Evaluation of TTV replication as a biomarker of immune checkpoint inhibitors efficacy in melanoma patients.

Torque Teno Virus (TTV) is a small, non-enveloped, single-stranded and circular DNA virus that infects the majority of the population worldwide. Increased levels of plasma TTV viral load have been observed in various situations of immune deficiency or dysregulation, and several studies have suggested that TTV levels may be inversely correlated with immune competence. The measurement of TTV viremia by qPCR has been proposed as a potential biomarker for the follow-up of functional immune competence in immunosuppressed individuals, particularly hematopoietic stem cell transplant recipients. We hypothesized that TTV viral load could be used as a prognostic marker of immune checkpoint inhibitor (ICI) efficacy, and therefore investigated the TTV viral load in melanoma patients treated with nivolumab or pembrolizumab before and after 6 months of treatment. In the present study, TTV viral load was not different in melanoma patients before anti-PD-1 introduction compared to healthy volunteers, was not modified by ICI treatment and did not allowed to distinguish patients with treatment-sensitive tumor from patients with treatment-resistant tumor.

No correlation between Torque Teno virus viral load and BK virus replication after kidney transplantation.

BACKGROUND: Assessment of the intensity of immunosuppression in transplant recipients to estimate the risk of rejection and infection is not entirely satisfactory at the present time. Determination of Torque teno virus (TTV) viral load appears to be a promising tool in this setting. OBJECTIVES: We evaluated the level of replication and kinetics of TTV during the first three months after kidney transplantation compared to BK virus replication. RESULTS: In a retrospective cohort of 116 renal transplant recipients, TTV viral load gradually increased during the first three months post-transplantation with no significant difference or discriminatory threshold between patients with and without BK virus replication. However, the level of TTV replication appeared to be indirectly related to the risk of BK virus replication, particularly according to the induction treatment used (antithymocyte globulin: ATG or basiliximab). Among patients receiving ATG, those receiving cyclosporine had significantly lower TTV viral loads (p < 0.01) with threefold lower reactivation of BKPyV (13 vs 37%) 3 months post-transplantation. Similarly, among the women in our cohort, TTV viral load was significantly higher in women receiving ATG (6.58 ± 1.57 versus 4.62 ± 2.0 log10 copies/mL for basiliximab: p < 0.01), also with threefold higher BKPyV reactivation frequencies (40 vs 13,3%). CONCLUSION: The multiparametric variation of TTV viral load does not appear to be individually appropriate for the early detection or monitoring of possible post-transplant BKPyV virus reactivation in renal transplant recipients.

Role of BK polyomavirus (BKV) and Torque teno virus (TTV) in liver transplant recipients with renal impairment.

PURPOSE: Renal impairment is a common complication after liver transplantation (LT). While BK polyomavirus (BKV) has been linked to renal failure in kidney transplant recipients, Torque teno virus (TTV) is a surrogate marker for immunosuppression that does not have a clear association with any human disease. The impact of BKV and TTV on renal impairment after LT is unknown. METHODOLOGY: In this retrospective study, urine and serum samples from 136 liver transplant recipients were screened for BKV and TTV by quantitative PCR. In addition, serum was screened for BKV-specific antibodies and the VP1 typing region was sequenced for BKV genotyping. All parameters were correlated with clinical data.Results/Key findings. BK viruria was detected up to 21 years after transplantation in 16.9 % of cases. BK viraemia was detected in 8.7 % of patients with BK viruria up to 4 years after LT. BKV-specific antibodies were detected in 93.6 % of all LT recipients and correlated with BKV viral load in urine. There was no correlation between renal impairment and the detection of BK DNA in urine (OR 0.983). TTV DNA was detected in 84.6 % of serum samples and in 66.6 % of urine samples. The TTV viral load in serum correlated with the BKV viral load but had no impact on renal impairment. CONCLUSION: Our data indicate that the detection of BKV and TTV is not a risk factor for renal impairment after LT. A correlation of TTV and BKV viral load seems to be an indicator for the immune status of the host.

High-dose zinc oral supplementation after stem cell transplantation causes an increase of TRECs and CD4+ naïve lymphocytes and prevents TTV reactivation.

