EP4240873A1 - Utilisation du virus torque teno (ttv) en tant que marqueur pour determiner la capacite de proliferation des lymphocytes t - Google Patents

Utilisation du virus torque teno (ttv) en tant que marqueur pour determiner la capacite de proliferation des lymphocytes t

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Publication number
EP4240873A1
EP4240873A1 EP21815241.1A EP21815241A EP4240873A1 EP 4240873 A1 EP4240873 A1 EP 4240873A1 EP 21815241 A EP21815241 A EP 21815241A EP 4240873 A1 EP4240873 A1 EP 4240873A1
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European Patent Office
Prior art keywords
ttv
lymphocytes
subject
patient
load
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EP21815241.1A
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German (de)
English (en)
French (fr)
Inventor
Karen Brengel-Pesce
Sophie ASSANT-TROUILLET
William MOUTON
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Biomerieux SA
Universite Claude Bernard Lyon 1 UCBL
Hospices Civils de Lyon HCL
Original Assignee
Biomerieux SA
Universite Claude Bernard Lyon 1 UCBL
Hospices Civils de Lyon HCL
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Application filed by Biomerieux SA, Universite Claude Bernard Lyon 1 UCBL, Hospices Civils de Lyon HCL filed Critical Biomerieux SA
Publication of EP4240873A1 publication Critical patent/EP4240873A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • TITLE USE OF TORQUE TENO VIRUS (TTV) AS A MARKER TO DETERMINE THE PROLIFERATIVE CAPACITY OF T LYMPHOCYTES
  • the immune system defends the body against attacks such as pathogenic infections, cell transformation and physical or chemical damage. An immunocompetent individual is thus able to trigger a protective immune response against antigenic stimulation.
  • an immunodeficiency manifests itself. This takes various forms and can affect the innate or adaptive immune system, or both, depending on the source of the deficiency.
  • immunodeficiency is acquired during life. It can result from a pathology, infectious or not (for example an HIV infection), or be produced by a therapy, such as radiotherapy or chemotherapy.
  • the immunodeficiency state is particularly dangerous, because the patient then has an increased susceptibility to secondary infections by pathogens such as bacteria, viruses, parasites or fungi.
  • pathogens such as bacteria, viruses, parasites or fungi.
  • the use of immunosuppressive therapies in transplants including hematopoietic stem cell transplants (HSCTs)
  • HSCTs hematopoietic stem cell transplants
  • a set of techniques is available for measuring immunocompetence, none of which is completely satisfactory. These techniques measure, among other things, the cell-mediated immune response and include in particular the analysis of lymphocyte populations, in particular the count of CD4 + T lymphocytes or the measurement of the ratio of CD4 + /CD8 + T lymphocytes, the measurement of the capacity of lymphocyte proliferation, measurement of the cytotoxic activity of T lymphocytes, measurement of the antibody response, labeling of tetramers, detection of cytokines produced, ELISpot, etc.
  • Some of these techniques such as for example the counting of T lymphocytes, give a result which does not necessarily reflect the activity of these cells and therefore the activity of the immune system. An absolute number of T cells says nothing about the ability of these cells to multiply.
  • TTV Torque Teno Virus
  • UTR well-conserved non-coding region
  • TTV produces chronic infections with no clearly associated clinical manifestation. We speak of an apathogenic or orphan virus. Numerous studies have thus focused on the involvement of TTV in human pathology, in particular in certain hepatic pathologies, without a clear role being identified for this virus.
  • TTV load is higher in subjects with immunodeficiency.
  • significant levels of TTV are found in patients who have received immunosuppressive treatments in the context of organ transplants (Rezahosseini et al., Transplant Rev (Orlando). 33(3): 137-144, 2019) or of GCSH (Albert et al., Med Microbiol Immunol. 208(2):253-258, 2019).
  • GCSH GCSH
  • TTV load may be a marker of immunocompetence (8-11).
  • immunocompetence was only assessed by the number of immune cells or by the occurrence of clinical adverse events (11-14).
  • the quantity of cells is not necessarily associated with the quality of the cells, that is to say that the activity of the T cells is not reflected by their number (12).
  • the present invention relates to a method for determining whether T lymphocytes are functional in a subject. More specifically, the inventors have shown that the proliferative capacity of T lymphocytes is inversely correlated to the load of torque teno virus (TTV for Torque Teno Virus), in particular in a patient having received a transplant, more particularly an HSCT.
  • TTV viral load is inversely correlated to the proliferation of T lymphocytes: thus the greater the TTV load, the less the lymphocytes are able to proliferate.
  • Viral load is not specifically correlated with any other parameter, be it the number of T cells or a clinical criterion, underlining the relevance of the identified correlation.
  • the TTV load is a specific marker of T cell activity, particularly in patients who have received a transplant and in particular an HSCT.
  • the evolution of the functionality of the immune system can therefore be monitored in a subject by a simple measurement of its TTV load. It is thus possible to quickly assess the functional state of the immune system without having to implement the cumbersome technical steps usually used to assess this parameter.
  • the subject of the present description is a method for determining the proliferative capacity of T cells in a subject, said method comprising measuring the TTV load in the patient.
  • the proliferative capacity of T cells is not necessarily correlated with the number of said T cells. Therefore, the known methods of counting T cells are in no way informative about the proliferative capacity of T cells and therefore, on the functionality of the immune system. He It is therefore to the merit of the inventors to have identified a new parameter, easily measured routinely, which is advantageously correlated with the proliferative capacity of the T cells, thus making it possible to evaluate the functionality of the immune system.
  • T lymphocytes or “T cells” are essential cells of the immune system responsible for boosting or slowing down the immune response.
  • T lymphocytes are characterized by the expression of a membrane marker called CD3 and of a specific receptor, the TCR (for “T cell receptor”), which is directly involved in antigenic recognition.
  • the T lymphocytes can express other surface markers, in particular CD4 and CD8, which correspond to specific functional categories of T lymphocytes. residual cells from the recipient or the donor T lymphocytes present in the graft. They can also be na ⁇ ve T lymphocytes resulting from the differentiation of donor stem and progenitor cells in the recipient's thymus.
  • T cell activation or “T cell activation” here refers to the process by which na ⁇ ve T cells become able to participate in the immune response.
  • the activation of T cells leads in particular to their proliferation.
  • the activation of the T cells is thus advantageously evaluated by measuring the proliferation of the T cells.
  • the measurement of the proliferation of the T cells is usually carried out by techniques well known to those skilled in the art but requiring a heavy implementation.
  • a mitogen such as concanavalin A (Con A), pokeweed mitogen (PWM for "pokeweed mitogen” and phytohaemagglutinin ( PHA), independently of the specificity of their TCR.
  • Con A concanavalin A
  • PWM pokeweed mitogen
  • PHA phytohaemagglutinin
  • the methods of the prior art evaluate the proliferative capacity of T lymphocytes by measuring the DNA synthesis of T lymphocytes after their stimulation by a mitogen.
  • these methods require a cumbersome implementation and can therefore hardly be used routinely.
  • the method described here is particularly simple and robust.
  • Assay by flow cytometry for example from culture pellets, in order to determine the proliferation of T lymphocytes.
  • a method for determining the proliferative capacity of T cells in a subject, said method comprising: a) measuring the TTV load from a sample of said subject; and b) determining the proliferative capacity of the subject's T cells in view of the viral load measured in a).
  • a high TTV load indicates that the T cells have a low proliferative capacity. Conversely, a low TTV load indicates that the T cells have a high proliferative capacity.
  • a reference TTV load may be the TTV viral load measured in the same individual.
  • the present method is more particularly suitable for evaluating the proliferative capacities of T cells in a subject likely to present an immunodeficiency state.
  • immunodeficiency refers to the reduction or suppression of immune system function.
  • An “immunodeficiency state” therefore designates here a state in which the immune system of a subject is reduced or absent.
  • the humoral and/or cellular immune response towards infectious pathogens is defective in subjects with an immunodeficiency state. More preferentially, the state of immunodeficiency is manifested at least by a reduction in the cellular response.
  • Immunodeficiency can be primary or secondary.
  • Primary immunodeficiencies include innate deficiencies of the immune system with increased susceptibility to infections.
  • secondary immunodeficiency or acquired immunity is a loss of immune function that occurs throughout life, such as, but not limited to, following exposure to pathogens, disease (eg, lymphoma or leukemia), therapy to treat disease (eg, radiotherapy or chemotherapy), immunosuppression, or aging.
