Use of Torctenovirus (TTV) as a marker to measure the proliferative capacity of T lymphocytes

Abstract

The subject of the present invention is a method for determining the proliferative capacity of T lymphocytes in a subject, said method comprising the following steps: a) measuring the amount of Torque Teno virus from a biological sample of said subject; and b) determining the proliferative capacity of T lymphocytes based on the amount of virus measured in a).

Description

[Technical Field]

[0001] The immune system defends the body from attacks such as pathogenic infection, cell transformation, and physical or chemical damage. Thus, an immune individual can trigger a protective immune response to antigenic stimuli.

[0002] However, when the immune system is weakened or completely absent, immunodeficiency develops. This can take various forms and, depending on the cause of the deficiency, may affect the innate immune system, the adaptive immune system, or both. In many cases, immunodeficiency is acquired throughout life. It can be caused by pathology, whether infectious or not (e.g., HIV infection), or it can be induced by treatments such as radiation therapy or chemotherapy. Immunodeficiency is particularly dangerous because patients subsequently show increased susceptibility to secondary infections by pathogens such as bacteria, viruses, parasites, and fungi. For example, immunosuppressive therapy for grafts, especially the use of hematopoietic stem cell transplantation (HSCT), can cause recurrent, or in some cases fatal, microbial infections in patients.

[0003] Therefore, it is important to be able to determine the function of the target immune system. This allows for the adaptation of an appropriate therapeutic response, especially when immunodeficiency is identified.

[0004] While various techniques are available for measuring immune function, none are completely satisfactory. These techniques measure immune responses to cell-mediated reactions, particularly the analysis of lymphocyte populations, especially CD4 + T cell count or CD4 + / CD8 + This includes measuring the ratio of T cells, measuring lymphocyte proliferation, measuring T cell cytotoxicity, measuring antibody response, labeling tetramers, detecting produced cytokines, and using ELISpot, etc.

[0005] For example, some of these techniques, such as T cell counting, give results that do not necessarily reflect the activity of these cells, and consequently, the activity of the immune system. The absolute number of T cells does not indicate anything about the ability of these cells to proliferate. For example, a small number of T cells after HSCT does not necessarily mean that these T cells are unable to proliferate and therefore unable to protect the patient from microbial infection. Furthermore, cell proliferation tests are difficult to perform routinely and can be difficult to standardize. Therefore, there is still a need for simple and easy-to-use methods to determine immune function.

[0006] Torquettenovirus (TTV) is a virus of the Anelloviridae family that was first identified in 1997 in Japanese patients with post-transfusion hepatitis (1-7). TTV is a virus with a small size (approximately 3.8 kb) of single-stranded circular DNA, containing a coding region with great genetic diversity and a well-conserved non-coding region (UTR). The use of primers to amplify the sequence of this non-coding region has shown the high global prevalence of TTV (approximately 90%). TTV causes chronic infection without clearly associated clinical findings. It is called a non-pathogenic or orphan virus. Thus, although many studies have dealt with the involvement of TTV in human pathology, particularly in certain liver pathologies, the clear role of this virus has not been identified.

[0007] However, high TTV levels have been observed in immunocompromised individuals. For example, in the context of organ transplantation, high levels of TTV are found in patients who have received immunosuppressive therapy (Non-Patent Literature 1) or HSCT (Non-Patent Literature 2). Furthermore, a correlation is thought to exist between TTV levels and immune system deficiencies associated with chronic infections and cancer (Non-Patent Literature 3), while TTV is also seen in association with immunosuppressive viral infections such as HIV and HCV infection.

[0008] These observations suggested that TTV levels may be a marker of immune function (8–11). However, in these studies, immune function was assessed only from the number of immune cells or the occurrence of undesirable clinical events (11–14). It is now already suggested that cell quantity is not necessarily related to cell quality, i.e., T cell activity is not reflected in their number (12).

[0009] Therefore, there is still a need for a simple and reliable method that allows us to determine whether T cells can be activated in a given subject. [Prior art documents] [Patent Documents]

[0010] [Non-Patent Document 1] Rezahosseini et al.,Transplant Rev(Orlando). 33(3):137-144,2019 [Non-Patent Document 2] Albert et al.,Med Microbiol Immunol.208(2):253-258,2019 [Non-Patent Document 3] Zhong et al.,Ann NY Acad Sci.945:84-92,2001;Fogli et al.,Clin Dev Immunol.2012:829584,2012;Beland et al.,J infect Dis.209(2):247-254,2014;Gorzer et al.,J Heart Lung Transplant.33(3):320-323,2014) [Modes for carrying out the invention]

[0011] The present invention relates to a method for determining whether T cells are functional in a subject. More precisely, the inventors have shown that, particularly in patients who have undergone transplantation, more specifically HSCT, the proliferative capacity of T cells is inversely correlated with the amount of torque teno virus (TTV). The amount of TTV virus is inversely correlated with the proliferation of T cells: thus, the higher the amount of TTV, the lower the proliferative capacity of T cells. The virus amount is not specifically correlated with another parameter, whether this is the number of T cells or a clinical criterion, emphasizing the relevance of the identified correlation. The amount of TTV is a specific marker of T cell activity, particularly in patients who have undergone transplantation, particularly HSCT. Thus, the development of the function of the immune system can be tracked in a subject by simply measuring the amount of TTV. Thus, it is possible to rapidly evaluate the functional state of the immune system without performing the cumbersome technical procedures usually employed to evaluate this parameter.

[0012] Method for determining the proliferative capacity of T cells This description relates to a method for determining the proliferative capacity of T cells in a subject, including the measurement of the amount of TTV in a patient.

[0013] As mentioned above, the proliferative capacity of T cells does not necessarily correlate with the number of said T cells. Thus, known methods of counting the number of T cells provide no information about the proliferative capacity of T cells and thus about the function of the immune system. Thus, the inventors have the advantage of identifying a new parameter that is easily measurable in daily life and that is advantageously correlated with the proliferative capacity of T cells, thereby making it possible to evaluate the function of the immune system.

[0014] T cells are essential cells of the immune system that play a role in amplifying or reducing the immune response. Preferably, T cells are characterized by the expression of a membrane marker called CD3 and a specific receptor, the TCR (meaning "T cell receptor"), which is directly involved in antigen recognition. Advantageously, T cells can also express other surface markers, particularly CD4 and CD8, which correspond to specific functional categories of T cells. In the context of HSCT, the T cells involved may be a small number of residual T cells in the recipient, or donor T cells present in transplantation. They may also be naive T cells resulting from the differentiation of donor stem cells and progenitor cells in the recipient's thymus.

[0015] In this specification, the terms "activation of the T cells" or "activation of T cells" refer to the process by which naive T cells become capable of participating in the immune response. T cell activation is particularly linked to their proliferation. Therefore, T cell activation is advantageously assessed by measuring T cell proliferation. While the measurement of T cell proliferation is usually performed by techniques known to those skilled in the art, these methods are cumbersome to implement. In particular, it is well known that T cells can proliferate in the presence of mitogens such as concanavalin A (Con A), pokeweed mitogen (PWM stands for "pokeweed mitogen"), and phytohemagglutinin (PHA), regardless of the specificity of their TCR. Prior art methods assess the proliferative capacity of T cells by measuring the synthesis of T cell DNA after mitogen stimulation. However, these methods are cumbersome to implement and may not be suitable for routine use. By comparison, the methods described herein are particularly simple and powerful.

[0016] Therefore, the T cell proliferation test involves the following steps: - A process of isolating peripheral blood mononuclear cells (PBMCs) from whole blood by centrifugation; - The isolated PBMCs are optionally incubated in a supplemental medium such as a cell culture plate; - A mitogen stimulation (preferably double) process; -Incubation process; - To determine T cell proliferation, for example, a flow cytometry analysis step from the culture pellet, Includes.

[0017] In particular, detailed protocols are provided in the examples of embodiments.

[0018] According to the first aspect, a method for determining the proliferative capacity of T cells in a subject is described herein, and the method is a) A step of measuring the amount of TTV from the target sample; b) a) step of determining the proliferative capacity of the target T cells according to the viral load measured in a), Includes.

[0019] In a preferred embodiment, a high TTV level indicates low T cell proliferative capacity. Conversely, a low TTV level indicates high cell proliferative capacity.

[0020] Of course, in order to determine whether the amount of TTV in a biological sample is high or low and to draw conclusions about the proliferative capacity of T cells, the amount of TTV can be favorably compared to a reference TTV amount or control amount as defined later in this description. For example, the reference TTV amount may be the amount of TTV virus measured in a single individual.

[0021] More specifically, this method is better suited to evaluating the proliferative capacity of T cells in subjects who may have an immunodeficiency state.

[0022] As used herein, the terms “immunodepression” (or “immunosuppression”) refer to a decrease or suppression of the function of the immune system. Therefore, “immunodeficiency” as used herein refers to a state in which the immune system of a subject is reduced or absent. Preferably, in a subject with an immunodeficiency, there is a deficiency in the humoral and / or cellular immune response to infectious pathogens. More preferably, the immunodeficiency is manifested by at least a decrease in the cellular response.

[0023] Immunodeficiency can be primary or secondary. Primary immunodeficiency includes congenital defects of the immune system that increase susceptibility to infection. In contrast, secondary (or acquired) immunodeficiency corresponds to loss of immune function that occurs in life, such as, but is not limited to, exposure to pathogens, disease (e.g., lymphoma or leukemia), therapy to treat disease (e.g., radiotherapy or chemotherapy), immunosuppression, or aging. Pathogens that can cause immunodeficiency include, in particular, human immunodeficiency virus (HIV) 1 (HIV-1), HIV-2, Treponema pallidum, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium vivax, hepatitis B virus (HBV), hepatitis C virus (HCV), prions, West Nile virus, parvovirus, Trypanosoma cruzi, coronaviruses such as SARS-CoV-1 and SARS-CoV-2, and / or cowpox virus. Furthermore, immunodeficiency can be intentionally induced by drugs to prevent graft rejection, for example in organ transplantation (e.g., kidney, liver, heart, lung, pancreas, intestine, etc.) and transplant preparation such as HSCT.

[0024] The immunodeficiency described herein is preferably secondary (or acquired) immunodeficiency. Therefore, the immunodeficiency described herein may be of any origin, but is not limited to, immunosuppressive side effects of therapies including immunosuppressive treatment, drug therapy or radiotherapy, hereditary immunosuppressive genetic traits or diseases, acquired immunosuppressive diseases (such as AIDS), cancer (such as leukemia or lymphoma), etc. In particular, immunodeficiency is associated with transplantation, especially HSCT.

[0025] In certain embodiments, the subject most likely to be immunocompromised is the transplant recipient. According to a more specific embodiment, this transplant is an HSCT.

[0026] More specifically, this description relates to a method for determining the proliferative capacity of T cells in subjects who have undergone HSCT, and the method consists of the following steps: a) A step of measuring the amount of TTV from the target sample; b) a) step of determining the proliferative capacity of the target T cells according to the viral load measured in a), Includes.

[0027] According to a preferred embodiment, a high TTV level indicates low cell proliferative capacity. Conversely, a low TTV level indicates high cell proliferative capacity.

[0028] In this specification, “stem cells” are intended to refer to undifferentiated but specialized cells that possess two main characteristics: the ability to self-renew and remain in place for very long periods, and the ability to generate all types of differentiated cells in a particular tissue, which defines their pluripotency. In this specification, “hematopoietic stem cells” or “HSCs” more specifically refer to stem cells that can give rise to different blood cells (especially erythrocytes, platelets, granulocytes, T cells or B cells, monocytes). HSCs can be favorably obtained from umbilical cord blood, or from peripheral blood, or from bone marrow.

[0029] As understood herein, "hematopoietic stem cell transplantation" or "HSCT" generally refers to a hematological treatment procedure involving the transplantation of HSCs derived from bone marrow, peripheral blood, or umbilical cord blood from a donor to a recipient.

[0030] HSCT is a potential therapeutic approach for a variety of diseases. These include hematological disorders, particularly malignant hematological disorders such as acute leukemia, spinal dysplasia, and lymphoma, as well as non-malignant hematological disorders with severe prognoses, including constitutional medullary hypoplasia and abnormal hemoglobinopathy, solid tumors, immune deficiencies, and hematopoietic enzyme deficiencies and immune deficiencies such as Gaucher disease. The pathology is preferably a hematological disorder, and more preferably a malignant hematological disorder.

[0031] HSCT can be autologous (using the patient's own stem cells, i.e., the donor and recipient are the same person) or allogeneic (hereinafter referred to as "allogeneic hematopoietic stem cell transplantation (allo-HSCT)," where the stem cells are derived from a donor other than the recipient). The HSCT described herein is preferably allogeneic.