INTRODUCTION: Zinc plays an important role in thymic function and immune homeostasis. We performed a prospective clinical trial using a high-dose zinc oral supplementation to improve the immune reconstitution after hematopoietic stem cell transplant (HSCT). PATIENTS AND METHODS: We enrolled 18 patients undergoing autologous HSCT for multiple myeloma. Nine patients were randomized to receive only a standard antimicrobial prophylaxis; whereas, nine patients received in addition 150 mg/day of zinc from day +5 to day +100 after transplant. RESULTS: CD4+ naïve lymphocytes and TRECs showed a significant increase from day +30 until day +100 only in the zinc-treated group. Moreover, the load of Torquetenovirus, a harmless virus that replicates in course of immunedepression, increased at day +100 only in the control group. No severe adverse events were reported during the zinc consumption. CONCLUSION: First data from the ZENITH trial suggest that high-dose zinc supplementation is safe and may enhance the thymic reconstitution after HSCT. Registered: http://Clinicaltrials.gov (NCT03159845); and EUDRACT: 2014-28 004499-47.

Torque teno virus dynamics during the first year of life.

BACKGROUND: Torque teno virus is a small chronically persisting circular negative ssDNA virus reaching near 100% prevalence. It is reported to be a marker for immune function in immunocompromised patients. The possibility of vertical maternal-fetal transmission remains controversial but incidence rate of TTV DNA in children increased with age. TTV dynamics well studied for allogeneic hematopoietic stem cell transplantation as a predictor of post-transplant complications but there is no viral proliferation kinetics data for other patient groups or healthy individuals. The aim of this study was to determine TTV dynamics during the first year of life of healthy infants. METHODS: Ninety eight clinically healthy breastfeeding infants (1-12 months of age) were analyzed by quantitative PCR for the whole blood TTV load with the test sensitivity of about 1000 viral copies per milliliter of blood (total number of samples including repeatedly tested infants was 109). RESULTS: 67% of all analyzed samples were TTV-positive demonstrating significant positive correlation between age and TTV load (r = 0.81, p < 0.01). CONCLUSIONS: This is the first study to suggest that viral load increases during the first year of life reaching a plateau after 6 months with strong proliferation for the first 60 days. Our data well correlates with TTV dynamics in patients following allogeneic hematopoietic stem cell transplantation.

Torquetenovirus Dynamics and Immune Marker Properties in Patients Following Allogeneic Hematopoietic Stem Cell Transplantation: A Prospective Longitudinal Study.

Torquetenovirus (TTV) has been proposed as a marker of immune function in patients receiving immunosuppression after solid organ transplantation. This study aimed to define TTV plasma dynamics and investigate clinical associations in patients following allogeneic hematopoietic stem cell transplantation (HSCT). This was a single-center prospective longitudinal study involving 50 consecutive patients treated with HSCT between March 2015 and April 2016. TTV plasma DNA levels were measured with quantitative PCR at 12 consecutive time points during the first year after HSCT. Forty of the 50 patients (80%) had detectable TTV viremia before HSCT (median level, 5.37 log10 copies/mL; interquartile range [IQR], 3.51-6.44 log10 copies/mL). All patients subsequently developed TTV viremia during the follow-up period. Plasma viral loads evolved dynamically over time, with a peak of 8.32 log10 copies/mL (IQR, 7.33-9.35 log10 copies/mL) occurring at 79 days (IQR, 50-117 days) following HSCT and a stable plateau toward the end of the follow-up period. The type of malignancy, the use of antithymocyte globulin during conditioning, and the occurrence of acute graft-versus-host disease requiring systemic therapy had temporary effects on TTV dynamics. TTV levels showed a significant correlation with absolute lymphocyte counts following engraftment (rs = -.27; P < .01) and with cytomegalovirus (CMV; rs=.39; P < .01) and Epstein-Barr virus (EBV; rs=.45; P = .02) viral loads during phases of viremia. Immune-related clinical events were not predicted by TTV levels. TTV viremia occurred universally and was sustained throughout the first year after HSCT. Several variables and events before and after HSCT were correlated with TTV levels and hint toward immune marker properties of TTV, but their complex interactions might perturb the capability of TTV to predict immune-related complications in this population.

Detection and Phylogenetic Analysis of Torque Teno Virus in Salivary and Tumor Biopsy Samples from Head and Neck Carcinoma Patients.