  • Pathogens that can cause immunodeficiency include but are not limited to Human Immunodeficiency Virus (HIV) 1 (HIV-1), HIV-2, Treponema pallidum, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium knowlesi, hepatitis B virus (HBV), hepatitis C virus (HCV), prions, West Nile virus, parvovirus, Trypanosoma cruzi, coronaviruses such as S ⁇ RS-COV-1 and S ⁇ RS- COV-2, and/or vaccinia virus.
  • HIV Human Immunodeficiency Virus
  • HIV-1 HIV-1
  • HIV-2 HIV-2
  • Treponema pallidum Plasmodium falciparum
  • Plasmodium malariae Plasmodium malariae
  • Plasmodium ovale Plasmodium ovale
  • Plasmodium vivax Plasmodium knowlesi
  • HBV
  • Immunodeficiency can also be deliberately induced with drugs, for example in preparation for a transplant, such as an organ transplant (eg, kidney, liver, heart, lung, pancreas, intestine, etc.) or HSCT, to prevent graft rejection.
  • a transplant such as an organ transplant (eg, kidney, liver, heart, lung, pancreas, intestine, etc.) or HSCT, to prevent graft rejection.
  • the immunodeficiency according to the present description is a secondary (or acquired) immunodeficiency.
  • the immunodeficiency described herein can be of any origin such as, for example, but not limited to, immunosuppressive treatment, immunosuppressive side effects of drugs or therapy including radiation therapy, inherited immunosuppressive genetic traits or diseases, acquired immunosuppressive diseases such as AIDS, cancers such as leukemia or lymphoma.
  • immunodeficiency is linked to a transplant, especially an HSCT.
  • the subject likely to present an immunodeficiency state is a subject having received a transplant.
  • this graft is a HSCT.
  • the description relates to a method for determining the proliferative capacity of T cells in a subject, the subject having received a HSCT, the method comprising the steps of: a) measuring the TTV load from a sample from subject ; and b) determining the proliferative capacity of the subject's T cells in view of the viral load measured in a).
  • a high TTV load indicates that the T cells have a low proliferative capacity. Conversely, a low TTV load indicates that the T cells have a high proliferative capacity.
  • stem cell we mean here undifferentiated but specialized cells having two main properties: the ability to self-renew and maintain themselves in place for very long periods, and the ability to generate all types of differentiated cells of a specific tissue, which defines their multipotency.
  • the “hematopoietic stem cells” or “HSCs” designate here more particularly the stem cells which can lead to the various blood cells (in particular red blood cells, platelets, granulocytes, T or B lymphocytes, and monocytes). HSCs can advantageously be obtained from umbilical cord blood. Alternatively, they can be obtained from peripheral blood. It is also possible to obtain them from bone marrow.
  • Hematopoietic stem cells transplant or "GCSH” or “HSCT” (for “hematopoietic stem cells transplant”) as understood herein is a therapeutic procedure in the field of hematology in which HSCs, generally derived from bone marrow, peripheral blood or umbilical cord blood, are transplanted from a donor to a recipient.
  • HSCT is a potentially curative approach for a variety of pathologies. These are in particular haemopathies, in particular malignant haemopathies, such as acute leukaemias, myelodysplasias and lymphomas, and non-malignant haemopathies with a severe prognosis, including constitutional aplastic anemia and haemoglobinopathies, solid tumours, deficiencies immune system and enzymatic deficiencies of hematopoietic tissue, such as Gaucher disease, for example.
  • the pathology is a hemopathy, more preferably, a malignant hemopathy.
  • HSCT can be autologous (the patient's own stem cells are used, i.e. the donor and the recipient are one and the same person) or allogeneic (hereafter “allo-HSCT”: stem cells come from a donor who is not the recipient).
  • the HSCT in the method described here is an allogeneic transplant.
  • the description relates to a method for determining the proliferative capacity of T cells in a subject, the subject having undergone allo-HSCT, the method comprising steps of: a) measuring the TTV load from a sample of the subject; and b) determining the proliferative capacity of the subject's T cells in view of the viral load measured in a).
  • a high TTV load indicates that the T cells have a low proliferative capacity. Conversely, a low TTV load indicates that the T cells have a high proliferative capacity.
  • preparation treatments are administered before the transplant to destroy or reduce the activity of the immune system of the recipient.
  • conditionings aim to prevent graft rejection and reduce tumor burden.
  • Myeloablative conditioning as used herein is conditioning that destroys the bone marrow cells of the recipient.
  • the myeloabalative conditioning also destroys the immune system of the recipient, thus facilitating graft take.
  • the myeloabalative conditioning may in particular comprise one or more stages of chemotherapy and/or radiotherapy.
  • two of the commonly used conditionings are busulfan-cyclophosphamide and cyclophosphamide-total body irradiation (TBI).
  • TBI busulfan-cyclophosphamide and cyclophosphamide-total body irradiation
  • the myeloabalative conditioning is applied to a patient under the age of 55, preferably under the age of 50.
  • the conditioning is non-myeloablative, attenuated, or otherwise referred to as “reduced-intensity” conditioning.
  • “Attenuated conditioning” is conditioning that does not totally destroy the bone marrow of the recipient, but leads to an inhibition of the immune system of the latter, thus facilitating the taking of the graft.
  • Such attenuated conditioning preferably includes administration of an immunosuppressant.
  • an attenuated conditioning protocol may include the combination of fludarabine, cyclophosphamide or another alkylating agent, and an ICT.
  • Another example of an attenuated conditioning protocol may include the combination of fludarabine, anti-lymphocyte serum (ALS), and busulfan.
  • an attenuated conditioning protocol may include the combination of fludarabine, idarubicin, and aracytin.
  • another example of an attenuated conditioning protocol may include the combination of fludarabine with mini full irradiation.
  • the attenuated conditioning is applied to a patient under the age of 75.
  • the term "donor”, as used herein, refers to the subject whose HSCs are transferred to the recipient.
  • the term “recipient” or “patient” means the subject who receives the CSH from the donor.
  • the recipient is affected by a pathology to which the HSCT must provide a therapeutic benefit, whether complete or partial.
  • the term "subject” refers to a vertebrate, preferably a mammal, and most preferably a human.
  • a human can for example be a patient.
  • the subject is a patient.
  • patient refers to a human being who has come into contact with a health professional, such as a doctor, a medical structure or a health establishment such as a hospital for example.
  • a health professional such as a doctor, a medical structure or a health establishment such as a hospital for example.
  • biological sample is understood here to mean any sample which can be taken from a subject. In general, the biological sample must allow the determination of the TTV load.
  • the biological sample includes, but is not limited to, whole blood, serum, plasma, sputum, nasopharyngeal swabs, urine, stool , skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, tears, tissue or spinal fluid, cerebral fluid, sample of trigeminal ganglion, sample of sacral ganglion, tissue adipose tissue lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, genital tissue, and central nervous system tissue.
  • the sample to be tested can be used directly from the biological source or following a pre-treatment to modify the character of the sample.
  • pretreatment may include the preparation of plasma from blood, the dilution of viscous fluids, and so on.
  • Pretreatment processes may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, lysis, etc.
  • it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.
  • the biological sample is blood or a derivative thereof, such as plasma or serum.
  • the biological sample is preferably blood, plasma or serum from the subject being tested.
  • TTV Torque Teno virus
  • TTV genome reference is here made to the genomes of all the Anelloviridae, including the alphatorqueviruses (TTV), the betatorqueviruses (TTMV), the gammatorqueviruses (TTMDV).
  • TTV-1 a the genome of the prototype strain of the Torque Teno virus, TTV-1 a. More specifically, an example of a TTV genome is a sequence such as for example that which is represented by SEQ ID No 1 and whose Genbank accession number is AB017610.
  • the TTV genome has a size of about 3.8 kb.
  • the structure and the genomic organization of the TTVs is well known (cf. for example refer to Biagini, Curr Top Microbiol lmmunol.33 ⁇ : 21 -33, 2009) and is exemplified in [Fig 1].
  • the TTV genome can thus be divided into an untranslated region (UTR) of approximately 1 to 1.2 kb and a potential coding region of approximately 2.6 to 2.8 kb.
  • the coding region contains in particular two large open reading frames: ORF1 and ORF2, coding two proteins of 770 and 202 residues respectively.
  • the open reading frames ORF1 and ORF2 are between nucleotides 589-2901 and 107-715, respectively.
  • the TTV genome may have other open reading frames.
  • the TTV genome may include two additional reading frames, ORF3 and ORF4 [Fig 1].
  • the UTR untranslated region is well conserved. It comprises in particular a GC-rich sequence capable of forming a secondary structure. The amplification of selected sequences in the UTR-5' untranslated region has demonstrated that the prevalence of the virus is very high throughout the entire world population (Hu et al., J Clin Microbiol. 43(8) : 3747-3754, 2005). This region comprises in particular a sequence of 128 bp which can be amplified by the TTV R-GENE® diagnostic kit (bioMérieux, France).