[0032] According to this specific embodiment, this description relates to a method for determining the proliferative capacity of T cells in a subject who has undergone allogeneic hematopoietic stem cell transplantation, the method comprising the following steps: a) A step of measuring the amount of TTV from the target sample; b) a) step of determining the proliferative capacity of the target T cells according to the viral load measured in a), Includes.

[0033] According to a preferred embodiment, a high TTV level indicates low cell proliferative capacity. Conversely, a low TTV level indicates high cell proliferative capacity.

[0034] In allogeneic transplantation, preparatory treatment (or conditioning treatment) is performed before the transplant surgery to disrupt or reduce the activity of the recipient's immune system. This conditioning aims to prevent graft rejection and reduce the amount of tumor tissue.

[0035] Pre-conditioning can be "myeloablative." "Myeloablative" pre-conditioning as understood herein is pre-conditioning that destroys cells in the recipient's bone marrow. Advantageously, myeloablative pre-conditioning also destroys the recipient's immune system, thus facilitating graft acceptance. Myeloablative pre-conditioning may include one or more steps of chemotherapy and / or radiotherapy, in particular. For example, two commonly used pre-conditioning combinations are busulfan-cyclophosphamide and cyclophosphamide-total body irradiation (TBI). Preferably, myeloablative pre-conditioning is applied to patients under 55 years of age, preferably under 50 years of age.

[0036] Alternatively, the conditioning regimen may be non-myeloablative conditioning, which is attenuated and also called “reduced intensity.” “Attenuated conditioning” is conditioning that inhibits the recipient’s immune system and promotes graft acceptance, rather than completely destroying the recipient’s bone marrow. This attenuated conditioning preferably includes the administration of immunosuppressants. For example, a protocol for attenuated conditioning may include a combination of fludarabine, cyclophosphamide, or other alkylating agents with TBI. Another example of an attenuated conditioning protocol may include a combination of fludarabine, anti-lymphocyte serum (ALS), and busulfan. Another example of an attenuated conditioning protocol may include a combination of fludarabine, idarubicin, and cytarabine. Finally, another example of an attenuated conditioning protocol may include a combination of fludarabine and complete mini-irradiation. In a preferred embodiment, the attenuated conditioning is applied to patients under 75 years of age.

[0037] As used herein, the term “donor” refers to the person who transplants HSCs into the recipient. Herein, “recipient” or “patient” means the person who receives HSCs from the donor. In certain embodiments, the recipient is affected by a pathology, whether complete or partial, to which the HSCs should provide a therapeutic benefit.

[0038] As used herein, the term “subject” refers to a vertebrate, preferably a mammal, most preferably a human. For example, a human is a patient.

[0039] Preferably, in all embodiments of the methods described below, the subject is a patient.

[0040] The term "patient" refers to, for example, a person who has come into contact with medical professionals such as doctors, medical organizations, or medical facilities such as hospitals.

[0041] In this specification, “biological sample” means any sample that can be taken from a subject. Generally, the biological sample must enable the determination of the TTV amount. As understood herein, biological samples include, but are not limited to, whole blood, serum, plasma, sputum, nasopharyngeal samples, urine, feces, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal plasma, tears, spinal cord tissue or cerebrospinal fluid, cerebral fluid, trigeminal ganglion samples, sacral lymph node samples, adipose tissue, lymphoid tissue, placental tissue, tissue of the upper reproductive system, tissue of the gastrointestinal tract, reproductive tissue, and tissue of the central nervous system. Test samples may be used directly from the biological source or after pretreatment to modify the characteristics of the sample. For example, this pretreatment includes the preparation of plasma from blood, dilution of viscous fluids, etc. Pretreatment methods may also include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of inhibitory components, addition of reagents, dissolution, etc. Furthermore, it may be beneficial to modify the solid test sample to form a liquid culture medium or to release the analyte. The biological sample is preferably blood or a derivative thereof (such as plasma or serum). Therefore, the biological sample is preferably blood, plasma, or serum derived from the subject being tested.

[0042] "Torque Teno virus or TTV" refers to viruses of the Anelloviridae family. As understood herein, TTV is a non-enveloped virus with a negatively polarized circular single-stranded DNA genome. In this specification, "TTV genome" refers to the genomes of all Anelloviridae species, including alphatorquevirus (TTV), betatorquevirus (TTMV), and gammatorquevirus (TTMDV). For example, this specification refers to the genome of TTV-1a, a prototype strain of tolketenovirus. More specifically, an example of a TTV genome is a sequence represented, for example, by sequence number 1, with Genbank acceptance number AB017610.

[0043] The size of the TTV genome is preferably about 3.8 kb. The structure and genomic composition of TTV are well known (see, e.g., Biagini, Curr Top Microbiol Immunol. 331:21-33, 2009) and are illustrated in Figure 1. Thus, the TTV genome can be divided into an untranslated region (UTR) of about 1–1.2 kb and a potential coding region of about 2.6–2.8 kb. The coding region includes, in particular, two large open reading frames, ORF1 and / or ORF2, which encode two proteins having 770 and 202 residues, respectively. In the TTV genome represented by Sequence ID No. 1, the open reading frames ORF1 and / or ORF2 are located between nucleotides 589–2901 and 107–715, respectively. The TTV genome may have other open reading frames. For example, the TTV genome may include two additional reading frames, ORF3 and / or ORF4 (Figure 1).

[0044] In contrast, the untranslated region UTR is well-conserved. It contains, in particular, GC-rich sequences capable of forming secondary structures. Amplification of selected sequences in the untranslated region UTR-5' allows for the demonstration of a very high prevalence of the virus across the global population (Hu et al., J Clin Microbiol. 43(8):3747-3754, 2005). This region specifically contains a 128 bp sequence that can be amplified with the TTV R-GENE® diagnostic kit (bioMerieux, France).

[0045] As understood herein, “viral load” is the number of viral particles present in a biological sample. Viral load reflects the severity of a viral infection. Viral load can be determined by measuring the amount of one of the viral components (genomic DNA, mRNA, protein, etc.) in the biological sample. Therefore, viral load preferably refers to the proportion of nucleic acid sequences belonging to the virus in question in the biological sample. More preferably, viral load represents the copy number of the viral genome in question in the biological sample.

[0046] In this case, the viral load represents the TTV load. More specifically, “TTV load” as used herein corresponds to the viral load of TTV, i.e., the number of TTV viral particles present in the biological sample. The TTV load in a subject means the viral load of all TTV present in the subject. The TTV load can be determined by measuring the amount of TTV components such as nucleic acids or proteins in the biological sample. Preferably, the TTV load corresponds to the amount of TTV nucleic acid sequences present in the biological sample. Therefore, determining the TTV load in a subject according to the present invention involves estimating the total number of TTV sequences in the biological sample from the subject. In particular, according to the present invention, there is no selection of a specific strain of TTV to be measured in the biological sample. Determining the TTV load preferably involves determining the amount of active and / or inactive viral copies. This consists of determining the amount of incorporated or potentially circulating viral copies.

[0047] The level of TTV, i.e., the amount of TTV, can be determined by measuring the levels of TTV DNA, TTV RNA, or TTV protein. Therefore, the method according to the present invention may include other preliminary steps between obtaining a sample from a patient and step a) defined above, corresponding to the conversion of the biological sample into a sample of double-stranded DNA, mRNA (or corresponding cDNA), or protein, which is then ready for use in in vitro detection of TTV in step a). The preparation or extraction of double-stranded viral DNA, mRNA (and reverse transcription of the latter into cDNA), or protein from a cell sample is merely a routine procedure well known to those skilled in the art. The double-stranded DNA may correspond to the entire TTV genome or only a portion thereof. Once double-stranded DNA, mRNA (or corresponding cDNA), or readily available protein samples become available, TTV can be detected at the genomic DNA level (i.e., based on the presence of at least one sequence consisting of at least a portion of the TTV genome), the mRNA level (i.e., based on the TTV mRNA content in the sample), or the protein level (i.e., based on the TTV protein content in the sample), depending on the type of sample and transformation.

[0048] Preferably, the level of TTV is determined by measuring the level of TTV nucleic acid, more preferably TTV DNA.

[0049] Methods for detecting nucleic acids in biological samples include, in particular, amplification including PCR amplification, sequencing, hybridization with labeled probes, and all other methods known to those skilled in the art.

[0050] According to the first embodiment, the TTV quantity is determined by amplification of the TTV sequence.

[0051] A preferred approach consists of amplifying sequences known to be specific to the TTV genome. In this specification, “TTV genome-specific sequences” means sequences present in the majority of known TTVs but not in the vast majority of other viruses, particularly the vast majority of other anelloviruses. It is preferable that TTV-specific sequences are present in at least 90%, 95%, 96%, 97%, 98%, or 99% of the genomes of known TTVs. More preferably, they are present in 100% of the genomes of known TTVs. Alternatively, TTV-specific sequences may be present in less than 10%, 5%, 4%, 3%, 2%, or 1% of the genomes of known anelloviruses other than TTVs. It is preferable that no TTV-specific sequences are present in the entire genome of known anelloviruses other than TTVs. Such sequences are, for example, sequences contained in the untranslated region (UTR). More specifically, such sequences correspond to a 128 bp sequence of the untranslated region 5'-UTR amplified using the TTV R-GENE® diagnostic kit (bioMerieux, France).

[0052] Accordingly, according to this embodiment, the method described herein involves the use of primers and probes for amplification of sequences known to be specific to the TTV genome. In the art, these primers are usually preferably oligonucleotides. For example, these primers may include less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, or less than 12 nucleotides. Alternatively, these primers may include at least 12, 15, 20, 25, or 30 nucleotides. The primers used are preferably 12-20 nucleotides, 12-25 nucleotides, 15-20 nucleotides, or 15-25 nucleotides. Those skilled in the art will know how to determine the length and sequence of amplification primers to be used after a TTV-specific sequence has been selected. For example, it may be possible to use the same primers provided in the TTV R-GENE® diagnostic kit (bioMerieux, France).

[0053] In particular, amplification techniques include isothermal methods and techniques based on PCR (polymerase chain reaction). There are numerous isothermal amplification methods. The most commonly used methods for detecting pathogens are LAMP (Loop-Mediated Amplification) and RPA (Recombinase Polymerase Amplification). Isothermal amplification methods include, for example, NASBA (nucleic acid sequence-based amplification), HDA (helicase-dependent amplification), RCA (rolling cycle amplification), SDA (strand displacement amplification), EXPAR (exponential amplification reaction), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids), and SMART (signal-mediated amplification of RNA technology) (see, for example, Asiello and Baeumner, Lab Chip 11(8):1420-1430, 2011). The PCR technique used preferably quantitatively measures the initial amount of DNA, cDNA, or RNA. Examples of PCR-based techniques that can be used in the methods described herein include, but are not limited to, real-time PCR (Q-PCR), reverse transcription PCR (RT-PCR), multiplex reverse transcription PCR, real-time reverse transcription PCR (QRT-PCR), and digital PCR. These techniques are well known to those skilled in the art and readily available, and therefore do not need to be described in detail herein.

[0054] The amount of TTV is preferably determined by real-time quantitative PCR. Numerous methods for the detection and quantification of TTV have been described in the prior art (see, e.g., Maggi et al., J Virol. 77(4):2418-2425, 2003). In particular, the method described by Kulifaj et al. is referred to (J Clin Virol. 105:118-127, 2018). This method is particularly advantageous due to its simplicity and robustness. It is based on the amplification of a sequence contained in the non-coding region UTR. Since this sequence is present in all known TTVs, it gives this method very high specificity. Furthermore, it is particularly versatile and can be performed on any type of PCR platform. It is particularly advantageous to use the TTV R-GENE® diagnostic kit (bioMerieux, France) to perform this method.

[0055] Alternatively, viral load determination is performed by digital PCR. Digital PCR involves several PCR analyses in extremely diluted nucleic acids, so that most positive amplifications reflect the signal of a single matrix molecule. Therefore, digital PCR can measure individual model molecules. The proportion of positive amplifications among the total number of PCRs analyzed allows for the estimation of the matrix concentration in the original or undiluted sample. This technique was proposed to enable the detection of various genetic phenomena (Vogelstein et al., Proc Natl Acad Sci USA 96:9236-924, 1999). Digital PCR, like real-time PCR, can potentially distinguish subtle quantitative differences in target sequences between samples.

