On 2014 Jan 08, Daniele Focosi commented:

Anelloviruses and immune competence.

We read with interest the recent article by De Vlaminck et al showing marked human plasma virome compositional dynamics in 96 heart and lung transplant recipients (De Vlaminck et al., 2013). Since most fluctuations in plasma virome were due to expansions and contractions of members of the family Anelloviridae, specifically torquetenovirus (TTV) species, these authors suggested that levels and fluctuations of these viruses in plasma might serve as indicators of immune system functionality These findings and hypotheses are not unexpected to us. In 2008, using a quantitative real-time PCR assay targeting conserved TTV genome regions, we reported very similar dynamics in myeloma and lymphoma patients receiving high-dose chemotherapy supported by autologous hematopoietic stem cells transplantation. Here we found a clear correlation between peaks of TTV viremia and expansion of senescent CD8+CD57+ T lymphocytes, which are known to soothe immune responses and are associated with CMV infections (Maggi et al., 2008 ). We then demonstrated that the time needed for TTV viremia to return to baseline levels after the peak that inexorably followed conditioning predicts how long is the time for recovery of the immune system function, and hence we proposed TTV viremia as a surrogate marker of functional immune competence (Focosi et al., 2010 ). We finally confirmed the preeminent role of hematopoietic cells in controlling plasma TTV viremia in fully myeloablated recipients of haploidentical stem cell transplantations (Maggi et al., 2010). We also found TTV viremia kinetics similar to those reported by De Vlaminck et al in 114 kidney and pancreas transplant recipients followed-up for 1 year (Focosi et al., 2013; manuscript in preparation). CMV positive (donor or recipient) transplant cases were treated with anti-CMV prophylaxis, valganciclovir. Here again we found that the hematopoietic cells played a pivotal role in regulating plasma TTV viremia, and demonstrated a massive drop of TTV viremia in the immediate days after lympho-depleting induction regimens. TTV viremia rebounded after day 7, peaked at month 3-6 to decline thereafter during maintenance immunosuppression. Interestingly, although statistical limitations did not allow to demonstrate a correlation between TTV viremia levels and graft rejections, we found a positive association with CMV reactivations. In their paper, De Vlaminck et al. used shotgun sequencing to determine human virome composition in cell-free plasma DNA and concluded that this data offer a potential application to monitor the effect of pharmacological treatment and predict immunocompetence. Shotgun sequencing is a powerful technique but is time-consuming, expensive, and poorly apt for routine diagnostics. In our papers we have instead shown that a fast and cheap quantitative, TTV-universal real-time PCR can provide an estimate of immunocompetence and be a simple and practical tool for tailor-made maintenance immune suppression. In conclusion, although we should be aware that the factors that can impact on TTV viremia levels and complexity are numerous (including polymorphisms in innate immunity genes and superinfections with different TTV genotypes) and largely uninvestigated, we totally agree with De Vlaminck et al that replication of TTV in humans is a promising marker to monitor functional activity of immune system. We declare that we don’t have any conflict of interest related to this comment.

Dr. Fabrizio Maggi, MD^1,2,*,# Dr. Daniele Focosi, MD^3,* Prof. Mauro Bendinelli, MD, PhD³ Prof. Ugo Boggi, MD, PhD³ Prof. Mauro Pistello, PhD³

¹ Virology Section, Pisa University Hospital, Pisa, Italy ² Chair, Anelloviridae Study Group, International Committee on Taxonomy of Viruses ³ Department of Translational Research, University of Pisa, Pisa, Italy

^* both authors contributed equally to this manuscript. ^# corresponding author : Virology Section, Pisa University Hospital, via Paradisa 2, 56124 Pisa, Italy. E-mail: fabrizio.maggi63@gmail.com. Phone : +39 050 997055

References : 1. De Vlaminck, I., Khush, K.K., Strehl, C., Kohli, B., Luikart, H., Neff, N.F., Okamoto, J., Snyder, T.M., Cornfield, D.N., Nicolls, M.R., et al. (2013). Temporal response of the human virome to immunosuppression and antiviral therapy. Cell 155, 1178-1187. 2. Focosi, D., Macera, L., Santi, M., Vistoli, F., Pistello, M., Scatena, F., Maggi, F., and Boggi, U. (2013). TTV kinetics as a novel marker of functional immune deficiency. Paper presented at: European Society for Organ Transplantation (ESOT) (Vienna). 3. Focosi, D., Maggi, F., Albani, M.M., L, Ricci, V.G., S, Di Beo, S.G., M, Antonelli, G., Bendinelli, M.P., M, and Ceccherini-Nelli, L.P., M (2010 ). Torquetenovirus viremia kinetics after autologous stem cell transplantation are predictable and may serve as a surrogate marker of functional immune reconstitution. J Clin Virol 47, 189-192. 4. Maggi, F., Focosi, D., Albani, M., Lanini, L., Vatteroni, M.L., Petrini, M., Ceccherini-Nelli, L., Pistello, M., and Bendinelli, M. (2010). Role of hematopoietic cells in the maintenance of chronic human torquetenovirus plasma viremia. J Virol 84, 6891-6893. 5. Maggi, F., Focosi, D., Ricci, V., Paumgardhen, E., Ghimenti, M., Bendinelli, M., Ceccherini-Nelli, L., Papineschi, F., and Petrini, M. (2008 ). Changes in CD8+57+ T lymphocyte expansions after autologous hematopoietic stem cell transplantation correlate with changes in torquetenovirus viremia. Transplantation 85, 1867-1868.