  • “Viral load” as understood here is the number of viral particles present in a biological sample. Viral load reflects the severity of a viral infection.
  • the viral load can be determined by measuring the amount of one of the components of the virus (genomic DNA, mRNA, protein, etc.) in this biological sample.
  • the viral load thus refers to the proportion of nucleic acid sequences belonging to said virus in a biological sample. More preferentially, the viral load represents the number of copies of the genome of said virus in a biological sample.
  • the viral load represents the TTV load.
  • the “TTV load” corresponds here more particularly to the TTV viral load, that is to say the number of TTV viral particles present in a biological sample.
  • TTV load in a subject means the viral load of any TTV harbored by said subject.
  • TTV load can be determined by measuring the amount of a TTV component, such as a nucleic acid or protein, in that biological sample.
  • the TTV load corresponds to the quantity of TTV nucleic acid sequences present in a biological sample.
  • the determination of the TTV load in a subject according to the invention comprises the estimation of the number of sequences of all the TTVs in a biological sample of said subject.
  • the determination of the TTV load comprises the determination of the quantity of active and/or inactive viral copies. It consists of determining the quantity of circulating, integrated or latent viral copies.
  • TTV levels - and therefore TTV load - can be determined by measuring levels of TTV DNA, TTV RNA, or TTV protein.
  • the method according to the invention can thus comprise another preliminary step, between the taking of the sample from the patient and step a) as defined above, corresponding to the transformation of the biological sample into a double-stranded DNA sample, or into an mRNA (or corresponding cDNA) sample, or into a protein sample, which is then ready to be used for the in vitro detection of TTV in step a).
  • the preparation or extraction of viral double-stranded DNA, mRNA (as well as the reverse transcription of this into cDNA) or proteins from a cell sample are only well-known routine procedures of the person in the trade.
  • the double-stranded DNA can correspond either to the entire TTV genome, or only to a part of it.
  • detection of TTVs can be performed, depending on the type of sample or transformation, either by genomic DNA (i.e. based on the presence of at least one sequence consisting of at least part of the TTV genome), or by mRNA (i.e. based on the TTV mRNA content in the sample), or at the protein level (i.e. based on the TTV protein content in the sample).
  • TTV levels are determined by measuring levels of TTV nucleic acid, more preferably TTV DNA.
  • Methods of detecting a nucleic acid in a biological sample include, but are not limited to, amplification, including PCR amplification, sequencing, hybridization with a labeled probe, and any other methods known to the person responsible for job.
  • the TTV load is determined by amplifying the TTV sequences.
  • a preferred approach is to amplify sequences which are known to be specific to the TTV genome.
  • specific sequence of the TTV genome is meant here a sequence which is present in the majority of the known TTVs, but which is absent from the majority of the other viruses, in particular from the majority of the other anelloviruses.
  • a TTV specific sequence is present in at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of known TTV genomes. More preferably, it is present in 100% of known TTV genomes.
  • a TTV-specific sequence is present in less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of known non-TTV anellovirus genomes.
  • a TTV-specific sequence is absent from all known anellovirus genomes other than TTVs.
  • Such a sequence is for example a sequence comprised in the untranslated region UTR. More particularly, such a sequence corresponds to the 128 bp sequence of the 5′-UTR untranslated region which is amplified using the TTV R-GENE® diagnostic kit (bioMérieux, France).
  • the method described here comprises the use of primers and probes for the amplification of sequences known to be specific for the TTV genome.
  • these primers are preferably oligonucleotides.
  • these primers can comprise less than 30 nucleotides, at least 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides or less than 12 nucleotides.
  • these primers comprise at least 12, 15, 20.25 or 30 nucleotides.
  • the primers used comprise between 12 and 20 nucleotides, between 12 and 25 nucleotides, between 15 and 20 nucleotides or even between 15 and 25 nucleotides.
  • One skilled in the art will be able to determine the length and sequence of amplification primer to use once the TTV specific sequence has been chosen. It could, for example, use the same primers as those supplied in the TTV R-GENE® diagnostic kit (bioMérieux, France).
  • the amplification techniques include in particular isothermal methods and techniques based on PCR (Polymerase Chain Reaction).
  • Isothermal amplification methods include a large number of methods. The most used to detect pathogens are the LAMP (Loop-Mediated Amplification) and RPA (Recombinase Polymerase Amplification) methods.
  • Isothermal amplification methods also include methods such as for example the NASBA method (nucleic acid sequence-based amplification), HDA (helicasedependent amplification), RCA (rolling circle amplification) and SDA (strand displacement amplification), EXPAR (exponential amplification reaction), ICANs (isothermal and chimeric primer-initiated amplification of nucleic acids), SMART (signal-mediated amplification of RNA technology), etc.
  • the PCR technique used quantitatively measures the initial amounts of DNA, cDNA or RNA.
  • PCR-based techniques that may be used in the methods described herein include, without limitation, techniques such as real-time PCR (Q-PCR), reverse PCR (RT-PCR), multiplex reverse PCR , real-time reverse PCR (QRT-PCR) and digital PCR (or digital PCR). These techniques are technologies well known and readily available to the person skilled in the art. It is not necessary to detail them here.
  • the determination of the TTV load is carried out by real-time quantitative PCR.
  • Numerous methods for the detection and quantification of TTVs have been described in the art (see for example Maggi et al., J Virol. 77(4): 2418-2425, 2003). Reference will be made in particular to the method described by Kulifaj et al. (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 included in the non-coding region UTR. This sequence is present in all known TTVs, thus conferring a very high specificity to the method. In addition, it is particularly versatile and can be implemented with any type of PCR platform. It is particularly advantageous to use the TTV R-GENE® diagnostic kit (bioMérieux, France) to implement this method.
  • viral load determination is performed by digital PCR.
  • Digital PCR involves multiple PCR runs on extremely dilute nucleic acids such that most positive amplifications reflect the signal from a single template molecule. Digital PCR thus allows the counting of individual model molecules. The proportion of positive amplifications among the total number of PCRs analyzed allows an estimate of the template concentration in the original or undiluted sample. This technique has been proposed to allow the detection of a variety of genetic phenomena (Vogelstein et al., Proc Natl Acad Sci USA 96: 9236-924, 1999). Digital PCR, like real-time PCR, potentially allows the discrimination of fine quantitative differences in target sequences between samples.
  • TTV DNA levels are measured by sequencing.
  • sequencing is taken in its broadest sense and refers to any technique known to those skilled in the art for determining the sequence of a polynucleotide molecule (DNA or RNA), i.e. that is to say, to determine the succession of nucleotides composing this molecule.
  • TTV DNA can thus be sequenced by any technique known in the art. Sequencing as understood herein includes but is not limited to Sanger sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing (including 454 sequencing, Solexa Genome sequencing Analyzer), sequencing with capillary electrophoresis, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing (MPSS), terminator sequencing of reversible dye, pairwise sequencing, short term sequencing, sequencing with exonucleases, ligation sequencing, single molecule sequencing, sequencing by synthesis, sequencing by electron microscopy real-time sequencing, sequencing with reverse termination, nanopore sequencing, reversible terminator sequencing, semiconductor sequencing, SOLiD(R) sequencing, Single Molecule Real-Time Analysis (SMRT) sequencing, MS-PET sequencing, mass spectrometry, and their combinations.
  • SOLiD(R) sequencing Single Molecule Real-Time Analysis (SMRT) sequencing
  • MS-PET sequencing mass spectrometry
  • a particular embodiment uses high-throughput DNA sequencing, using for example the MiSeq, NextSeq 500 platforms, and the HiSeq series developed by Illumina (Reuter et al., Mol Cell, 58: 586-597, 2015 Bentley et al. Nature; 456: 53-59, 2008), the Genome Sequencer platform from 454 and Roche (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 Hélicos single molecule sequencing platform (Harris et al.
  • High-throughput sequencing also includes methods such as SMRT real-time sequencing (Roads et al., Genomics, Proteomics & Bioinformatics, 13(5): 278-289, 2015), Ion Torrent sequencing (WO 2010/008480; Rothberg et al., Nature, 475: 348-352, 2011) and sequencing using nanopores (Clarke J et al. Nat Nanotechnol: 4: 265-270, 2009).
  • Sequencing is performed on all of the DNA contained in the biological sample or on parts of the DNA contained in the biological sample. It will be immediately clear to the person skilled in the art that said sample contains at least a mixture of TTV DNA and host subject DNA. Additionally, TTV DNA will likely represent only a minor fraction of the total DNA present in the sample.