[0056] According to another embodiment, the level of TTV DNA is measured by sequencing. As used herein, the term “sequencing” is most widely accepted and refers to the techniques known to those skilled in the art for determining the sequence of a polynucleotide molecule (DNA or RNA), that is, for determining the sequence of the nucleotides that make up the molecule.

[0057] Therefore, TTV DNA can be sequenced by techniques known in the art. As understood herein, sequencing includes, among other things, Sanger sequencing, whole-genome sequencing, hybridization sequencing, pyrosequencing (especially 454 sequencing, Solexa Genome Analyzer sequencing), capillary electrophoresis sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, ultrafast sequencing, massively parallel signature sequencing (MPSS), reversible dye-terminator sequencing, pair sequencing, and short-term sequencing. This includes sequencing, exonuclease sequencing, ligation sequencing, single-molecule sequencing, synthesis sequencing, electron microscopy sequencing, real-time sequencing, reverse termination sequencing, nanopore sequencing, reversible terminator sequencing, semiconductor sequencing, SOLiD(R) sequencing, SMRT sequencing (single-molecule real-time analysis), MS-PET sequencing, mass spectrometry, and combinations thereof.Specific embodiments utilize ultrafast DNA sequencing using, for example, the MiSeq, NextSeq 500, and HiSeq series platforms developed by Illumina (Reuter et al., Mol Cell, 58:586-597, 2015; Bentley et al., Nature; 456:53-59, 2008), the 454 genome sequencer and Roche platform (Margulies et al., Nature; 437:376-380, 2005), the SOLiD platform from Applied Biosystems (McKernan et al., Genome Res; 19:1527-1541, 2009), the Polanator platform (Shendure et al., Science, 309:1728-1732), or the Helicos single-molecule sequencing platform (Harris et al., Science; 320:106-109, 2008). Ultrafast sequencing includes methods such as SMRT real-time sequencing (Roads et al., Genomics, Proteomics & Bioinformatics, 13(5):278-289, 2015), Ion Torrent sequencing (WO2010 / 008480; Rothberg et al., Nature, 475:348-352, 2011), and nanopore sequencing (Clarke J et al. Nat Nanotechnol:4:265-270, 2009).

[0058] Sequencing is performed on all or part of the DNA contained in a biological sample. Those skilled in the art will readily see that the sample contains a mixture of at least the host subject's DNA and TTV DNA. Furthermore, the TTV DNA likely represents only a small fraction of the total DNA present in the sample. Conveniently, DNA is generally fragmented randomly by physical means before sequencing.

[0059] The first approach involves sequencing a specific sequence of the genome of a TTV species. The other approach involves using a method that can quantitatively determine the genotype of nucleic acids obtained from biological samples with great accuracy. In certain embodiments, accuracy is achieved by analyzing a large number (e.g., millions or billions) of nucleic acid molecules without amplification using a protocol based on prior knowledge of the target sequence (i.e., the TTV sequence in this case).

[0060] In a preferred embodiment, the method of the present invention includes a step of quantifying the number of readings.

[0061] In certain embodiments, a random subset of nucleic acid molecules from a biological sample is subjected to ultrafast sequencing. Preferably, TTV sequences are identified by global sequencing by comparison with publicly deposited TTV sequences. This comparison is favorably based on the level of sequence identity with known TTV sequences, enabling the detection of even distantly related variants. Common software such as BLAST can be used to determine the level of identity between sequences.

[0062] Therefore, sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with known TTV sequences are identified as TTV sequences. Accordingly, according to this embodiment, the determination of the TTV quantity includes numbering the TTV sequences identified by sequencing in biological samples from the subject.

[0063] In another embodiment, the TTV amount is determined by measuring the amount of viral protein in a biological sample. Therefore, it is possible to use specific antibodies in well-known techniques, particularly immunoprecipitation, immunohistochemistry, Western blotting, dot blotting, ELISA or ELISPOT, electrochemical luminescence (ECLIA), protein chips, antibody chips, or tissue chips bound to immunohistochemistry. Other methods that may be used include microscopy or histochemistry methods, particularly FRET or BRET techniques, including confocal and electron microscopy; adaptive optical methods such as electrochemical methods (voltammetry and amperometry techniques) and methods based on the use of one or more excitation wavelengths; atomic force microscopy; and radiofrequency methods (multipolar, confocal, and non-confocal resonance spectroscopy, etc.); detection of fluorescence, emission, chemiluminescence, absorbance, reflectance, transmittance, and birefractive power or refractive index (e.g., by surface plasmon resonance, elliptometry, resonance mirror methods, etc.); flow cytometry; radioisotope or magnetic resonance imaging; electrophoresis on polyacrylamide gels (SDS-PAGE); mass spectrometry; and analysis by liquid chromatography (LC-MS / MS) coupled with mass spectrometry. All of these techniques are well known to those skilled in the art and do not need to be described in detail herein. Furthermore, specific antibodies for TTV protein are already available.

[0064] In a preferred embodiment, the method described herein includes an additional step of standardizing the amount of nucleic acid or viral protein measured.

[0065] In a preferred embodiment, it can be advantageous to standardize the level of TTV present in a biological sample, i.e., the amount of TTV DNA, TTV RNA, or TTV protein, to a specific parameter of the sample. Standardizing the measured amount of TTV to a specific parameter allows for a reduction in the error rate when comparing the viral loads of two different biological samples. Examples of parameters that can be useful for this standardization include physical parameters that are independent of the sample's content, such as volume. It is also possible to standardize the amount of TTV DNA, TTV RNA, or TTV protein to the total amount of DNA, RNA, or protein present in the sample. Alternatively, a specific DNA or RNA sequence, or a specific protein, can be used as a standardization tool. For example, this sequence or protein may be a human sequence or protein.

[0066] Alternatively, the amount of TTV DNA or RNA, or TTV protein in a given sample is compared to an internal control. Therefore, the amount of TTV nucleic acid or protein measured in a biological sample can be referenced to a defined amount of identifiable and quantifiable nucleic acid or appropriate protein, such as host or exogenous nucleic acid or protein. It is preferable that this identifiable and quantifiable nucleic acid or protein be treated as a target nucleic acid or protein (e.g., amplified, sequenced, etc.). Thus, a known amount of this identifiable and quantifiable nucleic acid or protein can be added to the sample from the outset, then treated as a target nucleic acid or protein, and undergoes all sample preparation steps before the measurement of the amount of this viral nucleic acid or protein. The preparation steps may include, for example, means of protecting the viral nucleic acid and disrupting the host nucleic acid using various nucleases. Alternatively, these steps may include means of protecting the viral protein and disrupting the host protein using, for example, various proteases. Internal controls allow for evaluation of the quality and degree of processing (e.g., amplification or sequencing) of the molecule under consideration (nucleic acid or protein) in the sample. Preferably, the internal control is a nucleic acid molecule of a known sequence, which is present in the sample at a known concentration. More preferably, this nucleic acid molecule is a single-stranded circular DNA molecule of a viral genome of a known sequence and concentration in the sample. This known virus can be, for example, a virus of the Circoviridae family. The absolute number of TTV genomes of known sequence and concentration can be estimated by the ratio of the number of sequences in the sample to the control. Alternatively, this internal control is a protein of a known sequence, which is present in the sample at a known concentration.

[0067] If the TTV amount is determined by measuring the amount of TTV nucleic acid or protein, and then optionally standardized, it may be advantageous to compare it to a reference TTV amount.

[0068] "Reference TTV amount" or "reference viral load" means, in the sense of this application, the amount of TTV used as a reference. This means that the reference TTV amount corresponds to the concentration of "reference level TTV nucleic acid (or protein)" or "reference level TTV nucleic acid (or protein)," i.e., the concentration of TTV nucleic acid (or protein) used as a reference. As understood herein, "reference concentration TTV nucleic acid (or protein)" is a baseline level obtained from a subject or group of subjects having a specific immunoqualification status, measured in a control sample equivalent to the test substance. For example, this could be a subject or group of subjects that are healthy or do not have a disease that leads to immunosuppression. Alternatively, it could be an immunosuppressed subject or group of subjects, for example, after immunosuppressive treatment. Finally, it could be the same individual that underwent transplant surgery, for example, before or immediately after immunosuppression.

[0069] The reference level can be determined in several ways. For example, the control may be a predetermined value that can take various forms. It can be a unique threshold, such as the median or mean. The "reference level" can be a unique value applicable to each patient. Alternatively, the reference level can vary as a function of a particular subpopulation of patients. For example, the reference level for TTV levels may differ between older men and younger men, and the reference level for this viral load may differ between women and men. Furthermore, the "reference level" can be established based on comparison groups, such as a group with low levels of TTV nucleic acid (or protein) and a group with high levels of TTV nucleic acid (or protein). Another example of a comparison group is a group with a disease, condition, or specific symptom and a group without the disease. For example, a predetermined value can be defined when the test population is divided equally (or unevenly) into groups such as low-risk, medium-risk, and high-risk groups.

[0070] Reference levels can also be determined by comparing levels of TTV nucleic acid (or protein) in a population of transplant recipients or patients with immunosuppressive diseases. This can be achieved, for example, by histogram analysis, where the entire cohort of subjects is shown graphically, with the first axis representing the level of the TTV nucleic acid (or protein) and the second axis representing the number of patients in the group expressing the TTV nucleic acid (or protein) at a given level. Two or more separate groups of subjects can be determined by identifying subpopulations of cohorts with the same or similar levels of TTV nucleic acid (or protein). Reference levels can then be determined based on the level that best distinguishes these separate groups. Reference levels can also represent the levels of two or more TTV nucleic acids (or proteins) present. Two or more markers can be represented, for example, by the ratio of the levels of each marker.

[0071] Furthermore, clearly healthy populations will have a "different" normal range than populations known to have conditions associated with high concentrations of the TTV nucleic acid (or protein). Therefore, the selected values ​​may take into account which category the individuals belong to. Appropriate ranges and categories can be selected simply by routine experiments by those skilled in the art. "High" or "elevated" means high relative to the selected control. Generally, the control is based on clearly healthy, normal individuals within an appropriate age group.

[0072] In a preferred embodiment, the reference concentration corresponds to the concentration of TTV nucleic acid (or protein) in the general population or the concentration of a combination of multiple TTV nucleic acids (or multiple TTV proteins).

[0073] It will be understood that the control in the methods described herein may be a biological sample measured in parallel with the test sample, in addition to a predetermined value. According to this embodiment, the reference level would be a reference level of TTV nucleic acid or multiple TTV nucleic acids (or protein or multiple proteins) in a sample obtained from a healthy subject.

[0074] The reference concentration of TTV nucleic acid (or protein) is preferably the concentration of this TTV nucleic acid (or protein) in a healthy subject or a population of healthy subjects. According to another preferred embodiment, the reference concentration of TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in an immunosuppressed subject or a population of immunosuppressed subjects (e.g., after immunosuppressive treatment). According to another preferred embodiment, the reference concentration of TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in the same individual that received the transplant at a specific point in time, for example, before or immediately after the latter.

[0075] Preferably, in all embodiments of the above method, the T cells are a population of CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD3+ and / or CD4+ and / or CD8+ T cells, preferably CD3+ T cells.

[0076] Methods for monitoring T cell activity The methods described herein enable rapid and easy assessment of the proliferative capacity of T cells in a given subject.

[0077] Several factors contribute to the severe immunosuppression in recipients of HSCT, particularly allogeneic hematopoietic stem cell transplantation. Pre-conditioning alters the recipient's lymphoid tissue, in particular. The presence and treatment of GvHD can lead to new immune complications. Finally, the small number of transplanted T cells compared to the size of the T cell compartment in immune-qualified individuals, and the extremely limited number of donor immune precursors present in the graft, also contribute to the delayed immune recovery in recipients. All of these factors make recipients susceptible to post-transplant complications such as microbial infections and GvHD.

[0078] Thanks to the methods described herein, T cell activity can be easily evaluated in situations where the immune system is impaired, such as after HSCT, particularly after allogeneic hematopoietic stem cell transplantation.

[0079] Therefore, other aspects of this disclosure relate to HSCT, in particular a method for monitoring T cell activity in patients who have undergone allogeneic hematopoietic stem cell transplantation. The method comprises the following steps: a) The step of measuring the proliferative capacity of T cells at the initial point in time using the method described above; b) A step of comparing the T cell proliferation capacity measured in a) with the T cell reference proliferation capacity; c) A step to determine the change in the patient's T cell activity in accordance with the comparison in step b), Includes.

[0080] As understood herein, “reference T cell proliferative capacity” refers to the T cell proliferative capacity estimated from the reference TTV amount described above. Naturally, the comparison in step b) can be performed simply by comparing the TTV amount of the patient sample determined in step a) with the reference TTV amount.