On 2014 Jan 08, Daniele Focosi commented:

Anelloviruses and immune competence.

We read with interest the recent article by De Vlaminck et al showing marked human plasma virome compositional dynamics in 96 heart and lung transplant recipients (De Vlaminck et al., 2013). Since most fluctuations in plasma virome were due to expansions and contractions of members of the family Anelloviridae, specifically torquetenovirus (TTV) species, these authors suggested that levels and fluctuations of these viruses in plasma might serve as indicators of immune system functionality These findings and hypotheses are not unexpected to us. In 2008, using a quantitative real-time PCR assay targeting conserved TTV genome regions, we reported very similar dynamics in myeloma and lymphoma patients receiving high-dose chemotherapy supported by autologous hematopoietic stem cells transplantation. Here we found a clear correlation between peaks of TTV viremia and expansion of senescent CD8+CD57+ T lymphocytes, which are known to soothe immune responses and are associated with CMV infections (Maggi et al., 2008 ). We then demonstrated that the time needed for TTV viremia to return to baseline levels after the peak that inexorably followed conditioning predicts how long is the time for recovery of the immune system function, and hence we proposed TTV viremia as a surrogate marker of functional immune competence (Focosi et al., 2010 ). We finally confirmed the preeminent role of hematopoietic cells in controlling plasma TTV viremia in fully myeloablated recipients of haploidentical stem cell transplantations (Maggi et al., 2010). We also found TTV viremia kinetics similar to those reported by De Vlaminck et al in 114 kidney and pancreas transplant recipients followed-up for 1 year (Focosi et al., 2013; manuscript in preparation). CMV positive (donor or recipient) transplant cases were treated with anti-CMV prophylaxis, valganciclovir. Here again we found that the hematopoietic cells played a pivotal role in regulating plasma TTV viremia, and demonstrated a massive drop of TTV viremia in the immediate days after lympho-depleting induction regimens. TTV viremia rebounded after day 7, peaked at month 3-6 to decline thereafter during maintenance immunosuppression. Interestingly, although statistical limitations did not allow to demonstrate a correlation between TTV viremia levels and graft rejections, we found a positive association with CMV reactivations. In their paper, De Vlaminck et al. used shotgun sequencing to determine human virome composition in cell-free plasma DNA and concluded that this data offer a potential application to monitor the effect of pharmacological treatment and predict immunocompetence. Shotgun sequencing is a powerful technique but is time-consuming, expensive, and poorly apt for routine diagnostics. In our papers we have instead shown that a fast and cheap quantitative, TTV-universal real-time PCR can provide an estimate of immunocompetence and be a simple and practical tool for tailor-made maintenance immune suppression. In conclusion, although we should be aware that the factors that can impact on TTV viremia levels and complexity are numerous (including polymorphisms in innate immunity genes and superinfections with different TTV genotypes) and largely uninvestigated, we totally agree with De Vlaminck et al that replication of TTV in humans is a promising marker to monitor functional activity of immune system. We declare that we don’t have any conflict of interest related to this comment.

Dr. Fabrizio Maggi, MD^1,2,*,# Dr. Daniele Focosi, MD^3,* Prof. Mauro Bendinelli, MD, PhD³ Prof. Ugo Boggi, MD, PhD³ Prof. Mauro Pistello, PhD³

¹ Virology Section, Pisa University Hospital, Pisa, Italy ² Chair, Anelloviridae Study Group, International Committee on Taxonomy of Viruses ³ Department of Translational Research, University of Pisa, Pisa, Italy

^* both authors contributed equally to this manuscript. ^# corresponding author : Virology Section, Pisa University Hospital, via Paradisa 2, 56124 Pisa, Italy. E-mail: fabrizio.maggi63@gmail.com. Phone : +39 050 997055

References : 1. De Vlaminck, I., Khush, K.K., Strehl, C., Kohli, B., Luikart, H., Neff, N.F., Okamoto, J., Snyder, T.M., Cornfield, D.N., Nicolls, M.R., et al. (2013). Temporal response of the human virome to immunosuppression and antiviral therapy. Cell 155, 1178-1187. 2. Focosi, D., Macera, L., Santi, M., Vistoli, F., Pistello, M., Scatena, F., Maggi, F., and Boggi, U. (2013). TTV kinetics as a novel marker of functional immune deficiency. Paper presented at: European Society for Organ Transplantation (ESOT) (Vienna). 3. Focosi, D., Maggi, F., Albani, M.M., L, Ricci, V.G., S, Di Beo, S.G., M, Antonelli, G., Bendinelli, M.P., M, and Ceccherini-Nelli, L.P., M (2010 ). Torquetenovirus viremia kinetics after autologous stem cell transplantation are predictable and may serve as a surrogate marker of functional immune reconstitution. J Clin Virol 47, 189-192. 4. Maggi, F., Focosi, D., Albani, M., Lanini, L., Vatteroni, M.L., Petrini, M., Ceccherini-Nelli, L., Pistello, M., and Bendinelli, M. (2010). Role of hematopoietic cells in the maintenance of chronic human torquetenovirus plasma viremia. J Virol 84, 6891-6893. 5. Maggi, F., Focosi, D., Ricci, V., Paumgardhen, E., Ghimenti, M., Bendinelli, M., Ceccherini-Nelli, L., Papineschi, F., and Petrini, M. (2008 ). Changes in CD8+57+ T lymphocyte expansions after autologous hematopoietic stem cell transplantation correlate with changes in torquetenovirus viremia. Transplantation 85, 1867-1868.