverse independent association with TTV log level and ABMR risk (relative risk of 0.94 per TTV log level; 95% CI, 0.90-0.99; P = .02). Finally, TTV load was found to inversely correlate with mean fluorescence intensity of peak DSA, a marker that is shown to be associated with ABMR diagnosis.³²

In another study, Strassl and associates³³ investigated 1010 consecutive patients from the prospective Vienna Kidney Transplant Cohort Study for availability of allograft biopsies and adequately stored sera for TTV quantification by PCR. Results showed that patients with acute biopsy-proven alloreactivity according to the Banff classification (n = 33) showed lower levels of TTV in the peripheral blood compared with patients without rejection (n = 80) at a median of 43 days before biopsy. The risk of alloreactivity decreased by 10% per log level of TTV copies/mL (P = .005). They found that TTV levels >1 × 10⁶ copies/mL excluded rejection with a sensitivity of 94%. Multivariable generalized linear modeling suggested an independent association between TTV level and alloreactivity.³³

In light of the above-mentioned data from various studies, TTV could be able to join the current list of predictive biomarkers in transplantation to detect whether the patient is either over- or under-immunosuppressed. Several studies are needed to confirm or disprove the use of TTV as a biomarker.

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