  • the DNA is randomly fragmented, generally by physical methods, prior to sequencing.
  • a first approach consists in sequencing specific sequences of the genome of a species of TTV.
  • Another approach is to use a method that allows quantitative genotyping of nucleic acids obtained from the biological sample with high precision.
  • the precision is achieved by analyzing large numbers (e.g., millions or billions) of nucleic acid molecules without any amplification using protocols that rely on prior knowledge of the target sequences (i.e. i.e. in this case, the sequences of the TTVs).
  • the method of the invention comprises a step of quantifying the number of readings.
  • a random subset of nucleic acid molecules from the biological sample is subjected to high-throughput sequencing.
  • the TTV sequences are identified in the global sequencing data by comparison with publicly deposited TTV sequences. This comparison is advantageously based on the level of sequence identity with a known TTV sequence and makes it possible to detect even remote variants. Common software such as BLAST can be used to determine the level of identity between sequences.
  • the determination of the TTV load therefore comprises the numbering of the TTV sequences identified by sequencing in the biological sample of the subject.
  • the TTV load is determined by measuring the amount of a viral protein in the biological sample. It is thus possible to use specific antibodies, in particular in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, electrochemiluminescence (ECLIA) , protein arrays, antibody arrays, or tissue arrays coupled with immunohistochemistry.
  • specific antibodies in particular in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, electrochemiluminescence (ECLIA) , protein arrays, antibody arrays, or tissue arrays coupled with immunohistochemistry.
  • FRET or BRET techniques microscopy or histochemistry methods, including in particular confocal microscopy and electron microscopy methods, methods based on the use of one or several excitation wavelengths and a suitable optical method, such as an electrochemical method (the techniques of voltammetry and amperometry), the atomic force microscope, and radiofrequency methods, such as multipolar, confocal resonance spectroscopy and no- confocal, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or index of refraction (for example, by surface plasmon resonance, by ellipsometry, by resonant mirror method , etc.), flow cytometry, radioisotope or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); by mass spectrometry and by liquid chromatography coupled to mass spectrometry (LC-MS/MS). All these techniques are well known to those skilled
  • the method described here includes an additional step of normalizing the amount of nucleic acid or viral protein measured.
  • TTV i.e. the amount of TTV DNA, TTV RNA or TTV proteins present in the biological sample
  • a specific parameter of this sample Normalizing the measured TTV load to a specific parameter reduces the error rate when comparing viral loads from two different biological samples.
  • An example of a parameter that can be useful for this normalization can be a physical parameter, independent of the content of the sample, such as its volume, for example.
  • a particular DNA or RNA sequence or a particular protein as a standardization tool.
  • this sequence or this protein can be a human sequence or protein.
  • the amount of TTV DNA or RNA or TTV protein in a given sample is compared to an internal control.
  • the quantity of nucleic acid or of TTV protein measured in the biological sample can be related to a defined quantity of a nucleic acid or of an appropriate protein which can be identified and quantified, such as an acid nucleic acid or a host or exogenous protein.
  • this identifiable and quantifiable nucleic acid or protein is processed (eg, amplified, sequenced, etc.) as the target nucleic acid or protein.
  • the preparation steps can include means for protecting the viral nucleic acid and destroying the host nucleic acids, for example by using different nucleases. Alternatively, these steps may include means to protect viral proteins and destroy host proteins, for example by using different proteases.
  • the internal control makes it possible to evaluate the quality and the extent of the processing (for example an amplification or a sequencing) of the molecules considered (nucleic acids or proteins) in the sample.
  • said internal control is a nucleic acid molecule of known sequence, this nucleic acid molecule being present in the sample at a known concentration. More preferably, this nucleic acid molecule is the genomic single-stranded circular DNA molecule of a virus of known sequence and concentration in the sample. Such a known virus may for example be a virus from the Circoviridae family. The ratio of the number of sequences of the sample to the control makes it possible to estimate the absolute number of TTV genomes of known sequence and concentration. Alternatively, this internal control is a protein of known sequence, which is present in the sample at a known concentration.
  • TTV load has been determined by measuring the quantity of nucleic acid or TTV protein determined, this being optionally normalized, it may be advantageous to compare it to a reference TTV load.
  • a reference TTV load or “a reference viral load” is meant within the meaning of the present application any TTV load used as a reference.
  • the reference TTV load corresponds to "a reference level of nucleic acid (or protein) of TTV” or "a control level of nucleic acid (or protein) of TTV", i.e. ie at a concentration of a TTV nucleic acid (or protein) used as a reference.
  • a TTV nucleic acid (or protein) reference concentration is a baseline level measured in a control sample comparable to that tested, and which is obtained from a subject or group of subjects with a specific immunocompetent status.
  • It can be for example a subject or a group of healthy subjects or those without disease leading to immunosuppression. It can also be a subject or a group of immunocompromised subjects, for example following an immunosuppressive treatment. Finally, it may be the same individual who underwent the transplant, for example before or just after it.
  • the reference level can be determined by a plurality of methods.
  • the control can be a predetermined value, which can take various forms. It can be a single threshold value, such as a median or mean.
  • the “reference level” can be a single value, also applicable to each patient individually. Alternatively, the baseline may vary based on specific patient subpopulations. So, for example, older men might have a different baseline than younger men for TTV load, and women might have a different baseline than men for this viral load.
  • the "reference level” can be established on the basis of comparative groups, such as groups not having a high level of TTV nucleic acid (or protein) and groups having levels of nucleic acid (or protein) of elevated TTVs.
  • comparison groups would be groups with a particular disease, condition, or symptoms and groups without disease.
  • the predetermined value may be set, for example, when a test population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group, and a high-risk group.
  • the baseline level can also be determined by comparing the nucleic acid (or protein) level of TTV in populations of transplant patients or patients with diseases leading to immunosuppression. This can be accomplished, for example, by histogram analysis, in which an entire cohort of subjects is presented graphically, with a first axis representing the level of said TTV nucleic acid (or protein), and a second axis representing the number of patients in the group of patients expressing TTV nucleic acid (or protein) at a given level. Two or more distinct groups of subjects can be determined by identifying subpopulations of the cohort that have the same or similar TTV nucleic acid (or protein) levels. The determination of the reference level can then be made on the basis of a level that best distinguishes these distinct groups. A reference level can also represent the levels of two or more of the present nucleic acids (or proteins) of TTV. Two or more markers can be represented, for example, by a ratio of values for the levels of each marker.
  • an apparently healthy population will have a different 'normal' range than a population known to exhibit a condition associated with a high concentration of said TTV nucleic acid (or protein).
  • the selected predetermined value may take into account the category into which an individual falls. Appropriate ranges and categories can be selected with just routine experimentation by the skilled person. By “high”, “increased”, we mean high relative to a selected control. Generally, control will be based on normal, apparently healthy individuals in an appropriate age range.
  • the reference concentration corresponds to the concentration of TTV nucleic acid (or protein) or combination of TTV nucleic acids (or proteins) in the general population.
  • the controls in the method described here can be, in addition to predetermined values, biological samples measured in parallel with the samples tested.
  • the reference level will be that of the TTV nucleic acid(s) (or proteins) in a sample from a healthy subject.
  • the reference concentration of TTV nucleic acid (or protein) will be the concentration of this TTV nucleic acid (or this protein) in a healthy subject or in a population of healthy subjects.
  • the reference concentration of the nucleic acid (or protein) of TTV will be the concentration of this nucleic acid (or this protein) of TTV in an immunocompromised subject or in a population of immunocompromised subjects (for example, following immunosuppressive therapy).
  • the reference concentration of the TTV nucleic acid (or protein) will be the concentration of this TTV nucleic acid (or this protein) in the same individual having undergone the transplant at a specific time. , for example before or just after this one.
  • the T cells are CD3+ T lymphocytes, T lymphocytes CD4+, CD8+ T lymphocytes or a population of CD3+ and/or CD4+ and/or CD8+ T lymphocytes, preferably CD3+ T lymphocytes.
  • the methods described here make it possible to quickly and easily assess the proliferative capacity of T cells in a subject.
  • Another aspect of the present disclosure therefore relates to a method for monitoring the activity of T lymphocytes in a patient who has received an HSCT, in particular an allo-HSCT.
  • This method comprises steps of: a) measuring the proliferative capacity of the T lymphocytes according to the methods described above at a first instant; b) comparison of the proliferative capacity of the T lymphocytes measured in a) with a proliferative capacity of the reference T lymphocytes; and c) determining the change in the activity of the patient's T lymphocytes in view of the comparison of step b).
  • a “reference T cell proliferation capacity” as understood herein is a T cell proliferation capacity estimated from a reference TTV load as described above. It is understood that the comparison of step b) can be done simply by comparing the load in TTV in the patient sample determined in step a) with a reference TTV load.
  • the proliferative capacity of the lymphocytes in the patient at this first instant with the proliferative capacity of the reference T lymphocytes of an immunocompromised individual, it is possible to estimate, at this first instant, the reconstitution of the patient's immunocompetence after HSCT, especially allo-HSCT.
  • immunocompetence is meant here the acquisition of functionality by immune cells.
  • an increased capacity for T cell proliferation in the patient compared to that of an immunocompromised individual indicates that the reconstitution of the patient's immunocompetence is in progress.
  • such an increased capacity for proliferation of T lymphocytes in the patient compared to the immunocompromised subject corresponds to a lower TTV load.
  • the baseline T cell proliferative capacity may be that of a healthy individual.
  • a diminished T cell proliferative capacity in the patient compared to the baseline T cell proliferative capacity indicates that immunocompetence has not been fully restored in the patient. It will immediately be understood that a decrease in the proliferative capacity of T lymphocytes in the patient compared to the healthy subject corresponds to an increase in the load of TTV.
  • increased means a greater amount, e.g., an amount slightly greater than the original amount, or e.g., an amount in great excess of the original quantity, including all quantities in between.
  • “increase” may 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% more than the quantity or activity for which the increased quantity or activity is being compared.
  • the terms “increased”, “greater than”, “greater” and “increased” are used interchangeably herein.
  • diminished means a lesser amount, e.g., an amount slightly less than the original amount, or e.g., a greatly deficient amount relative to the original quantity, including all quantities in between.
  • decrease may 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 quantity or activity for which the reduced quantity or activity is being compared.
  • the terms “diminished”, “less than”, “less significant” and “reduced” are used interchangeably herein.
  • T cell proliferation capacity in the same patient at a second time.
  • the evolution of the activity of these T lymphocytes in this patient after the transplant can thus be followed simply, without implementing a heavy and complicated experimental device, by a simple measurement of the TTV load in the patient at the first and at the second moment.
  • the present method for monitoring the activity of the T lymphocytes in a patient who has received a HSCT comprises steps of: a) measuring the proliferation capacity of the T lymphocytes according to the methods described above at a first moment; b) measurement of the proliferative capacity of the T lymphocytes according to the methods described above at a second instant, this second instant being later than the first instant of step a); c) comparison of the proliferative capacities of the T lymphocytes measured in a) and b); and d) determining the change in the activity of the patient's T lymphocytes in view of the comparison of step c).
  • the method for monitoring the activity of the T lymphocytes in a subject having received a transplant therefore comprises the steps of: a) determining the viral load in TTV from a biological sample of the subject taken at a first instant; b) determination of the TTV viral load from a biological sample of the subject taken at a second instant, this second instant being later than the first instant of step a); c) comparison of viral loads in TTV measured in a) and b); and d) determination of the change in TTV load of the subject in view of the comparison of step c).
  • the viral load in TTV being inversely correlated to the proliferative capacity of T lymphocytes
  • the variation of the load in TTV makes it possible to determine whether the proliferative capacity of T lymphocytes is increased or decreased, thus providing an indication of the activity T lymphocytes and in particular on the reconstitution or not of the subject's immunocompetence.
  • the first instant of step a) is at the time of the graft.
  • the proliferative capacity of the T cells is measured in step b) using a sample taken at least 30 days, 60 days, 90 days, 100 days, 120 days, 150 days, 180 days , 210 days, 240 days, 270 days, 300 days, 330 days, 360 days, 720 days or 1080 days after the GCSH.
  • this sample is taken at 30 days, 60 days, 90 days, 100 days, 120 days, 150 days, 180 days, 210 days, 240 days, 270 days, 300 days, 330 days, 360 days, 720 days or 1080 days after HSCT.
  • T cells have an increased capacity for proliferation at the second instant compared to the first, their activity is itself increased at this instant.
  • an increased proliferative capacity of the T lymphocytes at this second moment means that there are more active T lymphocytes and that the patient is then better able to defend himself, in particular against threats.
  • Such a change in the proliferative capacity of T lymphocytes will result in a change in the opposite direction of the TTV load: a decrease in the latter over time thus reflects an increase in the proliferative capacity of T lymphocytes, i.e. that is, a reconstitution of the patient's immunocompetence.
  • the present method therefore makes it possible to estimate the reconstitution of the immune system of the recipient.
  • the evolution of the proliferative capacity of T cells makes it possible to assess the reappearance of immunocompetence in the patient after the transplant.
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • HSCT especially allo-HSCT
  • a patient who has received an HSCT in particular an allo-HSCT, remains susceptible to microbial infections, whether bacterial, viral, parasitic or fungal, as long as his immune system has not been reconstituted.
  • the present description relates to a method for determining the susceptibility to microbial infections in a patient who has received a HSCT, in particular an allo-HSCT.
  • This method comprises steps of: a) measuring the proliferative capacity of the T lymphocytes from a sample of the patient according to the methods described above at a first instant; b) comparison of the proliferative capacity of the T lymphocytes with a proliferative capacity of the reference T lymphocytes; and c) determining the patient's susceptibility to microbial infections from the comparison in step b).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • the methods described herein may further comprise one or more specific diagnostic steps for the presence of one or more of the infectious agents, such as, for example, the bacteria, viruses, parasites or yeasts and filamentous fungi mentioned in more detail below. The detection of these agents is practiced routinely in the clinic, in particular in relation to HSCTs, and the corresponding techniques are well known to those skilled in the art. It is therefore not necessary to detail them here.
  • Viral infections are in particular viral, bacterial, parasitic or fungal infections.
  • Viral infections are in particular infections by viruses of the Herpesviridae family, such as, for example, the HSV, VZV, HHV-6 viruses, the Epstein-Bar virus (EBV) or the human cytomegalovirus (CMVH).
  • viruses of the Herpesviridae family such as, for example, the HSV, VZV, HHV-6 viruses, the Epstein-Bar virus (EBV) or the human cytomegalovirus (CMVH).
  • EBV Epstein-Bar virus
  • CMVH human cytomegalovirus
  • These viral infections can also be caused by adenoviruses, respiratory syncytial virus (RSV), influenza virus (also called influenza virus or Myxovirus influenzae) or BK virus.
  • RSV respiratory syncytial virus
  • influenza virus also called influenza virus or Myxovirus influenzae
  • the bacteria responsible for bacterial infections can be, among others, staphylococci such as Staphylococcus aureus or coagulase-negative staphylococci, encapsulated bacteria such as Streptococcus pneumoniae, Neisseria meningitidis or Haemophilus influenzae, Legionella sp. or even strict aerobic non-fermentative Gram-negative bacilli such as the genera Pseudomonas, Acinetobacter, Stenotrophomonas, Burkholderia, Alcaligenes... Atypical mycobacterial infections can also be observed.
  • staphylococci such as Staphylococcus aureus or coagulase-negative staphylococci
  • encapsulated bacteria such as Streptococcus pneumoniae, Neisseria meningitidis or Haemophilus influenzae, Legionella sp.
  • strict aerobic non-fermentative Gram-negative bacilli such as the genera Pseudomonas, A
  • Parasitic infections with high morbidity and mortality can occur, as can toxoplasmosis (caused by Toxoplasma gondii).
  • yeasts such as Candida or Cryptococcus, but also filamentous fungi such as Aspergillus are responsible for invasive fungal infections which are among the major causes of infectious mortality after HSCT.
  • the reference T lymphocyte proliferation capacity corresponds to the proliferation capacity of the T lymphocytes estimated from a reference TTV load as described above.
  • the comparison of step b) can be done simply by comparing the TTV load in the sample from the patient determined in step a) with a reference TTV load.
  • This reference TTV load can for example be that of a healthy, non-immunocompromised individual.
  • the proliferation capacity of the reference T lymphocytes is then that of this healthy, non-immunocompromised individual.
  • the description relates to a method for determining the susceptibility to microbial infections in a patient who has received a HSCT, in particular an allo-HSCT.
  • This method comprises steps of: a) measuring the proliferative capacity of the T lymphocytes from a sample of the patient according to the methods described above at a first instant; b) comparison of the proliferative capacity of T lymphocytes with that of a healthy subject; and c) determining the patient's susceptibility to microbial infections from the comparison in step b).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • a reduced capacity for T cell proliferation in the patient compared to the healthy subject indicates that the patient's immune system is not fully functional.
  • a patient's T lymphocyte proliferation capacity lower than that of a healthy subject corresponds to a viral load measured in the patient's sample greater than the TTV load in the healthy subject.
  • the subject then presents a deficiency of its immune system, which leaves it within the reach of the attack of pathogens.
  • the patient is at risk of being infected with microbial organisms.
  • the patient is less susceptible to microbial infections when his T lymphocytes have substantially the same capacity for proliferation as those of a healthy subject.
  • the method for determining the susceptibility to microbial infections in a patient who has received a HSCT comprises steps of: a) measuring the proliferation capacity of the T lymphocytes according to the methods described higher at first; b) measurement of the proliferative capacity of the T lymphocytes according to the methods described above at a second instant, this second instant being later than the first instant of step a); c) comparison of the proliferative capacities of the T lymphocytes measured in a) and b); and d) determining the patient's susceptibility to microbial infections based on the comparison in step c).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • step b) An increased capacity for proliferation of the T lymphocytes in step b) compared with step a) reflects an increase in their activity and therefore a decrease in the patient's susceptibility to microbial infections between the two instants.
  • the present method makes it possible in particular to verify that the more time passes after the transplant, the less the patient becomes susceptible to microbial infections, that is to say that his immune system becomes increasingly functional.
  • the patient's susceptibility to microbial infections can therefore be determined using the methods described here, allowing specific treatment to be tailored to the patient's needs.
  • the preliminary determination of the immunocompromised state of the patient with the method of the invention thus leads to a safer treatment than the treatments designed on the basis of the methods of the prior art.
  • the proliferative capacity of the T lymphocytes corresponds to the proliferative capacity of the CD3+ T lymphocytes, of the CD4+ T lymphocytes, of the CD8+ T lymphocytes or of a population of CD3+ T and/or CD4+ T and/or CD8+ T lymphocytes, preferably to the proliferative capacity of CD3+ T lymphocytes.
  • the present invention also relates to a method for designing a treatment for microbial infections in a patient who has undergone CSH, in particular allo-GCSH, said method comprising: a) determining the patient's susceptibility to microbial infections according to the methods described above ; b) decide on a treatment based on the result of step a).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • the treatment can be decided on a preventive basis, that is to say that it can be ordered in view of the patient's immunodeficiency state to prevent an infection from breaking out.
  • it may be decided to prescribe the curative or prophylactic treatments commonly used after an HSCT, in particular an allo-HSCT, which are described below.
  • the present methods thus make it possible to be able to decide and to administer a preventive or curative treatment of a viral, bacterial, parasitic or fungal infection if a risk of such an infection is identified.
  • the present description therefore also relates to a method for treating an infection in a patient who has received a GCSH, in particular an allo-GCSH, said method comprising steps of: a) determining the susceptibility of the patient to microbial infections according to the methods described above; and b) administering appropriate treatment to said subject.
  • the present invention thus proposes a treatment intended for use in the treatment of an infection in a subject having received a HSCT, in particular an allo-HSCT, the use comprising the steps of: a) determining the susceptibility of the patient to microbial infections according to the methods described above; and (b) administering appropriate treatment to said subject.
  • the invention relates to the use of a treatment in the preparation of a medicament for treating an infection in a subject having received a HSCT, in particular an allo-HSCT, said use comprising the steps of: a) determination of the patient's susceptibility to microbial infections according to the methods described above; and (b) administering appropriate treatment to said subject.
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • the infections encountered after an HSCT are viral, bacterial, parasitic or fungal infections.
  • the treatments for these infections are well known and have been used clinically for many years (see for example Tomblyn et al., Biol Blood Marrow Transplant. 15(10): 1143-1238, 2009).
  • viral infections can be prevented by antiviral compounds such as aciclovir, ganciclovir, cidofovir, entecavir, fludarabine, lamivudine, tenofovir, ribavarin or valaciclovir, or specific monoclonal antibodies such as palivizumab (against RSV).
  • Antibiotics usually treat bacterial infections.
  • broad-spectrum antibiotics are used, such as beta-lactam antibiotics, glycopeptides, fosfomycin, macrolides, tetracyclines, aminoglycosides, chloramphenicol, quinolones, rifampicin and sulfonamides.
  • parasitic infections they are usually treated by administration of antiparasitic compounds such as cotrimoxazole, pyrimethamine and sulfadiazine.
  • antifungal compounds which can be administered are well known and notably include fluconazole and echinocandins.
  • the methods described here also have the advantage of being able to estimate the risk of GvHD appearing in a patient who has undergone CCSH, in particular allo-GCSHD.
  • the present description therefore also relates to a method for determining the susceptibility to GvHD in a patient who has received a HSCT, in particular an allo-HSCT, this method comprising steps of: a) measuring the proliferative capacity of the T lymphocytes from a sample from the patient at a first instant according to the methods described above; and b) determination of the susceptibility to GvHD in the patient from the measurement of step a).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • GvHD raft versus host disease
  • Acute GvHD refers to the onset of an allogeneic inflammatory response in three organs exclusively: skin, liver, and gastrointestinal tract.
  • chronic GvHD can affect at least one of the following eight organs: skin, mouth, eyes, gastrointestinal tract, liver, lungs, muscles, joints, fascia, and genitals.
  • GvHD is usually diagnosed by a clinical examination which may include histological analysis of a biopsy of the organ concerned (Schoemans et al. Bone Marrow Transplant 53: 1401-1415 2018 ).
  • the present method is particularly useful, for example because it predicts the possibility of weaning patients from immunosuppressive treatment and therefore their survival.
  • the present method offers the possibility of easily differentiating an active GvHD which requires the continuation of an immunosuppressive treatment from a GvHD which is no longer active at all and for which the treatment could be stopped (cf. Magro et al., Bull Cancer. 104S: S145-S168, 2017).
  • acute GvHD occurs within the first month (30 days) after transplantation, while chronic GvHD appears between 100 and 400 days after transplantation. Both are characterized by the activation of donor T lymphocytes present in the graft. The interaction between host antigens and donor T cells results in allogeneic T cell activation, proliferation, and differentiation into effector cells that attack host epithelial cells.
  • the recipient of an HSCT is likely to develop GvHD if the T cells in the patient's sample are able to proliferate.
  • the proliferation of the patient's T lymphocytes at a time when the immune system is not yet reconstituted is a strong indication that the patient is likely to be affected by GvHD.
  • This can be easily assessed by measuring the load in TTV, using the methods described above.
  • the indication given by this test can be supplemented if necessary by a clinical examination of the patient, in particular by the histological analysis of one or more biopsies of one or more organs of the patient.
  • the sample from the subject is taken less than 400 days after the transplant.
  • the sample is taken less than 100 days after the transplant; alternatively, the sample is taken between 100 and 400 days after the transplant.
  • the GvHD is an acute GvHD; in another particular embodiment, the GvHD is chronic GvHD.
  • step b) it may be useful to compare the measurement of step b) with a reference having a known capacity for proliferation of T lymphocytes, that is to say a capacity for proliferation of reference T lymphocytes .
  • the reference T cell proliferation capacity corresponds to the T cell proliferation capacity estimated from a reference TTV load as described above.
  • the comparison in step b) can be done simply by comparing the TTV load in the patient sample determined in step a) with a reference TTV load.
  • This reference TTV load may for example be that of a healthy, non-immunocompromised individual.
  • the proliferation capacity of the reference T lymphocytes is then that of this healthy, non-immunocompromised individual.
  • the baseline TTV load may be that of an immunocompromised individual.
  • the description relates to a method for determining the susceptibility to GvHD in a patient who has received a GCSH, in particular an allo-GCSH.
  • This method comprises steps of: a) measuring the proliferative capacity of the T lymphocytes from a sample of the patient according to the methods described above at a first instant; b) comparison of the proliferative capacity of T lymphocytes with that of an immunocompromised subject; and c) determining the patient's GvHD susceptibility from the comparison in step b).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • an increased capacity for proliferation of T lymphocytes in the patient compared to the immunocompromised subject indicates that the patient's immune system becomes functional again.
  • a patient's T cell proliferation capacity greater than that of an immunocompromised subject corresponds to a viral load measured in the patient's sample lower than the TTV load in the immunocompromised subject.
  • the subject then presents with active T lymphocytes, which can potentially attack the cells of the graft and trigger GvHD.
  • the patient is not likely to develop GvHD when his T lymphocytes have substantially the same capacity for proliferation as those of an immunocompromised subject.
  • T cell proliferation capacity in the same patient at a second time.
  • the person skilled in the art can thus monitor the evolution of the risk of occurrence of GvHD over time after the transplant.
  • an anti-GvHD treatment can be adapted according to the patient's real susceptibility to GvHD, which limits the risk of resistance appearing, while improving the patient's comfort of life.
  • the method for determining the susceptibility to GvHD in a patient who has received a HSCT comprises steps of: a) measuring the proliferative capacity of the T lymphocytes according to the methods described higher at first; b) measurement of the proliferative capacity of the T lymphocytes according to the methods described above at a second instant, this second instant being later than the first instant of step a); c) comparison of the proliferative capacities of the T lymphocytes measured in a) and b); and d) determining the patient's susceptibility to GvHD based on the comparison in step c).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • the patient's susceptibility to GvHD can therefore be determined using the methods described here, allowing the design of a specific treatment to be tailored to the patient's needs.
  • the present invention also relates to a method for designing a treatment for GvHD for a subject having received a CSH, in particular a GCSH, said method comprising: a) determining the patient's susceptibility to GvHD according to the methods described below above ; b) decide on a treatment based on the result of step a).
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • the present description also relates to a method for treating GvHD in a patient who has received a GCSH, in particular an allo-GCSH, said method comprising steps of: a) determining the patient's susceptibility to GvHD according to the methods described above; and b) administering appropriate treatment to said subject.
  • the present invention thus proposes a treatment intended to be used in the treatment of GvHD in a subject having received a GCSH, in particular an allo-GCSH, the use comprising the steps of: a) determining the susceptibility of the patient to the GvHD according to the methods described above; and (b) administering appropriate treatment to said subject.
  • the invention relates to the use of a treatment in the preparation of a medicament for treating GvHD in a subject having received a GCSH, in particular an allo-GCSH, said use comprising the steps of: a) determination of the patient's susceptibility to GvHD according to the methods described above; and (b) administering appropriate treatment to said subject.
  • the method for monitoring the activity of the T lymphocytes described above is implemented in a patient who has undergone conditioning before receiving a HSCT, in particular an allo-HSCT.
  • the conditioning is myeloablative.
  • the conditioning is attenuated.
  • Treatments for GvHD are well known and have been recommended by clinicians (see, for example, Magro et al., BullCancer. 104S: S145-S168, 2017; Penack et al., Lancet Haematol. 7 (2): e157-e167; 2020). Treatments for GvHD can be used prophylactically and most often consist of immunosuppressive treatments such as cyclosporine or tacrolimus.
  • GvHD treatments When GvHD treatments are used curatively, they vary depending on the severity of the complication. Nevertheless, these treatments most generally include immunosuppressants, corticosteroids, in particular prednisolone and methylprednisolone. Anti-lymphocyte serum (S ⁇ L) is also used in case of failure of corticosteroids. Finally, other second line drugs can be used such as mycophenolate mofetil (Cellcept®), monoclonal antibodies (anti TNF ⁇ or anti IL2 receptors).
  • Cellcept® mycophenolate mofetil
  • monoclonal antibodies anti TNF ⁇ or anti IL2 receptors
  • the description also relates to the use of the measurement of the variation of the TTV load in a subject to determine the proliferation capacity of the T lymphocytes of said subject.
  • the variation of the load can be determined by comparing the load in TTV measured in a sample taken at a first instant and in a sample taken at a second instant, the second instant being later than the first.
  • a decrease in the TTV load over time reflects, through the increase in the proliferative capacity of the T lymphocytes, an increase in the activity of said T lymphocytes.
  • Embodiment 1 Method for determining the proliferative capacity of T cells in a subject, the method comprising the steps of: a) measuring the TTV load from a biological sample of said subject; and b) determination of the proliferative capacity of the T lymphocytes in view of the viral load measured in a).
  • Embodiment 2 Method according to embodiment 1, characterized in that the TTV load is measured by amplification, sequencing or hybridization of a TTV sequence, preferably by amplification, more preferably by real-time PCR.
  • Embodiment 3 Method according to embodiment 1 or 2, characterized in that the biological sample is a sample of whole blood, plasma or serum.
  • Embodiment 4 Method according to any one of embodiments 1 to 3, characterized in that the determination of step b) comprises the comparison of the TTV load measured in a) with a reference TTV load .
  • Embodiment 5 Method according to any one of embodiments 1 to 4, characterized in that the patient has received a transplant.
  • Embodiment 6 Method according to embodiment 5, characterized in that the patient has received a hematopoietic stem cell transplant (HSCT), preferably an allo-HSCT.
  • HSCT hematopoietic stem cell transplant
  • Embodiment 7 Method according to embodiment 5 or 6, characterized in that the patient has undergone conditioning, preferably myeloablative or attenuated, before the transplant.
  • Embodiment 8 Method for monitoring the activity of T lymphocytes in a patient having received an allo-HSCT, the method comprising the steps of: a) measuring the proliferative capacity of T lymphocytes in the patient at a first instant according to any one of embodiments 1 to 6; b) comparison of the proliferative capacity of the T lymphocytes measured in a) with the proliferative capacity of the reference T lymphocytes; and c) determining the change in the activity of the patient's T lymphocytes in view of the comparison of step b).
  • Embodiment 9 Method for determining the susceptibility to microbial infections in a patient having received an allo-HSCT, the method comprising the steps of: a) measuring the proliferative capacity of the T lymphocytes in the patient at a first instant according to any of Embodiments 1 to 6; b) comparison of the proliferative capacity of the T lymphocytes measured in a) with the proliferative capacity of the reference T lymphocytes; and c) determining the patient's susceptibility to microbial infections based on the comparison in step b).
  • Embodiment 10 Method according to embodiment 9, characterized in that the microbial infection is a viral, bacterial, protozoan or fungal infection.
  • Embodiment 11 Method for determining susceptibility to graft versus host disease (GvHD) in a patient who received allo-HSCG, the method comprising steps of: a) measuring the proliferative capacity of the lymphocytes T in the patient at a first time according to any one of embodiments 1 to 6; b) comparison of the proliferative capacity of the T lymphocytes measured in a) with the proliferative capacity of the reference T lymphocytes; and c) determining the patient's GvHD susceptibility in view of the comparison of step b).
  • GvHD graft versus host disease
  • Embodiment 12 Method according to any one of embodiments 8 to 11, characterized in that the proliferation capacity of the reference T lymphocytes is the proliferation capacity of the T lymphocytes of a healthy individual or the proliferation capacity T cells from an immunocompromised individual.
  • Embodiment 13 Method according to any one of embodiments 8 to 11, characterized in that the reference T cell proliferation capacity is the T cell proliferation capacity measured in the patient at a second instant.
  • FIG 1 Representation of the genome structure of a TTV isolate.
  • TTV-1a isolation TTV-1a
  • the arrows represent the major ORFs (longer than 50 amino acids).
  • the GC-rich region and an N22 region are indicated.
  • the untranslated region UTR corresponds to the region going from the 3' end of ORF4 to the 5' end of ORF2 According to Biagini, Curr Top Microbiol Immunol. 331:21-33, 2009.
  • FIG 2 TTV viral load from plasma samples of allo-GCSH recipients and healthy volunteers.
  • the TTV viral load of 41 allo-GCSH recipients (black) and 54 healthy volunteers (white) was quantified.
  • the TTV viral load was quantified using the TTV R-GENE® kit (available only for research and not for diagnosis, Ref#69-030, bioMérieux. Marcy-l'Etoile , France).
  • the lowest viral load detected was 0.46 Log copy/mL (log cp/mL). Log copy/mL are used to describe the expression of TTV viral load between the two populations.
  • the variance was compared using an F-test (## p ⁇ 0.01).
  • Mean TTV viral load (black line) was compared using an unpaired t-test with Welch's correction (***p ⁇ 0.001).
  • DNA Deoxyribonucleic acid ; Hello, allogenic; GCSH. Hematopoietic stem cell transplant; TTV. torque teno virus
  • the CD3 + T-cell proliferative capacity of 41 allo-GCSH recipients (black) and 20 healthy volunteers (white) was quantified after 3-day stimulation with mitogen (PHA) and measured by flow cytometry using the Click-11® EdU AF488 kit.
  • the variance of the two populations was compared using an F-test (##. p ⁇ 0.01).
  • the comparison of means (black line) was performed using an unpaired t-test with Welch's correction (***. p ⁇ 0.001).
  • FIG 3A [Fig 3B] [Fig 3C] [Fig 3D]: Correlation between the TTV viral load and the number of T cells as well as the proliferation capacity of T-CD3 + lymphocytes.
  • TTV viral load expressed in Log copies/mL of plasma of 41 allo-HSCT recipients according to: (B) the proliferative capacity of T-CD3 + lymphocytes, (C) the absolute lymphocyte count and (D) CD3 + T-cell count. Patients are represented by dots. Extreme patients: "A" (square) and “B” (triangle), as well as linear regression (black line) are shown. Lymphocyte counts were measured by flow cytometry in the immunology laboratory using a large panel of T cell membrane markers. Proliferative capacity of CD3 + T-lymphocytes was determined after 3 days of stimulation with a mitogen (PHA) and measured by flow cytometry using the Click-lt® 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 Pearson's correlation coefficient (shown on each graph).
  • TTV plasma viral load expressed in Log cp/mL from 41 allo-HSCT recipient plasmas with T cell immunophenotyping and CD3 + T cell proliferative capacity A.
  • TTV viral load expressed in Log cp/mL of 41 plasmas of allo-GCSH recipients with respect to the proliferative capacity of T-CD3 + lymphocytes (B), the absolute number of lymphocytes (C) and the number of T-CD3 + cells (D). Lymphocyte subtypes and absolute counts were measured by flow cytometry in the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon) using a large panel of T cell membrane markers.
  • CD3 + proliferation was determined after 3-day stimulation with mitogen (PHA) and measured by flow cytometry using the Click-lt® EdU AF488 flow kit.
  • the correlation of TTV viral load (abscissa) and cell count or CD3 + T-cell proliferative capacity (ordinate) was determined using Pearson's correlation coefficient (shown on each graph).
  • A The values -0.5 and 0.5 represented by black dotted lines correspond to the bounds of the correlation confidence interval.
  • the Pearson rho and the 95% confidence interval (CI) for all parameters are represented by dots and a black line respectively.
  • B, C and D Patients are represented by black dots, extreme patients by a square and a triangle, linear regression is represented by a black line.
  • FIG 4 Chronological descriptive follow-up of patients with extreme values of TTV viral load. Chronological description of the main clinical stages (in black) and infectious episodes (in grey) between HSCT and inclusion for patient "A" and patient "B". Patient "A” had the lowest TTV viral load and conversely patient “B” had the highest TTV viral load.
  • CMV Cytomegalovirus
  • EBV Epstein-Barr virus
  • GCSH Hematopoietic stem cell transplant
  • GvHD Graft versus host disease; Mr. Month.
  • TTV plasma viral load expressed in Log cp/mL of 41 plasmas from allo-HSCT recipients and the time between HSCT and inclusion expressed in months.
  • the correlation of TTV viral load (abscissa) and delays (ordinate) was determined using the Pearson correlation coefficient (shown on each graph). Patients are represented by black dots and linear regression is represented by a black line.
  • HSCT hematopoietic stem cell transplant
  • TTV Torque Teno Virus
  • Heparinized whole blood samples and EDTA-treated plasma samples from patients receiving allo-HSCT were obtained from the previously described prospective Vaccheminf cohort (13).
  • the cohort was approved by a regional review board (Comotti de protection des brought Sud-Est V, Grenoble, France, number 69HCL17_0769) and is registered in ClinicalTrial.gov (NCT03659773).
  • Consecutive adult patients who underwent allo-HSCT in the hematology department of the Lyon University Hospital were prospectively included, once the patient's written consent was obtained.
  • PBMC Peripheral blood mononuclear cells
  • PBMC culture supernatants were collected to perform an IFN ⁇ secretion assay (IGRA, for “IFN ⁇ -release assay”) using Simple Plex cartridges on the ELLA nanofluidic system (ProteinSimple, San Jose, CA , USA) according to the manufacturer's instructions.
  • T cell proliferation was analyzed in pellets with the Click-iTTM Plus EdU Alexa FluorTM 488 Flow Cytometry Assay Kit (C10420; Life Technologies, Carlsbad, CA, USA), which measures the incorporation of 5 -ethynyl-2'-deoxyuridine (EdU), according to published protocol (16).
  • the percentage of EdU + proliferating cells was obtained by flow cytometry analyzes performed on a BD LSR FortessaTM flow cytometer (BD Biosciences, San Jose, CA, USA). For each experiment, at least 2.5 ⁇ 10 3 CD3 + cells were measured. Data were analyzed using BD FACSDiva software (version 8.0.3, BD Biosciences).
  • CD4 + and CD8 + T lymphocytes were counted in the immunology laboratory of Edouard Herriot Hospital (Hospices Civils de Lyon). In addition, a large panel of T cell membrane markers were measured by whole blood flow cytometry. We thus counted naive CD4 + and CD8 + T cells (CD45 + CCR7 + ), CD4 + and CD8 + T cells with central memory (CD45RA CCR7 + ), CD4 + and CD8 + T cells with effector memory (CD45 CCR7 + ) and CD4 + and CD8 + T cells with differentiated memory (CD45RA + CCR7) as previously described (14). The results were expressed in cells/pL.
  • TTV viral load and T cell proliferative capacity are expressed as mean (range).
  • the TTV load transformed into Log format was used for the analysis (Log copy/mL).
  • Differences between healthy and allo-GCSH recipients were calculated using an unpaired parametric t-test with Welch's correction. Correlations were assessed using a parametric Pearson's rho correlation coefficient.
  • Regression analyzes were performed to assess the association between the dependent variable (TTV viral load) and the independent variables (percentage of proliferating cells, absolute lymphocyte count, and CD3 + T cell count). The analysis of variance has was performed using F-tests. Differences in plasma TTV burden with respect to different clinical characteristics were estimated using the Mann-Whitney test. A value of p ⁇ 0.05 was considered significant.
  • Statistical analyzes were performed using GraphPad Prism® software (version 5; GraphPad software, La Jolla, CA, USA) and R (version 3.5.1).
  • the median duration [El] after transplantation at enrollment was 6 [5-8] months for allo-HSCT recipients.
  • 78% of allo-HSCT recipients received immunosuppressive drugs (corticosteroids, calcineurin inhibitors, others...), and 17% had chronic graft versus host disease [Table 1]. .
  • TTV viral load in TTV was studied in plasma samples from 80 healthy recipients and 41 allo-HSCT recipients. TTV viral load was detected by real-time PCR in 68% of healthy samples (54/80). Regarding allo-HSCT recipients, all patients included in the study had a detectable TTV viral load value. The mean (range) TTV viral load was significantly higher in allo-HSCT recipients compared to healthy subjects (3.9 (0.7-7.7) vs 2.1 (0.5-4. 3) Log copy/ml respectively, p ⁇ 0.0001) [Fig 2].
  • CD3 + T cells (VN, 521 -1772/pL) 915 (175-3406)
  • CD3 + CD4 + T cells VN, 336-1126/pL 273 (38-876)
  • CD4 + memory effectors CD45RA CCR7 (VN, 59-
  • CD45RATCR7 VN, 11 -102/pL
  • CD3 + CD8 + T cells VN, 125-780/pL
  • 602 60-2779
  • CD8 + memory effectors CD45RA CCR7 ) (VN, 15-
  • CD20 + B cells VN, 64-593/pL 287 (14-1299)
  • Patient (A) received a stem cell transplant from a geno-identical donor while patient (B) received a peripheral blood cell transplant from a pheno-identical donor.
  • Patient (A) had a simple post-transplant evolution without any particular clinical facts between transplantation and enrolment, i.e. no infectious episode, no GvHD and no immunosuppressive treatment [Fig 4].
  • patient (B) received heavy immunosuppressive therapy and suffered from acute GvHD and multiple severe bacterial/viral infections [Fig 4].
  • TTV the plasma viral load of TTV was significantly higher for the allo-HSCT recipient (at 6 months after transplantation) compared to healthy subjects, thus confirming observations of Tyagi and al., 2013 (post-transplant delay not specified) or Masouridi et al., 2016 (2-3 months after the transplant) (10,18).
  • T cells the main cells of the immune response against viral infection (19,20)
  • TTV replication 21,22.
  • T cell multiplication is ongoing, as the immune system is rebuilding, providing a large number of cells where the virus can replicate.
  • TTV load peaks around 3–6 months after transplantation before returning to a so-called normal value (23,24).
  • TTVs would use the growth of T cells, which are still naive and non-functional, to replicate before being finally regulated by the immune system and by functional T cells, thus suggesting an important link between the TTV viral load and immune function.
  • TTV viral load has also been associated with the number of CD8 + /CD57 + T cells, a lymphocyte subtype described as a potential marker of immunosenescence and increased during certain pathologies such as acquired immunodeficiency states , transplants or persistent viral infections. All these results suggest that there is therefore a potentially important link between TTVs and the function of the immune system, in particular with regard to its ability to regulate viral load.
  • this study establishes the existence of a correlation between TTV viral load and T cell function, and shows that this correlation is independent of cell number.
  • the number of CD3+ T lymphocytes can be significantly different (Patient 1 vs Patient 2) and conversely, for a significantly different viral load (Patient 3 vs Patient 4), the number of CD3+ T cells may be similar. Therefore, this confirms that the viral load in TTV is not correlated with the number of CD3+ T cells.
  • TTV Torque Teno Virus

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