OBJECTIVES: Because torque teno virus (TTV) has been implicated in tumorigenesis as a cocarcinogen, we studied TTV prevalence in saliva and biopsy samples from head and neck cancer (HNCC) patients, patients with premalignant lesions of oral cancer, and controls. We also wished to determine the TTV genotypes in HNCC patients. METHODS: A seminested polymerase chain reaction (PCR) amplifying the N22 region of the TTV genome, as well as direct sequencing of PCR fragments, was used. RESULTS: TTV prevalence was higher in HNCC patients (saliva: 27/71, 38%; tumor biopsy: 22/74, 30%) than in controls (saliva: 8/56, 14%; oral mucosa: 1/19, 5%). TTV prevalence was also high in patients with premalignant lesions of oral carcinoma (saliva: 9/18, 50%; biopsy: 5/21, 24%). By phylogenetic analysis, TTV belonging mostly to genotypes 1 and 2 was found in HNCC patients. In most of the cases, identical TTV strains were present in the biopsy and salivary sample of the same HNCC patient. In addition, the same TTV strain was detected in 2 laryngeal carcinoma biopsies obtained from 2 independent patients. CONCLUSIONS: Our data are compatible with the idea that TTV might act as a cocarcinogen in certain cases of HNCC. Alternatively, HNCC may facilitate either TTV replication or TTV entry into the saliva.

Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and Torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain.

In this study we detail the detection and genetic analysis of a novel porcine boca-like virus (PBo-likeV) in archival sera and tissue samples from pigs from farms in Great Britain. We also investigate the distribution of porcine circovirus type 2 (PCV2) genotypes and Torque teno sus virus (TTSuV) genogroups 1 and 2 in combination with this novel PBo-likeV. PBo-likeV was detected in over 70% of all tissues investigated. Over 24% of all tissues recovered from PMWS-affected animals had all viruses present and 25% of tissues recovered from non-PMWS-affected pigs were positive for all 4 viruses.

Hepatitis viruses (other than hepatitis B and C viruses) as causes of hepatocellular carcinoma: an update.

Chronic hepatitis B and C virus infections are universally accepted as causes of hepatocellular carcinoma in humans. Hepatitis A and E viruses cause only acute self-limiting infections of the liver. Of the remaining hepatitis viruses - Delta hepatitis, hepatitis G (GB-C), TT and SEN - all have at some time been incriminated as causes of hepatocellular carcinoma. Delta hepatitis virus requires helper functions from hepatitis B virus to become invasive. Chronic Delta/hepatitis B viral co-infection runs a more severe course than that resulting from chronic hepatitis B virus infection alone, with progression to cirrhosis being more likely and more rapid. A substantial majority of the early studies did not find an increased incidence of hepatocellular carcinoma in co-infected individuals. But more recently, an increased incidence of the tumour has been recorded more often than no increase. Further studies are needed to draw a firm conclusion with regard to the hepatocarcinogenic effect of dual Delta/hepatitis B virus co-infection. With one exception, no published study (of 13) has incriminated chronic infection with hepatitis G virus as a cause of hepatocellular carcinoma. The dissenting study, published in 1999, was the only one performed in the United States. Fewer studies of the hepatocarcinogenic effect of TT virus have been performed. Apart from one study, published in 1999, no convincing evidence is available that supports a causal role for TT virus in hepatocarcinogenesis. The exception was in Japanese patients with high hepatitis C viral loads but independent of chronic hepatitis C virus infection. No evidence has been produced to indicate that SEN virus causes hepatocellular carcinoma.

[TTV and HPV co-infection in cervical smears of patients with cervical lesions in littoral of Zhejiang province].

OBJECTIVE: To investigate the prevalence of transfusion-transmitted virus (TTV) and human papillomavirus (HPV) co-infection in cervical smears of patients with cervical lesions in littoral of Zhejiang province and analysis of transmitted route. METHODS: Nested polymerase chain reaction (nPCR) was established. TTV DNA were tested by nPCR in cervical smears of 95 patients with cervical lesions and 55 healthy women, paired serum samples were available from 55 and 42 women, and their viral titer. The genotypes of 95 specimens of cervical cytology were detected with HybriMax. The phylogenetic group of TTV was determined by means of nPCR with N22 primers. RESULTS: The prevalence of TTV DNA in cervical smears of patients with cervical lesions and healthy women was 52.7% (29/55) and was comparable with that in paired serum sample (50%). Symptomatic women had significantly higher prevalence of TTV DNA in cervical smears (74.7%) than healthy controls (P = 0.005). The TTV DNA prevalence in patient serum samples was 51%. The phylogenetic groups of TTV serum isolates were concordant with those of TTV from cervical smears of the same subjects, and genotype was G1b. The TTV viral titer in cervical smears were 10 to 1000 times as high as in serum. The total infection rate of HPV was 98.9% in patients, and was 27.3% in healthy women. The frequently detected genotype was HPV16, 18, 33 of HSIL, and HPV6 of LSIL. The HPV positive study subjects had significantly higher TTV DNA prevalence than HPV negatives (P = 0.02). CONCLUSION: High prevalence of TTV in cervical smears suggests that sexual transmission is another mode of expansion of TTV infection among the population. The higher viral titer in cervical smears than in the respective serum samples might indicate active TTV replication in the female genital tract. Nevertheless, cooperation between TTV and HPV needs to be further investigated.