[0081] For example, by comparing the proliferative capacity of the patient's T cells at this initial point in time with the baseline proliferative capacity of T cells in an immunosuppressed individual, it is possible to estimate the recovery of the patient's immune function at this initial point in time, particularly after HSCT, or allogeneic hematopoietic stem cell transplantation. In this specification, "immune function" refers to the acquisition of function by immune cells. Therefore, an increase in the proliferative capacity of the patient's T cells compared to that of an immunosuppressed individual indicates that the patient's immune function is recovering. As mentioned above, an increase in the proliferative capacity of the patient's T cells compared to an immunosuppressed subject corresponds to a low TTV level.

[0082] Alternatively, the baseline T cell proliferation rate can be that of a healthy individual. In this case, a decrease in the patient's T cell proliferation rate relative to the baseline rate indicates that the patient's immune function has not fully recovered. It will be immediately apparent that a decrease in the patient's T cell proliferation rate relative to a healthy subject corresponds to an increase in TTV levels.

[0083] As used herein in certain embodiments, the term “increased” means a greater amount, for example, slightly more than the original amount, or for example, a very large amount compared to the original amount, in particular all amounts within the range. As a variation, “increased” can mean an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the amount or activity being compared. In this specification, the terms “enhanced,” “greater,” “even greater,” and “increased” are used interchangeably.

[0084] As used herein in certain embodiments, the term “reduced” means a smaller amount, for example, a slightly less than the original amount, or for example, a very small amount compared to the original amount, in particular all amounts within the range. As a variation, “reduced” can refer to an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than the amount or activity being compared. In this specification, the terms “reduced,” “less than,” “even lower,” and “decreased” are used interchangeably.

[0085] The T cell proliferation capacity of the same patient at a second time point can also be used as a baseline for T cell proliferation. Therefore, by simply measuring the patient's TTV levels at the first and second time points without using expensive and complex testing equipment, changes in the activity of these T cells in this patient after transplantation can be easily monitored.

[0086] According to this particular embodiment, the method for monitoring T cell activity in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, involves the following steps: a) The step of measuring the proliferative capacity of T cells at the initial point in time using the method described above; b) A step of measuring the proliferative capacity of T cells at a second time point later than the first time point in step a) using the method described above; c) A step of comparing the proliferative capacity of T cells measured in a) and b); d) A step to determine the change in the patient's T cell activity in accordance with the comparison in step c), Includes.

[0087] According to other specific embodiments, this method for monitoring T cell activity in subjects who have undergone transplantation, preferably HSCT, and particularly allogeneic hematopoietic stem cell transplantation, involves the following steps: a) A step to determine the amount of TTV virus from the target biological sample collected at the initial point in time; b) A step of determining the amount of TTV virus from the subject's biological sample taken at a second time point, which is later than the first time point in step a): c) A step of comparing the viral load of TTV measured in a) and b); d) A step to determine the change in the target TTV amount in accordance with the comparison in step c), Includes.

[0088] As explained above, since the TTV viral load is inversely correlated with the T cell proliferation capacity, changes in the TTV load can be used to determine whether the T cell proliferation capacity is increasing or decreasing, thus providing an indicator of T cell activity, particularly the recovery (or opposite) of the target immune function.

[0089] Therefore, this method is particularly useful for monitoring the activity of a patient's T cells over time. In a preferred embodiment, the first time point in step a) is at the time of transplantation. In another preferred embodiment, the T cell proliferative capacity is measured in step b) using samples collected at least 30, 60, 90, 100, 120, 150, 180, 210, 240, 270, 300, 330, 360, 720, or 1080 days after HSCT. Alternatively, samples are collected 30, 60, 90, 100, 120, 150, 180, 210, 240, 270, 300, 330, 360, 720, or 1080 days after HSCT.

[0090] If cells have increased proliferative capacity at a second time point compared to an initial time point, it is clear that the activity of those cells themselves has increased at this time. For example, if the initial time point is the time of transplantation, an increase in T cell proliferative capacity at this second time point means that more T cells are activated, and the patient has a particularly high ability to resist threats. These changes in T cell proliferative capacity translate in the opposite direction to a change in TTV quantity: therefore, a decrease in TTV quantity over time reflects an increase in T cell proliferative capacity, i.e., a recovery of the patient's immune function. Thus, the current method makes it possible to estimate the recovery of the recipient's immune system. In other words, changes in T cell proliferative capacity make it possible to assess the reappearance of immune function in patients after transplantation.

[0091] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method for monitoring T cell activity described above is employed in patients who have undergone pre-conditioning prior to HSCT, especially allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0092] Methods for assessing the risk of microbial infection HSCT, particularly allogeneic hematopoietic stem cell transplantation, can cause early or late complications that vary considerably from patient to patient.

[0093] In particular, patients who have undergone HSCT, especially allogeneic hematopoietic stem cell transplantation, remain susceptible to microbial infections, whether bacterial, viral, parasitic, or fungal, until their immune system recovers. By evaluating the T-cell proliferation capacity at a predetermined time after HSCT, especially allogeneic hematopoietic stem cell transplantation, the susceptibility to microbial infections in transplanted patients can be determined.

[0094] In this particular embodiment, this description relates to a method for determining susceptibility to microbial infections in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation. The method comprises the following steps: a) The first step is to measure the proliferative capacity of T cells from the patient's sample using the method described above; b) A step of comparing the proliferative capacity of T cells with the normal proliferative capacity of T cells; c) A step to determine the susceptibility of patients to microbial infections based on the comparison in step b), Includes.

[0095] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0096] The methods described herein may further include one or more steps of a specific diagnostic of the presence of one or more infectious pathogens (such as bacteria, viruses, parasites or yeasts, and filamentous fungi), for example, as described in detail below. The detection of these pathogens is a routine clinical practice, particularly in relation to HSCT, and the corresponding techniques are familiar to those skilled in the art and therefore do not need to be described in detail herein.

[0097] These microbial infections include viral infections, bacterial infections, parasitic infections, and fungal infections. Viral infections are particularly caused by viruses of the Herpesviridae family, such as HSV, VZV, HHV-6, Epstein-Barr virus (EBV), or human cytomegalovirus (HCMV). These viral infections can also be caused by adenoviruses, polynuclear respiratory virus (RSV), influenza virus (also called influenza virus or Myxovirus influenzae), or BK virus. Among these, bacteria that cause bacterial infections can include staphylococci such as Staphylococcus aureus or coagulase-negative staphylococci, Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae, and obligate aerobic, non-fermenting, Gram-negative rod bacteria such as Legionella sp., Pseudomonas, Acinetobacter, Stenotrophomonas, Burkholderia, and Alkaligenes. Infections by atypical mycobacteria can also be observed. In particular, parasitic infections caused by Neisseria pneumoniae can occur, resulting in high morbidity and mortality rates, and toxoplasmosis (caused by the protozoan Toxoplasma gondii) can also occur. Finally, not only yeasts such as Candida and Cryptococcus, but also filamentous fungi such as Aspergillus are causes of invasive fungal infections, which are one of the major causes of infectious mortality after HSCT.

[0098] The baseline T cell proliferative capacity corresponds to the T cell proliferative capacity determined from the baseline TTV amount as described above. The comparison in step b) can be performed simply by comparing the TTV amount of the patient sample determined in step a) with the baseline TTV amount.

[0099] This baseline TTV level can be, for example, the TTV level of a healthy individual that is not immunosuppressed. Next, the baseline T cell proliferative capacity can be this TTV level of a healthy individual that is not immunosuppressed.

[0100] In this particular embodiment, this description relates to a method for determining susceptibility to microbial infections in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation. The method comprises the following steps: a) The first step is to measure the proliferative capacity of T cells from the patient's sample using the method described above; b) A step of comparing the proliferative capacity of T cells with that of healthy subjects; c) A step to determine the susceptibility of patients to microbial infections based on the comparison in step b), Includes.

[0101] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0102] In this case, the reduced T cell proliferation capacity in patients compared to healthy subjects indicates that the patient's immune system is not functioning properly. A lower T cell proliferation capacity in patients than in healthy subjects corresponds to a higher viral load measured in patient samples than the TTV amount in healthy subjects. In other words, the subject has a weakened immune system and is therefore vulnerable to attack by pathogens. Thus, patients are at risk of microbial infection. However, if T cells have substantially the same proliferation capacity as those in healthy subjects, patients are less susceptible to microbial infection.

[0103] The T cell proliferation capacity in the same patient at a second time point can also be used as the baseline T cell proliferation capacity. Therefore, those skilled in the art can monitor the development of the risk of microbial infection over time after transplantation. Thus, anti-infective therapy can be adapted as a function of the patient's actual susceptibility to microbial infection, limiting the risk of resistance development while improving the patient's quality of life.

[0104] In this particular embodiment, a method for determining susceptibility to microbial infection in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, involves the following steps: a) The step of measuring the proliferative capacity of T cells at the initial point in time using the method described above; b) A step of measuring the proliferative capacity of T cells at a second time point later than the first time point in step a) using the method described above; c) A step of comparing the proliferative capacity of T cells measured in a) and b); d) A step to determine the patient's susceptibility to microbial infection based on the comparison in step c), Includes.

[0105] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0106] Therefore, it is possible to track the temporal development of susceptibility to microbial infections in patients. The increased T cell proliferation capacity in step b) compared to step a) reflects increased activity and thus reflects a decrease in the patient's susceptibility to microbial infections between the two time points. The current method makes it possible to verify, in particular, that the longer the time elapsed since transplantation, the less susceptible the patient becomes to microbial infections, i.e., the more functional the immune system becomes.

[0107] Therefore, a patient's susceptibility to microbial infections can be determined using the methods described herein, enabling the adaptation of treatment to meet the patient's needs. Thus, preliminary determination of a patient's immunosuppressed state by the methods of the present invention leads to safer treatment than treatments designed based on prior art methods.

[0108] Preferably, in all embodiments of the above method, the T cell proliferative capacity corresponds to the proliferative capacity of CD3+ T cells, CD4+ T cells, CD8+ T cells, or the proliferative capacity of a population of CD3+ and / or CD4+ and / or CD8+ T cells, preferably the proliferative capacity of CD3+ T cells.

[0109] Therefore, the present invention also relates to a method for designing a treatment for microbial infection in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the method comprising the following steps: a) A step of determining the patient's susceptibility to microbial infection by the method described above. b) A step of determining the treatment according to the results of step a), Includes.

[0110] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0111] Treatment can be determined prophylactically, that is, prescribed considering the patient's immunocompromised state in order to prevent the development of infection. In this context, it may be decided to prescribe therapeutic or prophylactic treatments commonly used after HSCT, particularly allogeneic hematopoietic stem cell transplantation, as described below. This method enables the determination and implementation of prophylactic or therapeutic treatment for viral, bacterial, parasitic, or fungal infections when such infection risks are identified.

[0112] Therefore, this description also relates to a method for treating infection in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the method comprising the following steps: a) A step of determining the patient's susceptibility to microbial infection by the method described above; b) A step of administering a treatment suitable for the subject, Includes.

[0113] Therefore, the present invention proposes a treatment intended for use in the treatment of infection in subjects who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the use of which involves the following steps: a) A step of determining the patient's susceptibility to microbial infection by the method described above; b) A step of administering a treatment suitable for the subject, Includes.

[0114] In other words, the present invention relates to the therapeutic use in the preparation of pharmaceutical products for treating infections in subjects who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the said use involves the following steps: a) A step of determining the patient's susceptibility to microbial infection by the method described above; b) A step of administering a treatment suitable for the subject, Includes.

[0115] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0116] As described above, infections encountered after HSCT, particularly allogeneic hematopoietic stem cell transplantation, include viral, bacterial, parasitic, or fungal infections. Treatment for these infections is well-known and has been used in clinical practice for many years (see, for example, Tomblyn et al., Biol Blood Marrow Transplant. 15(10):1143-1238, 2009). Viral infections can therefore be prevented with antiviral agents such as acyclovir, ganciclovir, cidofovir, entecavir, fludarabine, lamivudine, tenofovir, ribavirin, or valacyclovir, or with specific monoclonal antibodies such as palivizumab (against RSV). Antibiotics typically enable the treatment of bacterial infections. In particular, broad-spectrum antibiotics such as β-lactam antibiotics, glycopeptides, fosfomycin, macrolides, tetracyclines, aminoglycosides, chloramphenicol, quinolones, rifampicin, and sulfamides are used. Parasitic infections are typically treated with antiparasitic agents such as cotrimoxazole, pyrimethamine, and sulfadiazine. Finally, well-known antifungal agents that can be administered include fluconazole and echinocandin.

[0117] Methods for assessing the risk of graft-versus-host disease (GvHD) The methods described herein also offer the advantage of being able to estimate the risk of developing GvHD in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation.

[0118] Therefore, this description also relates to a method for determining susceptibility to GvHD in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the method comprising the following steps: a) The step of measuring the proliferative capacity of T cells from the patient's sample at the initial point in time by the method described above; b) A step of determining the patient's susceptibility to GvHD from the measurements in step a), Includes.

[0119] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0120] As understood herein, “graft-versus-host disease” or “GvHD” is an inflammatory immune response to the recipient involving immunocompetent cells present in the graft. There are two clinical forms of GvHD: acute GvHD, which typically occurs around 100 days after transplantation, and chronic GvHD, which generally occurs beyond this timeframe. Acute GvHD refers to the appearance of an alloinflammatory response in only three organs: the skin, liver, and gastrointestinal tract. In contrast, chronic GvHD may affect at least one of eight organs: the skin, mouth, eyes, gastrointestinal tract, liver, lungs, muscles, joints, fascia, and genitals. Whether acute or chronic, GvHD is usually diagnosed by clinical examination, including histological analysis of a biopsy of the affected organ (Schoemans et al. Bone Marrow Transplant 53:1401-1415, 2018).

[0121] In this regard, it should be noted that this method is particularly useful, for example, in predicting the possibility of discontinuing a patient's immunosuppressive therapy and thereby ensuring the patient's survival. This method offers the possibility of easily distinguishing between active GvHD, which requires continued immunosuppressive therapy, and GvHD, which is no longer active and can be discontinued (see Magro et al., Bull Cancer. 104S:S145-S168, 2017).

[0122] More specifically, acute GvHD occurs in the first month (30 days) after transplant surgery, while chronic GvHD occurs between 100 and 400 days after transplant surgery. Both are characterized by the activation of donor T cells present in the graft. The interaction between host antigens and donor T cells leads to allogeneic activation of T cells, their proliferation, and differentiation into effector cells that attack host epithelial cells.

[0123] Recipients of HSCT, particularly allogeneic hematopoietic stem cell transplants, may develop GvHD if T cells in the patient's sample are able to proliferate. Proliferation of the patient's T cells, especially during a period when the immune system has not yet recovered, strongly indicates the patient's susceptibility to GvHD. This can be easily assessed by measuring TTV levels using the method described above. The signs indicated by this test can be supplemented, if necessary, by clinical examinations of the patient, particularly histological analysis of one or more biopsies taken from one or more of the patient's organs.

[0124] Patient samples are preferably collected less than 400 days after transplant surgery. More preferably, samples are collected less than 100 days after transplant surgery. Alternatively, samples are collected between 100 and 400 days after transplant surgery. In certain embodiments, GvHD is acute GvHD; in other specific embodiments, GvHD is chronic GvHD.

[0125] In certain embodiments, it may be useful to compare the measurements from step b) with a known baseline T cell proliferation capacity, i.e., the baseline T cell proliferation capacity. The baseline T cell proliferation capacity corresponds to the T cell proliferation capacity estimated from the baseline TTV amount as described above. The comparison in step b) can be performed simply by comparing the TTV amount of the patient sample determined in step a) with the baseline TTV amount.

[0126] This baseline TTV level is, for example, from a healthy individual that is not immunosuppressed. Next, the baseline T cell proliferation capacity is also from this healthy individual that is not immunosuppressed.

[0127] Alternatively, the reference TTV amount may be that of an immunosuppressed individual. In this particular embodiment, this description also relates to a method for determining susceptibility to GvHD in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation. The method comprises the following steps: a) The first step is to measure the proliferative capacity of T cells from the patient's sample using the method described above; b) A step of comparing the proliferative capacity of T cells with that of immunosuppressed subjects; c) A step of determining the susceptibility of patients to GvHD from the comparison in step b), Includes.

[0128] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0129] In this case, increased T cell proliferation in the patient compared to the immunosuppressed subject indicates that the patient's immune system is functioning again. A greater T cell proliferation rate in the patient than in the immunosuppressed subject corresponds to a lower viral load measured in the patient's sample than in the immunosuppressed subject's TTV load. In other words, the subject possesses active T cells that can attack the graft cells and induce GvHD. However, if the patient's T cells have substantially the same proliferation rate as the immunosuppressed subject, the patient is less likely to develop GvHD.

[0130] Furthermore, the T cell proliferation capacity of the same patient at a second time point can be used as the baseline T cell proliferation capacity. Therefore, those skilled in the art can monitor the development of the risk of GvHD development over time after transplantation. Thus, anti-GvHD therapy can be applied as a function of the patient's actual susceptibility to GvHD, while improving the patient's quality of life and limiting the risk of resistance development.

[0131] In this particular embodiment, a method for determining susceptibility to GvHD in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, involves the following steps: a) The step of measuring the proliferative capacity of T cells at the initial point in time using the method described above; b) A step of measuring the proliferative capacity of T cells at a second time point later than the first time point in step a) using the method described above; c) A step of comparing the proliferative capacity of T cells measured in a) and b); d) A step to determine the susceptibility to GvHD in patients according to the comparison in step c), Includes.

[0132] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0133] Therefore, it is possible to track the development of susceptibility to GvHD in patients over time. The increased T cell proliferative capacity in step b) compared to step a) reflects an increase in their activity, and thus reflects an increased susceptibility to developing GvHD in patients between the two time points. This risk is greater if it occurs immediately after transplantation, i.e., when the activated T cells are solely from the donor and there is a risk that they will attack the host organs.

[0134] Therefore, a patient's susceptibility to GvHD can be determined using the methods described herein, which makes it possible to develop treatments tailored to the patient's needs.

[0135] Therefore, the present invention also relates to a method for designing a treatment for GvHD for subjects who have undergone HSC, particularly HSCT, the method comprising the following steps: a) A step of determining the patient's susceptibility to GvHD by the method described above; b) A step of determining the treatment according to the results of step a), Includes.

[0136] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0137] This description also relates to a treatment method for GvHD in patients who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, and the said method consists of the following steps: a) A step of determining the patient's susceptibility to GvHD by the method described above; b) A step of administering a treatment suitable for the subject, Includes.

[0138] Therefore, the present invention proposes a treatment intended for use in the treatment of GvHD in subjects who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the use of which involves the following steps: a) A step of determining the patient's susceptibility to GvHD by the method described above; b) The process of administering a treatment suitable for the subject. Includes.

[0139] In other words, the present invention relates to the therapeutic use in the preparation of pharmaceutical products for treating GvHD in subjects who have undergone HSCT, particularly allogeneic hematopoietic stem cell transplantation, the said use comprising the following steps: a) A step of determining the patient's susceptibility to GvHD by the method described above; b) A step of administering a treatment suitable for the subject, Includes.

[0140] By monitoring changes in T cell activity over time, it is possible to monitor the recovery of post-transplant immune function, particularly in patients who have undergone pre-conditioning. According to this particular embodiment, the method of monitoring T cell activity described above is employed in HSCT, particularly in patients who have undergone pre-conditioning before allogeneic hematopoietic stem cell transplantation. In a preferred embodiment, the pre-conditioning is myeloablative. In another preferred embodiment, the pre-conditioning is attenuating.

[0141] Treatment for GvHD is well-known and is included in clinician recommendations (see, for example, Magro et al., BullCancer. 104S:S145-S168, 2017; Penack et al., Lancet Haematol. 7(2):e157-e167; 2020). Treatment for GvHD can be used prophylactically, most often beginning with immunosuppressive therapies such as cyclosporine or tacrolimus.

[0142] When GvHD is used therapeutically, the treatment varies depending on the severity of complications. However, these treatments most commonly involve immunosuppressants, corticosteroids, particularly prednisolone and methylprednisolone. If corticosteroids are ineffective, anti-lymphocyte serum (ALS) may also be used. Finally, other second-line drugs such as mycophenolate mofetil (CellCept®) and monoclonal antibodies (anti-TNFα or IL2 antireceptor) may be used.

[0143] This description also relates to the use of measuring fluctuations in TTV levels in a subject to determine the proliferative capacity of the target T cells.

[0144] The variation in quantity can be determined by comparing the TTV amount measured in a sample taken at the first time point with the TTV amount measured in a sample taken at a second time point, which is later than the first time point.

[0145] As mentioned above, an increase in TTV over time reflects a decrease in the activity of T cells through a decrease in their proliferative capacity. Conversely, a decrease in TTV over time reflects an increase in the activity of T cells through an increase in their proliferative capacity.

[0146] This description also relates to the following embodiments.

[0147] Embodiment 1: A method for determining the proliferative capacity of T cells in a subject, the method comprising the following steps: a) A step of measuring the amount of TTV from the target biological sample; b) a) step of determining the T cell proliferation capacity according to the viral load measured in a), Includes.

[0148] Embodiment 2: The method according to Embodiment 1, wherein the amount of TTV is measured by amplification, sequencing, or hybridization of the TTV sequence, preferably by amplification, and more preferably by real-time PCR.

[0149] Embodiment 3: The method according to any one of Embodiments 1 to 2, wherein the biological sample is a sample of whole blood, plasma, or serum.

[0150] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the determination in step b) includes comparing the TTV amount measured in a) with a reference TTV amount.

[0151] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the patient receives a transplant.

[0152] Embodiment 6: The method according to Embodiment 5, wherein the patient has undergone hematopoietic stem cell transplantation (HSCT), preferably allogeneic hematopoietic stem cell transplantation.

[0153] Embodiment 7: The method according to any one of Embodiments 5 to 6, wherein the patient receives pre-conditioning, preferably myeloablative or attenuating pre-conditioning, before the transplant surgery.

[0154] Embodiment 8: A method for monitoring T cell activity in a patient who has undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) In any of Embodiments 1 to 6, the first step is to measure the proliferative capacity of T cells in the patient; b) A step of comparing the T cell proliferation capacity measured in a) with the T cell reference proliferation capacity; c) A step of determining the change in the activity of the patient's T cells in accordance with the comparison in step b), Includes.

[0155] Example 9: A method for determining susceptibility to microbial infection in a patient who has undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) In any of Embodiments 1 to 6, the first step is to measure the proliferative capacity of T cells in the patient; b) A step of comparing the T cell proliferation capacity measured in a) with the T cell reference proliferation capacity; c) A step of determining the patient's susceptibility to microbial infections in accordance with the comparison in step b), Including.

[0156] Embodiment 10: The method according to Embodiment 9, wherein the microbial infection is a viral, bacterial, protozoan, or fungal infection.

[0157] Embodiment 11: A method for determining susceptibility to graft-versus-host disease (GvHD) in a patient who has undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) In any of Embodiments 1 to 6, the first step is to measure the proliferative capacity of T cells in the patient; b) A step of comparing the T cell proliferation capacity measured in a) with the T cell reference proliferation capacity; c) A step to determine the patient's susceptibility to GvHD based on the comparison in step b), Includes.

[0158] Embodiment 12: The method according to any one of Embodiments 8 to 11, wherein the reference proliferative capacity of the T cells is the proliferative capacity of T cells in a healthy individual or the proliferative capacity of T cells in an immunosuppressed individual.

[0159] Embodiment 13: The method according to any one of Embodiments 8 to 11, wherein the baseline proliferative capacity of the T cells is the proliferative capacity of the T cells measured in the patient at a second time point.

[0160] The present invention is more accurately described by the following embodiments. [Brief explanation of the drawing]

[0161] [Figure 1] Representation of the genome structure of a TTV isolate. Genomic structure of prototype TTV (TTV-1a isolate). Arrows indicate major ORFs (over 50 amino acids in length). GC-rich regions and region N22 (where TTV was originally isolated) are shown. The untranslated region UTR corresponds to the area from the 3' end of ORF4 to the 5' end of ORF2. From Biagini, Curr Top Microbiol Immunol. 331:21-33, 2009. [Figure 2]TTV viral load from plasma samples of allogeneic hematopoietic stem cell transplant recipients and healthy subjects. TTV viral loads were quantified from 41 allogeneic hematopoietic stem cell transplant recipients (black) and 54 healthy subjects (white). After DNA extraction, TTV viral load was quantified using the TTV R-GENE® kit (for investigational use only, not for diagnostic purposes, Ref#69-030, bioMerieux.Marcy-l'Etoile, France). The minimum viral load detected was 0.46 log copies / mL (log cp / mL). log copies / mL is used to describe the expression of TTV viral load between the two populations. Variances were compared using the F-test (##p<0.01). Mean TTV viral load (black line) was compared using the unpaired t-test and Welch correction (***p<0.001). Abbreviations: DNA Deoxyribonucleic acid; Allo Allo Allo Hematopoietic Stem Cell Transplantation (HSCT); TTV Torquetenovirus The proliferative capacity of CD3+ T cells from 41 allogeneic hematopoietic stem cell transplant recipients (black) and 20 healthy subjects (white) was quantified after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 kit. The variances of the two groups were compared using the F-test (##p<0.01). The mean values ​​(black line) were compared using an unpaired t-test with Welch correction (***p<0.001). Abbreviations: Allo Allo Allo Hematopoietic Stem Cell Transplantation (HSCT); PHA Phytohemagglutinin [Figure 3A]Correlation between TTV viral load and T cell count and CD3+ T cell proliferative capacity. Overall correlation of plasma TTV viral load (A) with the number of several T cell subtypes and CD3+ T cell proliferative capacity from 41 allogeneic hematopoietic stem cell transplant recipients. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters evaluated are shown as black dots and black lines, respectively. Detailed correlation of TTV viral load (B) as a function of CD3+ T cell proliferative capacity, (C) absolute lymphocyte count, and (D) number of CD3+ T cells, expressed as Log copies / mL of plasma from 41 allogeneic hematopoietic stem cell transplant recipients. Patients are shown as dots. Extreme patients: “A” (square) and “B” (triangle), as well as linear regression (black line). Lymphocyte counts were measured by flow cytometry in the immunology laboratory using a broad panel of T cell membrane markers. The proliferative capacity of CD3+ T cells was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 kit. The correlation between TTV viral load (x axis) and lymphocyte count or CD3+ T cell proliferative capacity (y axis) was determined using the Pearson correlation coefficient (shown in each figure). Abbreviations: Allo: Allogeneic; HSCT: Hematopoietic stem cell transplantation; NK: Natural killer; PHA: Phytohemagglutinin; TTV: Torquetenovirus. Overall correlation (A) between T cell immunophenotyping and CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) obtained from the plasma of 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation (B) between CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) from 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to absolute lymphocyte count (C). Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to CD3+ T cell count (D).At the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon), subtypes and absolute lymphocyte counts were measured by flow cytometry using a broad panel of T cell membrane markers. CD3+ proliferative capacity was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 flow kit. The correlation between TTV viral load (horizontal axis) and CD3+ T cell proliferative capacity or cell count (vertical axis) was determined using the Pearson correlation coefficient (shown in each figure). (A) Values ​​from -0.5 to 0.5, represented by the black dotted line, correspond to the range of the correlation confidence interval. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters are represented by dots and black lines, respectively (B, C, and D). Patients are represented by black dots, and the most extreme patients are represented by squares and triangles. Linear regression is represented by the black line. [Figure 3B]Correlation between TTV viral load and T cell count and CD3+ T cell proliferative capacity. Overall correlation of plasma TTV viral load (A) with the number of several T cell subtypes and CD3+ T cell proliferative capacity from 41 allogeneic hematopoietic stem cell transplant recipients. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters evaluated are shown as black dots and black lines, respectively. Detailed correlation of TTV viral load (B) as a function of CD3+ T cell proliferative capacity, (C) absolute lymphocyte count, and (D) number of CD3+ T cells, expressed as Log copies / mL of plasma from 41 allogeneic hematopoietic stem cell transplant recipients. Patients are shown as dots. Extreme patients: “A” (square) and “B” (triangle), as well as linear regression (black line). Lymphocyte counts were measured by flow cytometry in the immunology laboratory using a broad panel of T cell membrane markers. The proliferative capacity of CD3+ T cells was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 kit. The correlation between TTV viral load (x axis) and lymphocyte count or CD3+ T cell proliferative capacity (y axis) was determined using the Pearson correlation coefficient (shown in each figure). Abbreviations: Allo: Allogeneic; HSCT: Hematopoietic stem cell transplantation; NK: Natural killer; PHA: Phytohemagglutinin; TTV: Torquetenovirus. Overall correlation (A) between T cell immunophenotyping and CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) obtained from the plasma of 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation (B) between CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) from 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to absolute lymphocyte count (C). Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to CD3+ T cell count (D).At the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon), subtypes and absolute lymphocyte counts were measured by flow cytometry using a broad panel of T cell membrane markers. CD3+ proliferative capacity was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 flow kit. The correlation between TTV viral load (horizontal axis) and CD3+ T cell proliferative capacity or cell count (vertical axis) was determined using the Pearson correlation coefficient (shown in each figure). (A) Values ​​from -0.5 to 0.5, represented by the black dotted line, correspond to the range of the correlation confidence interval. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters are represented by dots and black lines, respectively (B, C, and D). Patients are represented by black dots, and the most extreme patients are represented by squares and triangles. Linear regression is represented by the black line. [Figure 3C]Correlation between TTV viral load and T cell count and CD3+ T cell proliferative capacity. Overall correlation of plasma TTV viral load (A) with the number of several T cell subtypes and CD3+ T cell proliferative capacity from 41 allogeneic hematopoietic stem cell transplant recipients. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters evaluated are shown as black dots and black lines, respectively. Detailed correlation of TTV viral load (B) as a function of CD3+ T cell proliferative capacity, (C) absolute lymphocyte count, and (D) number of CD3+ T cells, expressed as Log copies / mL of plasma from 41 allogeneic hematopoietic stem cell transplant recipients. Patients are shown as dots. Extreme patients: “A” (square) and “B” (triangle), as well as linear regression (black line). Lymphocyte counts were measured by flow cytometry in the immunology laboratory using a broad panel of T cell membrane markers. The proliferative capacity of CD3+ T cells was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 kit. The correlation between TTV viral load (x axis) and lymphocyte count or CD3+ T cell proliferative capacity (y axis) was determined using the Pearson correlation coefficient (shown in each figure). Abbreviations: Allo: Allogeneic; HSCT: Hematopoietic stem cell transplantation; NK: Natural killer; PHA: Phytohemagglutinin; TTV: Torquetenovirus. Overall correlation (A) between T cell immunophenotyping and CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) obtained from the plasma of 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation (B) between CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) from 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to absolute lymphocyte count (C). Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to CD3+ T cell count (D).At the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon), subtypes and absolute lymphocyte counts were measured by flow cytometry using a broad panel of T cell membrane markers. CD3+ proliferative capacity was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 flow kit. The correlation between TTV viral load (horizontal axis) and CD3+ T cell proliferative capacity or cell count (vertical axis) was determined using the Pearson correlation coefficient (shown in each figure). (A) Values ​​from -0.5 to 0.5, represented by the black dotted line, correspond to the range of the correlation confidence interval. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters are represented by dots and black lines, respectively (B, C, and D). Patients are represented by black dots, and the most extreme patients are represented by squares and triangles. Linear regression is represented by the black line. [Figure 3D]Correlation between TTV viral load and T cell count and CD3+ T cell proliferative capacity. Overall correlation of plasma TTV viral load (A) with the number of several T cell subtypes and CD3+ T cell proliferative capacity from 41 allogeneic hematopoietic stem cell transplant recipients. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters evaluated are shown as black dots and black lines, respectively. Detailed correlation of TTV viral load (B) as a function of CD3+ T cell proliferative capacity, (C) absolute lymphocyte count, and (D) number of CD3+ T cells, expressed as Log copies / mL of plasma from 41 allogeneic hematopoietic stem cell transplant recipients. Patients are shown as dots. Extreme patients: “A” (square) and “B” (triangle), as well as linear regression (black line). Lymphocyte counts were measured by flow cytometry in the immunology laboratory using a broad panel of T cell membrane markers. The proliferative capacity of CD3+ T cells was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 kit. The correlation between TTV viral load (x axis) and lymphocyte count or CD3+ T cell proliferative capacity (y axis) was determined using the Pearson correlation coefficient (shown in each figure). Abbreviations: Allo: Allogeneic; HSCT: Hematopoietic stem cell transplantation; NK: Natural killer; PHA: Phytohemagglutinin; TTV: Torquetenovirus. Overall correlation (A) between T cell immunophenotyping and CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) obtained from the plasma of 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation (B) between CD3+ T cell proliferative capacity and plasma TTV viral load (Log cp / mL) from 41 allogeneic hematopoietic stem cell transplant recipients. Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to absolute lymphocyte count (C). Detailed correlation of TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients with respect to CD3+ T cell count (D).At the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon), subtypes and absolute lymphocyte counts were measured by flow cytometry using a broad panel of T cell membrane markers. CD3+ proliferative capacity was determined after 3 days of stimulation with mitogen (PHA) and measured by flow cytometry using the Click-It® EdU AF488 flow kit. The correlation between TTV viral load (horizontal axis) and CD3+ T cell proliferative capacity or cell count (vertical axis) was determined using the Pearson correlation coefficient (shown in each figure). (A) Values ​​from -0.5 to 0.5, represented by the black dotted line, correspond to the range of the correlation confidence interval. Pearson's Rho (ρ) and 95% confidence interval (CI95) for all parameters are represented by dots and black lines, respectively (B, C, and D). Patients are represented by black dots, and the most extreme patients are represented by squares and triangles. Linear regression is represented by the black line. [Figure 4] Descriptive time-series monitoring of patients with the most extreme TTV viral loads. Time-series description of major clinical stages (black) and infection episodes (gray) from HSCT to inclusion for patients "A" and "B". Patient "A" had the lowest TTV viral load, while patient "B" had the highest. Abbreviations: CMV Cytomegalovirus; EBV Epstein-Barr virus; HSCT Hematopoietic stem cell transplantation; GvHD Graft-versus-host disease; M Month [Figure 5] Correlation between TTV viral load and time since HSCT. Detailed correlation: Detailed correlation between plasma TTV viral load (expressed as Log cp / mL) in the plasma of 41 allogeneic hematopoietic stem cell transplant recipients and the time elapsed from HSCT to enrollment (expressed as months). The correlation between TTV viral load (horizontal coordinate) and delay (vertical coordinate) was determined using the Pearson correlation coefficient (shown in each figure). Patients are represented by black dots, and linear regression is represented by the black line. Abbreviations: HSCT: Hematopoietic stem cell transplantation; TTV: Torque tenovirus [Examples]

[0162] Example 1 This study evaluated and compared the correlation between TTV viral load and immune cell count, as well as the function of immune cells, during the immune recovery phase after transplantation in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients.

[0163] Materials and methods research group Heparinized whole blood samples and EDTA-treated plasma samples obtained from patients who underwent allogeneic hematopoietic stem cell transplantation were taken from the aforementioned prospective cohort Vaccheminf (13). This cohort was approved by the regional review board (Comite de protection des personnes Sud-Est V, Grenoble, France, number 69HCL17_0769) and registered with ClinicalTrial.gov (NCT03659773). Adult patients continuing treatment who underwent allogeneic hematopoietic stem cell transplantation at the Department of Hematology, CHU Lyon, were included prospectively once written consent was obtained from the patients.

[0164] Upon admission, collected data such as demographic characteristics (age, sex) and clinical data (type of transplant, immunophenotypic testing, immunosuppressive therapy, GvHD status, and GvHD treatment) were recorded using an electronic case report form (eCRF).

[0165] In parallel, 80 healthy individuals (HVs) were collected from donors at the Lyon Blood Bank (Etablissement Francais du Sang, EFS). In accordance with EFS's standardized procedures for blood donation and Articles R.1243-49 of the Public Health Code, written consent was obtained from these healthy individuals indicating they did not object to the use of donated blood for research purposes. The age and sex of the donors were anonymously sent to the laboratory. Regulatory permission for the handling and storage of these samples was obtained from the Regional Ethics Committee (Comite de protection des personnes Sud-Est II, Bron, France) and the French Ministry of Higher Education, Research and Innovation (Ministry of Higher Education, Research and Innovation, Paris, France).

[0166] Quantification of TTV viral load Following the manufacturer's instructions, a 50 μL viral DNA elution volume was extracted from a 200 μL plasma sample using an easyMag extractor (bioMerieux, France). The presence and amount of TTV were then determined using the aforementioned TTV R-GENE® kit (bioMerieux, Marcy-l'Etoile, France) (14, 15).

[0167] T-cell proliferation test Peripheral blood mononuclear cells (PBMCs) were separated from heparinized fresh blood samples (heparinized whole blood) using Ficoll density gradient centrifugation (U-04; Eurobio, Les Ulis, France). Next, 10 5The cells / wells were incubated for 24 hours in supplemented culture medium in a 96-well cell culture plate (RPMI 1640; Eurobio) at 37°C and 5% CO2. Next, PBMCs were stimulated twice with the mitogen, phytohemagglutinin (PHA) at 4 μg / mL (R30852801; Remel, Oxoid, Thermo Fisher Scientific, USA) and incubated for 72 hours. The culture supernatant of PBMCs was collected and an IFNγ secretion assay (IGRA, "IFNγ-release assay") was performed using a Simple Plex cartridge on an ELLA nanofluidic system (ProteinSimple, San Jose, CA, USA) according to the manufacturer's instructions. The Click-iT (trademark) Plus EdU Alexa Fluor (trademark) 488 Flow Cytometry Assay Kit (C10420; Life Technologies, Carlsbad, CA, USA) was used to measure the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) to analyze T cell proliferation in the pellet according to the published protocol (16). Briefly, flow cytometry analysis performed on a BD LSR Fortessa (trademark) flow cytometer (BD Biosciences, San Jose, CA, USA) determined the percentage of EdU+ proliferating cells among (CD3 + cells) (BD Biosciences, San Jose, California). For each experiment, at least 2.5×10 3 CD3 + cells were measured. Data were analyzed using BD FACSDiva (registered trademark) software (version 8.0.3, BD Biosciences).

[0168] Immunophenotyping of T cells after transplantation surgery Leukocytes, as well as CD4 + and CD8 +T cells were counted in the immunology laboratory at Edouard Herriot Hospital (Hospices Civils de Lyon). Furthermore, a broad panel of T cell membrane markers was measured in whole blood by flow cytometry. As previously described (14), the following were counted by this method: naive CD4 + and CD8 + T cells (CD45 + CCR7 + ), Central Memory CD4 + and CD8 + T cells (CD45RA - CCR7 + ), Effector Memory CD4 + and CD8 + T cells (CD45 - CCR7 + ), and the separated memory CD4 + and CD8 + T cells (CD45RA + CCR7 - The results are expressed as cells / μL.

[0169] statistical analysis Immunophenotypic test data, TTV viral load, and T cell proliferation capacity are expressed as mean values ​​(ranges). TTV load converted to log format was used for analysis (log copies / mL). The difference between healthy recipients and allogeneic hematopoietic stem cell transplant recipients was calculated using an unpaired parametric t-test with Welch correction. Correlations were evaluated using the parametric Pearson-Raw (ρ) correlation coefficient. Regression analysis was performed with the dependent variable (TTV viral load) and independent variables (proliferating cell percentage, absolute lymphocyte count, CD3). +This study was conducted to evaluate the association with T cell count. Analysis of variance was performed using the F-test. The Mann-Whitney test was used to determine the difference in plasma TTV volume for various clinical features. A value of p<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism® software (version 5; GraphPad software, La Jolla, CA, USA) and R (version 3.5.1).

[0170] result Cohort characteristics From May 2018 to April 2020, healthy subjects (n=80) and allogeneic hematopoietic stem cell transplant recipients (n=41) were enrolled. There were no significant differences in age (median [IR]: 56 [40-64] vs. 46 [31-53] years, respectively) or sex (sex ratio: 1.6 vs. 1.4, respectively) between healthy individuals and allogeneic hematopoietic stem cell transplant recipients. At enrollment, 78% of allogeneic hematopoietic stem cell transplant recipients were receiving immunosuppressant medications (corticoids, calcineurin inhibitors, etc.), and 17% had chronic graft-versus-host disease (Table 1).

[0171] TTV viral load in plasma samples from healthy subjects and allogeneic hematopoietic stem cell transplants The TTV viral load in plasma samples obtained from 80 healthy recipients and 41 allogeneic hematopoietic stem cell transplant recipients was studied. TTV viral load was detected by real-time PCR in 68% (54 / 80) of healthy samples. For allogeneic hematopoietic stem cell transplant recipients, all patients included in this study had detectable TTV viral loads. The mean (range) TTV viral load was significantly higher in allogeneic hematopoietic stem cell transplant recipients compared to healthy subjects (3.9 (0.7~7.7) vs. 2.1 (0.5~4.3) log copies / mL, p<0.0001, respectively) [Figure 2].

[0172] Correlation between TTV viral load and T cell number, and the latter's proliferation ability. Considering allogeneic hematopoietic stem cell transplant recipients, including those 6 months post-transplant, most lymphocyte subtypes were within the normal range (NV). However, T cell CD4 + Naive CD4 + Central Memory CD4 + , effector memory CD4 in final differentiation + Naive CD8 + , and Central Memory CD8 + The amount, and CD4 + / CD8 + The ratio was below the normal value (see [Table 1]).

[0173] [Table 1] Baseline characteristics of allogeneic hematopoietic stem cell transplant recipients All laboratory data was recorded when the recipient registered. * Based on the 2016 revised World Health Organization classification of myeloid and lymphoid tumors. #Immunosuppressive therapy included anti-thymocyte globulin, cyclosporine, tacrolimus, methotrexate, mycophenolate mofetil, cyclophosphamide, and corticosteroids ≥1 mg / kg >21 days. Total lymphocyte count and subtypes were measured by flow cytometry using a broad panel of T cell membrane markers at the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon). The normal values ​​shown are provided by the laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon). [Table 1-1] [Table 1-2] [Table 1-3]

[0174] Abbreviations: Allo (allogeneic); DLI (donor lymphocyte infusion); GvHD (graft-versus-host disease); HLA (human leukocyte antigen); HSCT (hematopoietic stem cell transplantation); IR (interquartile range); TBI (total body irradiation); IS (immunosuppressant); IVIG (intravenous polyclonal immunoglobulin); MAC (myeloablative conditioning); NK (natural killer); ECP (extracorporeal photochemotherapy); CR (complete remission); RIC (reduced-intensity pre-transplant treatment); NV (normal value)

[0175] CD3 in allogeneic hematopoietic stem cell transplant recipients compared to healthy subjects + Aside from significantly lower proliferative capacity in cells (40.5% vs. 21.3%, p<0.0001, respectively), larger and more significant heterogeneity was also observed ([2.9%–42.3%] and [29.7%–55.3%] in allogeneic hematopoietic stem cell transplant recipients and healthy subjects, respectively; F-test p=0.0040), indicating underlying inter-individual variability in immune recovery among allogeneic hematopoietic stem cell transplant recipients (not shown in figures / tables).

[0176] Using the Pearson correlation test (Rhoe(ρ)[CI95]), the highest correlation was observed between TTV viral load and T cell proliferative capacity ((3A) and (3B)). Note that the total number of lymphocytes or a subset of specific cells (e.g., CD3) + It should be noted that no significant correlation was observed between (Pearson's Rho (ρ) ρ=-0.39 [CI 95% -0.62~-0.09]) and (ρ=0.13 [-0.19~0.42]) and (ρ=0.09 [-0.23~0.38]) respectively (Figure 3C) and (Figure 3D).

[0177] Clinical characteristics of patients with the most extreme TTV values When the correlation between T cell proliferation capacity in response to PHA stimulation and TTV levels was analyzed at the individual level, patients with the lowest viral load (0.65 log copies / mL) (A, represented by a square) had a high percentage of proliferating cells (41.6%), while patients with the highest viral load (7.72 log copies / mL) (B, represented by a triangle) had a low percentage of proliferating cells (2.9%) (Figure 3B). These two patients (male and female) were in the same age group (50 < age < 60 years) and were in complete remission before transplantation. Patient (A) received stem cell transplantation from a geno-identical donor, while patient (B) received peripheral blood cell transplantation from a pheno-identical donor. Patient (A) did not experience any specific clinical events such as infection episodes, GvHD, or immunosuppressive therapy between transplantation and registration, and the post-transplant development was straightforward (Figure 4). Conversely, patient (B) was undergoing cumbersome immunosuppressive therapy and suffered from acute GvHD and multiple severe bacterial / viral infections (Figure 4).

[0178] Discussion First, we compared the prevalence of TTV and plasma viral load in two different groups: 80 healthy, immunocompetent subjects and 41 immunosuppressed allogeneic hematopoietic stem cell transplant recipients from the monocentral prospective cohort VaccHemInf(13).

[0179] Recent studies (18) have shown a 68% prevalence of TTV in samples from healthy subjects. In contrast, TTV was detected in 100% of samples from immunosuppressed patients. Furthermore, plasma viral loads of TTV were found to be significantly higher in allogeneic hematopoietic stem cell transplant recipients compared to HV (10, 19). Therefore, some patients are unable to regulate their TTV viral load 6 months after allogeneic hematopoietic stem cell transplantation, despite having a sufficient number of T cells. No correlation was found between post-transplant delay [5-8 months] and plasma TTV viral load. One of the main findings of this study is that plasma viral loads of TTV were significantly higher in allogeneic hematopoietic stem cell transplant recipients (6 months post-transplant surgery) compared to healthy subjects, confirming the observations of Tyagi et al. (2013) (post-transplant delay not described) and Masouridi et al. (2016) (2-3 months post-transplant surgery) (10, 18). These results may be explained by the fact that T cells (19, 20), the main cells in the immune response to viral infection, are one of the main replication sites (21, 22) for TTV. In allogeneic hematopoietic stem cell transplant recipients 6 months post-transplant, T cell proliferation is ongoing as the immune system recovers, supplying a large number of cells for viral replication. It has also been noted that TTV levels peak around 3–6 months post-transplant surgery and then return to so-called normal levels (23, 24) (23, 24). Therefore, it can be hypothesized that TTV replicates by utilizing the proliferation of still naive and non-functional T cells, and is ultimately regulated by the immune system and functional cells, suggesting an important relationship between TTV viral load and immune function. This study includes an evaluation of the correlation between plasma TTV levels and the quantitative marker of immune cell count, which has already been evaluated in several studies, but with conflicting results (17, 23–25), and also includes the measurement of T cell proliferation after nonspecific stimulation, a qualitative marker of immune recovery. No effect was observed from the time between allogeneic hematopoietic stem cell transplantation surgery and registration (Figure 5). Despite the heterogeneous distribution of T cell proliferation values, which highlights inter-individual variability in immunosuppressed populations, a stronger correlation was observed between TTV viral load and T cell proliferation than with immune cell count (Figure 3A).More precisely, and interestingly, there is an inverse correlation, suggesting that a greater number of functional immune cells is associated with lower TTV loads. This result is consistent with what De Vlaminck et al. described in 2013 (11) in solid organ transplantation, where a decrease in the level of immunosuppression correlated with a decrease in TTV viral load. This also corresponds to many descriptions of the dynamics of TTV viral load after transplantation (23, 26), where an initial phase of decline associated with immunosuppressive treatment is observed, followed by a growth phase associated with an expansion of immune cell capacity for TTV replication, then a stabilization phase, and finally a decrease in viral load to baseline levels, which reflects functional immune recovery. Six months after allogeneic hematopoietic stem cell transplantation, while there is an expansion in the number of immune cells sufficient to allow significant TTV replication, it is thought that they would not be functional enough to regulate TTV viral load. Therefore, immunosuppressed patients would not be able to regulate TTV viral load despite a sufficient number of T cells. This also applies to other viruses classically described in allogeneic hematopoietic stem cell transplant recipients, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV) (24, 27, 28). In our cohort, 24% and 37% of patients were infected with these two viruses, respectively. This is consistent with observations made in patients with the most extreme TTV viral loads.

[0180] TTV viral load is a subtype of lymphocyte, CD8, described as a potential marker of immunosenescence. + / CD57 + TTV is also associated with the number of T cells and increases during certain pathological processes such as acquired immunodeficiency, transplantation, or persistent viral infection. Therefore, all these results suggest a potentially important relationship between TTV and immune system function, particularly with respect to the ability to regulate viral load.

[0181] Our study did not observe any influence of various clinical features (e.g., condition, underlying disease, immunosuppressive therapy, or GvHD) on TTV levels (Table 2).

[0182] Table 2 Comparison of plasma TTV volume and clinical characteristics in 41 recipients of allogeneic hematopoietic stem cell transplantation. All data was recorded when the recipient was registered. * According to the 2016 revised World Health Organization classification of myeloid and lymphoid tumors. #Immunosuppressive therapy included antithymocyte globulin, cyclosporine, tacrolimus, methotrexate, mycophenolate mofetil, cyclophosphamide, and corticosteroids ≥1 mg / kg >21 days. Mann-Whitney test ( *** Using p<0.001, the median plasma TTV volume [IR] was compared with clinical data. [Table 2]

[0183] Abbreviations: GvHD - Graft-versus-host disease; IR - Interquartile range; IVIG - Intravenous polyclonal immunoglobulin; MAC - Myeloablative conditioning; RIC - Reduced intensity pre-transplant treatment; TBI - Whole-body irradiation; TTV - Torque tenovirus

[0184] In summary, this study demonstrates a correlation between TTV viral load and T cell function, and shows that this correlation is independent of cell number.

[0185] Example 2 Example 1 compares the number of T cells, the proliferative capacity of the T cells, and the plasma TTV viral load among multiple patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT).

[0186] The TTV viral load was quantified, proliferation tests were performed, and CD3+ T cells were measured according to the same protocol as in Example 1. The results are shown in Table 3 below. [Table 3] Table 3 Comparison of plasma TTV viral load, CD3+ T cell count, and lymphocyte proliferation capacity.

[0187] Comparing TTV viral load and CD3+ T cell count, we found that for patients with similar viral loads, the CD3+ T cell count can differ significantly (1 patient vs. 2 patients), while conversely, for significantly different viral loads (3 patients vs. 4 patients), the CD3+ T cell count can be similar. Therefore, this confirms that TTV viral load does not correlate with the CD3+ T cell count.

[0188] However, when comparing the number and proliferative capacity of CD3+ T cells, it can be seen that in patients with similar proliferative capacity, the number of these lymphocytes can differ significantly (patient 1 vs patient 2). Conversely, when the number of CD3+ T cells is similar, proliferative capacity can differ significantly (patient 3 vs patient 4).

[0189] As a result, this confirms that the number of CD3+ T cells is not exclusively correlated with their proliferative capacity, and that, in contrast to the TTV viral load, a high number of CD3+ T cells does not suggest the proliferative capacity of the lymphocytes after stimulation, nor does it indicate the function of the immune system.

[0190] In fact, when looking at the TTV virus load, CD3 + It was found to be inversely correlated with the proliferative capacity of T cells (3 patients vs. 4 patients).

[0191] reference 1. Nishizawa T, Okamoto H, Konishi K, Yoshizawa H, Miyakawa Y, Mayumi M. A Novel DNA Virus (TTV) Associated with Elevated Transaminase Levels in Posttransfusion Hepatitis of Unknown Etiology. Biochem Biophys Res Commun. dec 1997;241(1):92-7. 2. Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from bench to bedside. Clin Microbiol Infect. juill 2016;22(7):589-93. 3. Spandole S, Cimponeriu D, Berca LM, Mihaescu G. Human anelloviruses: an update of molecular, epidemiological and clinical aspects. Arch Virol. avr 2015;160(4):893-908. 4. de Villiers E-M, Borkosky SS, Kimmel R, Gunst K, Fei J-W. The diversity of torque teno viruses: in vitro replication leads to the formation of additional replication-competent subviral molecules. J Virol. juill 2011;85(14):7284-95. 5. Kosulin K, Kernbichler S, Pichler H, Lawitschka A, Geyeregger R, Witt V, et al. Post-transplant Replication of Torque Teno Virus in Granulocytes. Front Microbiol. 2018;9:2956. 6. Maggi F, Bendinelli M. Immunobiology of the Torque Teno Viruses and Other Anelloviruses. In: de Villiers E-M, Hausen H zur, editeurs. TT Viruses [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2009 [cite 20 aout 2019]. p. 65-90. Disponible sur: http: / / link.springer.com / 10.1007 / 978-3-540-70972-5_5 7. Hino S, Miyata H. Torque teno virus (TTV): current status. Rev Med Virol. fevr 2007;17(1):45-57. 8. Mitchell AB, Glanville AR. Kinetics of TTV-DNA Plasma Load: A Global Measure of Immune Suppression? Transplantation. avr 2019;103(4):660-1. 9. Focosi D, Maggi F, Albani M, Macera L, Ricci V, Gragnani S, et al. Torquetenovirus viremia kinetics after autologous stem cell transplantation are predictable and may serve as a surrogate marker of functional immune reconstitution. J Clin Virol. fevr 2010;47(2):189-92. 10. Masouridi-Levrat S, Pradier A, Simonetta F, Kaiser L, Chalandon Y, Roosnek E. Torque teno virus in patients undergoing allogeneic hematopoietic stem cell transplantation for hematological malignancies. Bone Marrow Transplant. mars 2016;51(3):440-2. 11. De Vlaminck I, Khush KK, Strehl C, Kohli B, Luikart H, Neff NF, et al. Temporal Response of the Human Virome to Immunosuppression and Antiviral Therapy. Cell. nov 2013;155(5):1178-87. 12. Hoshina T, Ohga S, Fujiyoshi J, Nanishi E, Takimoto T, Kanno S, et al. Memory B-Cell Pools Predict the Immune Response to Pneumococcal Conjugate Vaccine in Immunocompromised Children. J Infect Dis. 1 mars 2016;213(5):848-55. 13. Conrad A, Boccard M, Valour F, Alcazer V, Tovar Sanchez A-T, Chidiac C, et al. VaccHemInf project: protocol for a prospective cohort study of efficacy, safety and characterisation of immune functional response to vaccinations in haematopoietic stem cell transplant recipients. BMJ Open. fevr 2019;9(2):e026093. 14. Kulifaj D, Durgueil-Lariviere B, Meynier F, Munteanu E, Pichon N, Dube M, et al. Development of a standardized real time PCR for Torque teno viruses (TTV) viral load detection and quantification: A new tool for immune monitoring. J Clin Virol. aout 2018;105:118-27. 15. Kulifaj D, Essig M, Meynier F, Pichon N, Munteanu E, Moulinas R, et al. Torque teno virus (TTV) in immunosuppressed host: Performances studies of TTV R-Gene(registered trademark) kit and donors and recipients kidney samples genotyping. J Clin Virol. sept 2016;82:S103-4. 16. Poujol F, Monneret G, Friggeri A, Rimmele T, Malcus C, Poitevin-Later F, et al. Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2′deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement. J Immunol Methods. dec 2014;415:71-9. 17. Focosi D, Spezia PG, Macera L, Salvadori S, Navarro D, Lanza M, et al. Assessment of prevalence and load of torquetenovirus viraemia in a large cohort of healthy blood donors. Clin Microbiol Infect. janv 2020;S1198743X20300367. 18. Tyagi A, Pradier A, Baumer O, Uppugunduri CRS, Huezo-Diaz P, Posfay-Barbe KM, et al. Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma. Virol J. 2013;10(1):191. 19. Rosendahl Huber S, van Beek J, de Jonge J, Luytjes W, van Baarle D. T cell responses to viral infections - opportunities for Peptide vaccination. Front Immunol. 2014;5:171. 20. Sant AJ, McMichael A. Revealing the role of CD4+ T cells in viral immunity. J Exp Med. 30 juill 2012;209(8):1391-5. 21. Focosi D, Macera L, Boggi U, Nelli LC, Maggi F. Short-term kinetics of torque teno virus viraemia after induction immunosuppression confirm T cells as the main replication-competent cells. J Gen Virol. 1 janv 2015;96(Pt_1):115-7. 22. Maggi F, Fornai C, Zaccaro L, Morrica A, Vatteroni ML, Isola P, et al. TT virus (TTV) loads associated with different peripheral blood cell types and evidence for TTV replication in activated mononuclear cells. J Med Virol. juin 2001;64(2):190-4. 23. Albert E, Solano C, Gimenez E, Focosi D, Perez A, Macera L, et al. Kinetics of Alphatorquevirus plasma DNAemia at late times after allogeneic hematopoietic stem cell transplantation. Med Microbiol Immunol (Berl). avr 2019;208(2):253-8. 24. Wohlfarth P, Leiner M, Schoergenhofer C, Hopfinger G, Goerzer I, Puchhammer-Stoeckl E, et al. Torquetenovirus Dynamics and Immune Marker Properties in Patients Following Allogeneic Hematopoietic Stem Cell Transplantation: A Prospective Longitudinal Study. Biol Blood Marrow Transplant. janv 2018;24(1):194-9. 25. Schmitz J, Kobbe G, Kondakci M, Schuler E, Magorsch M, Adams O. The Value of Torque Teno Virus (TTV) as a Marker for the Degree of Immunosuppression in Adult Patients after Hematopoietic Stem Cell Transplantation (HSCT). Biol Blood Marrow Transplant. nov 2019;S1083879119307426. 26. Gilles R, Herling M, Holtick U, Heger E, Awerkiew S, Fish I, et al. Dynamics of Torque Teno virus viremia could predict risk of complications after allogeneic hematopoietic stem cell transplantation. Med Microbiol Immunol (Berl). oct 2017;206(5):355-62. 27. Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol Oncol Clin North Am. fevr 2011;25(1):151-69. 28. Liu L, Zhang X, Feng S. Epstein-Barr Virus-Related Post-Transplantation Lymphoproliferative Disorders After Allogeneic Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transplant. juill 2018;24(7):1341-9. 29. Bosch M, Khan FM, Storek J. Immune reconstitution after hematopoietic cell transplantation: Curr Opin Hematol. juill 2012;19(4):324-35.

Claims

1. A method for determining the proliferative capacity of T cells in a subject, wherein the method is: a) A step of measuring the amount of TTV virus from the target biological sample; b) Based on the fact that the amount of TTV virus and the proliferative capacity of T cells are inversely correlated, a step is made to determine the proliferative capacity of T cells according to the amount of TTV virus measured in step a), A method characterized by including the following.

2. The method according to claim 1, wherein the amount of TTV virus is measured by amplification, sequencing, or hybridization of TTV sequences, preferably by amplification, and more preferably by real-time PCR.

3. The method according to any one of claims 1 to 2, wherein the biological sample is a sample of whole blood, plasma, or serum.

4. The method according to any one of claims 1 to 3, wherein step b) includes a step of comparing the amount of TTV virus measured in step a) with a reference amount of TTV virus.

5. The method according to any one of claims 1 to 4, wherein the subject has undergone transplantation.

6. The method according to claim 5, wherein the subject has undergone hematopoietic stem cell transplantation (HSCT), preferably allogeneic hematopoietic stem cell transplantation.

7. The method according to any one of claims 5 to 6, wherein the subject has received pretreatment, preferably myeloablative or attenuating pretreatment, before transplant surgery.

8. A method for monitoring T cell activity in subjects who have undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) A step of determining the T cell proliferation capacity in the subject from a biological sample taken from the subject at the initial point in time, using the method of any one of claims 1 to 6; b) A step of comparing the T cell proliferation capacity determined in step a) with the reference T cell proliferation capacity; c) A step of determining the change in the activity of the target T cell in accordance with the comparison in step b), A method characterized by including the following.

9. A method for monitoring T cell activity in a subject who has undergone transplantation, preferably HSCT, more preferably allogeneic hematopoietic stem cell transplantation, wherein the method is a) A step of determining the T cell proliferation capacity in the subject from a biological sample taken from the subject at the initial point in time, using the method described in any one of claims 1 to 6; b) A step of determining the T cell proliferation capacity in the subject from a biological sample taken from the subject at a second time point, which is later than the first time point in step a), by the method of any one of claims 1 to 6; c) A step of comparing the TTV virus amounts measured in steps a) and b); d) A step to determine the activity of the target T cells based on the comparison in step c), A method characterized by including the following.

10. A method for determining susceptibility to microbial infection in subjects who have undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) A step of determining the T cell proliferation capacity in the subject from a biological sample taken from the subject at the initial point in time, using the method of any one of claims 1 to 6; b) A step of comparing the T cell proliferation capacity determined in step a) with the reference T cell proliferation capacity; c) A step of determining the susceptibility to microbial infection of the subject in accordance with the comparison in step b), A method characterized by including the following.

11. The method according to claim 10, wherein the microbial infection is a viral, bacterial, protozoan, or fungal infection.

12. A method for determining susceptibility to graft-versus-host disease (GvHD) in subjects who have undergone allogeneic hematopoietic stem cell transplantation, wherein the method is a) A step of determining the T cell proliferation capacity in the subject from a biological sample taken from the subject at the initial point in time, using the method of any one of claims 1 to 6; b) A step of comparing the T cell proliferation capacity determined in step a) with the reference T cell proliferation capacity; c) A step of determining the susceptibility of the subject to GvHD in accordance with the comparison in step b), A method characterized by including the following.

13. The method according to any one of claims 8, 10, 11, and 12, wherein the baseline proliferative capacity of T cells is the proliferative capacity of T cells in a healthy individual or the proliferative capacity of T cells in an immunosuppressed individual.

14. The method according to any one of claims 8, 10, 11, and 12, wherein the baseline proliferative capacity of T cells is the proliferative capacity of T cells measured in the subject at a second time point, and the second time point is later than the first time point.

15. A method characterized by determining the T cell proliferation ability of a target based on the fact that there is an inverse correlation between the TTV virus amount and the T cell proliferation ability, by measuring the change in the TTV virus amount in the target.

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