TT viruses: oncogenic or tumor-suppressive properties?

Torque teno (TT) viruses have been more frequently reported in malignant biopsies when compared to normal control tissue. The possible contribution of TT virus infection to human carcinogenesis or the potential oncolytic functions of these virus infections are being discussed based on available experimental evidence. The data could suggest an involvement of TT virus infections as an indirect carcinogen by modulating T cell immune responses. Significant oncolytic functions, potentially mediated by the inhibition of nuclear factor (NF)-kappaB transcription factor or by apoptin-like gene activities, are emerging to be less likely.

Apoptosis-inducing proteins in chicken anemia virus and TT virus.

Torque teno viruses (TTVs) share several genomic similarities with the chicken anemia virus (CAV). CAV encodes the protein apoptin that specifically induces apoptosis in (human) tumor cells. Functional studies reveal that apoptin induces apoptosis in a very broad range of (human) tumor cells. A putative TTV open reading frame (ORF) in TTV genotype 1, named TTV apoptosis inducing protein (TAIP), it induces, like apoptin, p53-independent apoptosis in various human hepatocarcinoma cell lines to a similar level as apoptin. In comparison to apoptin, TAIP action is less pronounced in several analyzed human non-hepatocarcinoma-derived cell lines. Detailed sequence analysis has revealed that the TAIP ORF is conserved within a limited group of the heterogeneous TTV population. However, its N-terminal half, N-TAIP, is rather well conserved in a much broader set of TTV isolates. The similarities between apoptin and TAIP, and their relevance for the development and treatment of diseases is discussed.

In vivo and in vitro intragenomic rearrangement of TT viruses.

The in vitro replication of the Torque teno virus (TT virus) tth8 full-length genome and particle formation in a Hodgkin's lymphoma-derived cell line after transfection with cloned viral DNA were demonstrated. Analyses of the transcription patterns of tth8 and tth7 TT virus isolates in a number of lymphoma and T-cell leukemia cell lines indicated differential additional splicing events and intragenomic rearrangement generating open reading frames which could not be deducted from the genomic sequence. We also demonstrated the presence of rearranged TT virus genomes in vivo in sera taken from pregnant mothers whose children later developed childhood leukemia, as well as sera from control mothers. Control experiments using religated cloned genomic tth8 DNA mixed with cellular DNA did not result in such subviral molecules. These subviral isolates ranged from 172 bp to full-length TT virus genomes. Possible in vivo selection for specific rearranged molecules was indicated by the presence of one isolate (561 bp) in 11 serum samples. It remains to be clarified whether selected rearranged subviral components resulting from specific TT virus types may contribute to the initiation of disease. These data demonstrate new features of TT viruses suggesting possible similarities to plant viruses of the family Geminiviridae, as well as raise questions about the documented plurality and diversity of anelloviruses.

Torque teno virus (TTV): current status.

Torque teno virus (TTV), currently classified into the family Circoviridae, genus Anellovirus, was first found in a patient with non-A-E hepatitis. TTV has a single stranded circular DNA of approximately 3.8 kb. TTVs are extraordinarily diverse, spanning five groups including SANBAN and SEN viruses. Torque teno mini virus (TTMV) with approximately 2.9 kb genome also has wide variants. Recently, two related 2.2- and 2.6-kb species joined this community. Recombinations between variants are frequent. This extensive TTV diversity remains unexplained; it is unclear how TTVs could be viable, and why they require such genetic variation. An unequivocal culture system is still not available. TTVs are ubiquitous in > 90% of adults worldwide but no human pathogenicity of TTV has been fully established. Epidemiological surveys need to specify the variants being studied and clinical targets, and must calibrate the sensitivity of the assay used. Potentially interesting observations include a higher viral load in patients with severe idiopathic inflammatory myopathies, cancer and lupus. Active replication was also found in infants with acute respiratory diseases. TTV/TTMV-related viruses were found in chimpanzees, apes, African monkeys and tupaias, and also in chickens, pigs, cows, sheep and dogs. Experimentally, rhesus monkeys were persistently infected by TTV, but only 1/53 chimpanzees. TTV transcribes three species of mRNAs, 3.0-, 1.2- and 1.0-kb in the ratio of 60:5:35. Recently, at least three mRNAs were shown in chicken anaemia virus. The genomic region -154/-76 contains a critical promoter. TTV seems to have at least three proteins; however, the definite functions of these proteins await further research work.

Replication of TT virus in hepatocyte and leucocyte cell lines.

The TT virus (TTV) is a non-enveloped, single-stranded, circular, DNA virus, first isolated from a patient with hepatitis of unexplained etiology. The much deliberated pathological role of the virus continues to be conjectural in the absence of a suitable in vitro replication model. So far, the liver and the bone marrow have been shown to be the main sites of TTV replication. In this study, the human cell lines HepG2 and Chang Liver, the rat hepatoma cell line MH1C1, phytohemagglutinin (PHA)-stimulated TTV-negative peripheral blood mononuclear cell (PBMC) cultures and the B lymphoblast cell line, Raji were investigated as potential in vitro replication systems for TTV. The cell lines were infected with an inoculum prepared by pooling TTV genotype1 DNA positive sera and monitored for virus replication. Of the three hepatocyte cell lines, while the HepG2 and MH1C1 cell lines did not support TTV replication, the Chang Liver cell line showed clear morphological changes as a result of the in vitro infection, which included clumping and granular degeneration of the entire cell sheet over a period of 6 days. The infected cells also showed presence of virus-specific mRNA representative of viral transcription. The consistent presence of infectious viral particles in the supernatant culture fluid at 24-hr fluid replacement intervals indicated limited extra-cellular release of viral particles. The PHA-stimulated TTV-negative PBMC cultures and the Raji cell line were also able to support TTV replication and released significant levels of infectious viral particles into the supernantant culture fluid.

TT virus has a ubiquitous diffusion in human body tissues: analyses of paired serum and tissue samples.

The tissue tropism and possible correlation with liver disease of the TT virus (TTV) as well as its prevalence and genotype distribution remain undefined. TTV-DNA was investigated in paired sera and tissue samples from 144 patients, and sera and cerebrospinal fluids (CSF) from additional six subjects. Of the 144 tissue samples, 128 were liver biopsy specimens from subjects with hepatic disease while 16 were surgically obtained nonliver specimens from patients with extrahepatic disease. TTV cloning, sequencing and genotype analyses were performed on isolates from sera, tissue specimens and peripheral blood mononuclear cells of two patients with hepatic and four patients with extrahepatic pathologies, as well as from sera and CSFs of two subjects. TTV was found in 100% of the examined tissues and in 60.1 and 50% of sera from patients with hepatic and extrahepatic pathologies, respectively. Moreover, TTV was detected in four of the six CSFs analysed but only in two correspondent sera. Genotyping revealed the coexistence of multiple TTV genotypes and genetic variants in each infected individual, and the analysis of TTV mRNA showed the presence of transcripts in all the six different tissues studied. These results indicate that the entire adult population in our area is more likely infected by TTV, although several subjects are not viraemic and that TTV infects many different human tissues and is able to invade the central nervous system.

TT virus replicates in stimulated but not in nonstimulated peripheral blood mononuclear cells.

TT virus (TTV) is an unenveloped, single-stranded, circular-DNA virus which resembles members of the Circoviridae, that is commonly found in humans and which lacks pathological consequences for the infected host. TTV replication has been demonstrated in bone marrow cells but not in peripheral blood mononuclear cells (PBMC), suggesting that hematopoietic cells must be activated to support TTV replication. To test this hypothesis, PBMC from two naturally TTV-infected individuals and from two healthy TTV-DNA negative donors infected in vitro with a TTV-DNA-positive serum were cultured in the presence (stimulated) or absence (unstimulated) of phytohemagglutinin, lipopolysaccharide, and interleukin-2. TTV-DNA was detected in both stimulated and unstimulated PBMC. However, TTV-DNA replicative intermediates and mRNA were detected only in stimulated PBMC. Furthermore, TTV-DNA and mRNA were detected in PBMC from two TTV negative donors reinfected with supernatants from TTV-infected stimulated cells but not when using culture supernatants from unstimulated cells. These results demonstrate that TTV replicates in PBMC only when stimulated.

Tissue tropism of the TTV in experimentally infected rhesus monkeys.

OBJECTIVE: To determine whether the transfusion-transmitted virus (TTV) is hepatotropic. METHODS: Total DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes. RESULTS: The double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues. CONCLUSIONS: As the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic.