(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`(10) International Publication Number
`
`g
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`(43) International Publication Date
`WO 2013/156627 A1
`24 October 2013 (24.10.2013) WIPOI PCT
`
`
`(51)
`
`International Patent Classification:
`CIZQ 1/70 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/EP2013/058231
`
`(22)
`
`International Filing Date:
`
`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
`
`19 April 2013 (19.04.2013)
`
`English
`
`English
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`Priority Data:
`123054629
`
`20 April 2012 (20.04.2012)
`
`EP
`
`Applicant: INSTITUT PASTEUR [FR/FR]; 25-28, rue
`du Docteur Roux, F-75015 Paris (FR).
`
`Inventors: ELOIT, Mare; 91 rue du Cherche-Midi, F-
`75006 Paris (FR). CHEVAL. Justine; 91 rue de la sante',
`F-75013 Paris (FR). HEBERT, Charles; 22 rue Desaix
`Escalier B, F-78800 Houilles (FR). LECUIT, Marc; 7 rue
`Boissonade, F-75014 Paris (FR).
`
`Agent: REGIMBEAU; 20, rue de Chazelles, F-75847 Pat-
`is Cedex 17 (FR).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BVV, BY,
`
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, Sv, SY, TH, TJ,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GVV,
`ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`ofinventorsnip (Rule 4.1 7(iv))
`Published:
`
`with international search report (Art. 21(3))
`
`with sequence listing part ofdescription (Rule 5.2(a))
`
`(54) Title: ANELLOVIRUS GENOME QUANTIFICATION AS A BIOMARKER OF IMMUNE SUPPRESSION
`
`(57) Abstract: The present invention relates to the use of the measure of anelloviral load for the detelmination of immunosuppres -
`sion. More precisely, the present invention provides a method for characterizing the immunosuppressed or non-immunosuppressed
`Status of a subject, comprising the steps of determining the anelloviral load from a biological Sample of the said subject, and deterrn —
`ining from the said comparison the immunosuppressed or non-immunosuppressed status, The determination of the immunosup-
`pressed status of the subj eet can then be used to design or adapt a therapeutic treatment.
`
`
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`ANELLOVIRUS GENOME QUANTIFICATION
`
`AS A BIOMARKER OF IMMUNE SUPPRESSION
`
`The present invention relates to the use of the measure of anelloviral load for the
`
`determination of immunosuppression. More specifically,
`
`the invention relates to a
`
`method for the diagnosis of immunosuppression in a subject based on anellovirus
`
`viral load .
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`The immune system defends an organism against aggressions such as pathogen
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`infection, cellular transformation, and physical or chemical damage. When the
`
`immune system is less active than normal, immunodeficiency or immunosuppression
`
`occurs, resulting in life-threatening infections or cancer.
`
`Immunosuppression is a
`
`condition in which the immune system's ability to fight diseases,
`
`for example
`
`infectious diseases or cancer, is compromised or entirely absent. Immunosuppression
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`takes various forms, and may affect either the innate or the adaptive immune
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`system, or both, depending of the source of the deficiency.
`
`It usually results in
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`recurring or life-threatening infections.
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`Immunosuppression can either be the result of diseases, or be produced by
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`pharmaceuticals or an infection, resulting in an increased susceptibility to secondary
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`infections by pathogens such as bacteria and viruses.
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`Many diseases
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`are
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`thus
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`characterized by
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`the development of progressive
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`immunosuppression in the patient. The presence of an impaired immune response in
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`patients with malignancies (e.g.
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`leukemia,
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`lymphoma, multiple myeloma) is well
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`documented. Progressive immunosuppression has also been observed in certain
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`chronic infection such as AIDS, sepsis, leprosy, cytomegalovirus infections, malaria,
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`lupus, and the like. Immunodeficiency is also a potential adverse effect of many
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`therapeutic treatments (radiotherapy or chemotherapy for example).
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`In such a
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`situation of non-deliberate immunosuppression, patients are usually treated with
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`immunostimulants (e.g. cytokines),
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`in order to boost the patient’s immune system.
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`However,
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`immunostimulants lack specificity,
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`in that
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`they activate the immune
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`system in general. If not administered cautiously, they may trigger an overactivation
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`of the immune system, resulting in poor tolerance
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`Alternatively, immunosuppression may result from deliberate intent to weaken the
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`immune system. In general, deliberately induced immunosuppression is performed by
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`administration of immunosuppressive drugs,
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`in order to prevent
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`the body from
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`rejecting an organ transplant or
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`for
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`the treatment of auto-immune diseases.
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`Immunosuppressive treatments, however, when inappropriate or inadequate, may
`
`lead to an over-immunosuppression state where the patient is extremely vulnerable
`
`to infections.
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`Indeed, opportunistic infections and malignancies remain a significant
`
`cause of death after
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`transplantation and are obvious consequences of over-
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`immunosuppression.
`
`Because of the great diversity of causes, and because each of those causes may
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`affect
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`the immune
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`system in
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`a different
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`aspect, different
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`diagnosis
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`for
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`immunosuppression have been developed. Some nonspecific and pathogen-specific
`
`measures of cell-mediated immune function are available (Fishman et al. N Engl J
`
`Med. 2007, Fishman et al. Liver Transpl. 2011). Cell mediated immune function
`
`assays include lymphocyte subset analysis, particularly CD4+ T cell numbering or
`
`measure of the CD4+/CD8+ T cell ratio, neutrophil function assay, NK activation
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`assay,
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`lymphocyte proliferation assay (Hutchinson et al. Nephrol Dial Transplant
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`2003).
`
`Those assays however are not sufficiently sensitive to detect slight changes in the
`
`immune system. Additionally, each of those assays focus on assaying the integrity of
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`a specific pathway or mechanism of the immune system. None of them is based on
`
`assaying the end result, which is the capability of the immune system to respond to
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`or control infections.
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`Currently there is a no universal method for the diagnosis of immunosuppression that
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`could be used universally, that is, for any suspected cause of immunosuppression.
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`There is thus a ongoing need for a rapid, reliable and non-invasive test assessing
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`precisely the immune status of a patient. Especially, a diagnosis method that would
`
`allow for evaluation of the capacity of the immune system to respond to infections
`
`could be used to fine-tune immunosuppressive treatments to the proper needs of the
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`patients, and avoid over-immunosuppression.
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`The inventors have found that the anelloviral load is a reliable marker of immune
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`status and can thus be used for the diagnosis of immunosuppression.
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`DETAILED DESCRIPTION
`
`Anelloviruses
`
`(ANV) are viruses that
`
`infect more than 90% individuals. Mixed
`
`infections with several strains and ANV species are frequent, and most subjects host
`
`at least one of them, but no pathological consequence has been attributed to ANV
`
`infection.
`
`In particular, no clear correlation between the anelloviral load and the
`
`immune state of the subject has been identified in the prior art. Patients on
`
`immunosuppressive treatment generally showed an increase in the load of specific
`
`viral strains (e.g. TLMV and TN). However, the inter-individual variations were such
`
`that the only relevant information could be obtained from examining the changes in
`
`the viral load of each individual. In particular, no conclusion could be drawn from the
`
`comparison of groups of patients , treated or not treated. Thus the methods of the
`
`prior art did not enable the determination of the immunosuppressive status of a
`
`subject and the design of a specific treatment thereof (Moen et al., J Med Virol,
`
`70(1): 177-182, 2003; Moen et al., AIDS, 16(12): 1679-1682, 2002; Christensen et al.,
`
`J Infect Dis, 181: 1796—1799, 2000; Shibayama et al., AIDS, 15: 563—570, 2001;
`
`Touinssi et al., J Clin Virol, 21: 135—141, 2001).
`
`In contrast, the present inventors have surprisingly found that the anelloviral load
`
`can be used as a marker for immunosuppression. Whereas previous studies were
`
`based on PCR, and thus were highly dependent upon primers design, resulting in
`
`missing variants of this highly variable virus (Moen et al., J Virol Methods,104(1): 59-
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`67, 2002),
`
`the present
`
`inventors used High Throughput Sequencing (HTS). This
`
`technique led to the identification of a broad and unbiased range of ANV sequences,
`
`enabling the present
`
`inventors to demonstrate the existence of a correlation
`
`between the anelloviral
`
`load and immunosuppression with a high degree of
`
`confidence.
`
`In particular, the inventors have found that immunosuppressed subjects
`
`have a higher anelloviral load by comparison with healthy subjects.
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`The measure of the global load of the viruses from the family of anelloviruses can
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`thus be used as a marker of the immune state of the subject. More precisely,
`
`according to the invention, a high anelloviral load in a subject indicates that the said
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`subject is immunosuppressed.
`
`Thus,
`
`in a first aspect,
`
`the invention relates to a method for characterizing the
`
`immunosuppressed or non-immunosuppressed status of a subject, comprising the
`
`steps of :
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`a) determining the anelloviral load from a biological sample of the said subject,
`
`and
`
`b) assessing from the determination of step a)
`
`the immunosuppressed or non-
`
`immunosuppressed status.
`
`The term “immunosuppression” (or “immunodepression” or “immunodeficiency”), as
`
`used herein, refers to the reduction or suppression of the immune system function,
`
`i.e. immunosuppression generally denotes a state when a subject’s specific and/or
`
`non-specific immune system function is
`
`reduced or absent. The whole immune
`
`response may be depressed, or a particular population of immunologically active
`
`lymphocytes may be selectively affected.
`
`In some cases,
`
`the effect may be
`
`preferentially on T cells rather than B cells.
`
`If B cells are affected, it may be on a
`
`specific subclass of antibody-producing cells. Antigen-specific immunosuppression
`
`may be the result of deletion or suppression of a particular clone of antigen-specific
`
`cells, or the result of enhanced regulation of the immune response by antigen-
`
`specific suppressor cells.
`
`It can also be the result of increased production of
`
`antiidiotypic antibody.
`
`Immunosuppression may result from certain diseases such as AIDS or lymphoma or
`
`from certain drugs such as some of those used to treat cancer. Immunosuppression
`
`may also be deliberately induced with drugs, as in preparation for bone marrow or
`
`other organ transplantation to prevent
`
`the rejection of
`
`the transplant. Thus
`
`immunosuppression according to the invention may be from any origin such as, for
`
`example, but not limited to, immunosuppressive treatment, immunosuppressive side
`
`effects of drugs or therapy including radiotherapy,
`
`inherited immunosuppressive
`
`genetic traits or diseases, acquired immunosuppressive diseases such as Ale, cancers
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`such as leukemia or lymphoma.
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`By “immunosuppressed status” it is herein referred to a condition where the immune
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`system function of a subject is reduced or absent. Thus, according to the invention
`
`the terms “immunosuppressed”, “immunodepressed” or “immunocompromised” are
`
`all deemed to carry the same meaning.
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`In
`
`a particular embodiment,
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`the
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`“immunosuppressed status” of the subject means the ability of the subject to control
`
`viral infection, that is to say, the ability of the subject to prevent viral amplification
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`from said viral infection.
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`It is difficult to precisely asses the immunosuppressed status of a subject with the
`
`methods of the prior art. This may lead to situations where too high a dose of an
`
`immunosuppressive treatment is given to a patient in need thereof. It is also possible
`
`that a dose not high enough of an immunostimulating treatment is administered to a
`
`patient, because the immunosuppressed status of the said patient was not correctly
`
`determined. The consequences for the patient’s health are potentially serious in
`
`either situation. For example, immunosuppressive treatments, when inappropriate or
`
`inadequate, may lead to an over-immunosuppression state, which leaves the patient
`
`highly susceptible to infections. On the other hand, it is important to be capable of
`
`identifying reliably chronically immunosuppressed patients, so as to provide them
`
`with the most adequate treatment.
`
`The method
`
`of
`
`the
`
`invention
`
`allows
`
`the
`
`precise determination
`
`of
`
`the
`
`immunosuppressed status of the subject, enabling a specific treatment to be tailored
`
`to the needs of the patient. The prior determination of the immunosuppressed status
`
`of the patient with the method of the invention thus lead to a treatment safer than
`
`the treatments designed on the basis of the methods of the prior art.
`
`Thus
`
`the present
`
`invention
`
`also
`
`relates
`
`to
`
`a method
`
`for
`
`designing
`
`an
`
`immunomodulation treatment for a subject, said method comprising:
`
`a) determining from a biological sample of a subject the anelloviral load, and
`
`b) assessing from the determination of step a)
`
`the immunosuppressed or non-
`
`immunosuppressed status, and
`
`c) designing the immunomodulation treatment according to said immunosuppressed
`
`or non-immunosuppressed status assessed in step b)
`
`The invention is also drawn to a method of treatment of a condition associated with
`
`immunodeficiency. As used herein; “conditions associated with immunodeficiency” or
`
`“immunodeficiency disorders” refer to a diverse group of conditions characterized
`
`primarily by an increased susceptibility to various opportunistic infections with
`
`consequent severe acute, recurrent or chronic disease.
`
`In a first embodiment,
`
`this
`
`increased susceptibility to infection results from immunosuppression due to one or
`
`more defects in the immune system.
`
`Immunosuppression in this case is non-
`
`deliberate.
`
`Immunodeficiency
`
`disorders
`
`encompass,
`
`without
`
`limitation,
`
`"immunodeficiency syndromes" wherein all features are the result of the immune
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`defect, and "syndromes with immunodeficiency", wherein some, even prominent
`
`features cannot be explained by the immune defect. By means of example and not
`
`limitation,
`
`diseases
`
`and
`
`conditions
`
`associated with
`
`immunodeficiency
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`or
`
`immunosuppression comprise: human immunodeficiency virus (HIV)
`
`infection and
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`acquired immune deficiency syndrome (AIDS), hypogammaglobulinemia, hematologic
`
`cancers such as leukaemia and lymphoma,
`
`, lymphocytopenia (lymphopenia) of any
`
`origin, lupus erythematosus, cachexia, opioids abuse, mastocytosis, rheumatic fever,
`
`trypanosomiasis, alcohol abuse.
`
`The group of immunodeficiency disorders also encompasses diseases and conditions
`
`associated with immunosuppression arising from an artificial, usually controlled
`
`diminution or prevention of a subject's immune response.
`
`Immunosuppression in
`
`subjects is thus deliberately induced.
`
`It may be caused by immunosuppressive
`
`treatment, or it may occur as a side effect of a therapy of other indications (e.g.,
`
`side effect of cancer chemotherapy). These latter conditions include such conditions
`
`as total bone marrow ablation, bone marrow transplantation, organ transplantation,
`
`treatment with immunosuppressive drugs such as inter alia tacrolimus, cyclosporine,
`
`methotrexate, mycophenolate, azathioprine, interferons, and immunoglobulins such
`
`anti-CD20 and anti-CD3; and treatments with: chemotherapy agents, corticosteroids,
`
`anti-TNF drugs, radiation.
`
`Thus the present
`
`invention also relates to a method for treating a condition
`
`associated with immunodeficiency in a subject, said method comprising:
`
`a)
`
`determining the immunosuppressed or non-immunosuppressed status of the said
`
`subject according to the methods of the invention, and
`
`b)
`
`adapting an immunomodulation treatment to the said subject.
`
`The present invention thus provides an immunomodulation treatment for use in
`
`treating a condition associated with immunodeficiency in a subject, wherein the use
`
`comprises the steps of:
`
`a)
`
`the immunosuppressed or non-immunosuppressed status of the said subject is
`
`determined according to the methods of the invention, and
`
`b)
`
`the said immunomodulation treatment is adapted to the said subject.
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`In other words, the invention relates to the use of an immunomodulation treatment
`
`in the preparation of a medicament
`
`for
`
`treating a condition associated with
`
`immunodeficiency in a subject, wherein:
`
`a) the immunosuppressed or non-immunosuppressed status of the said subject is
`
`determined according to the methods of the invention, and
`
`b) the said immunomodulation treatment is adapted to the said subject.
`
`By “immunomodulation treatment”, it is herein referred to any treatment intended
`
`to induce, enhance, inhibit or suppress an immune function. According to a preferred
`
`embodiment, an immunomodulation treatment is an immunosuppressive treatment.
`
`According to another preferred embodiment, an immunomodulation treatment is an
`
`immunostimulating treatment.
`
`By “immunosuppressive treatment” it is herein referred to any treatment intended to
`
`inhibit or suppress an immune function of a subject that would be adverse to a
`
`desired clinical outcome.
`
`Immunosuppressive treatments include for example
`
`treatments that are intended to induce immune function deficiency in a subject in
`
`order to treat the said subject with a transplant of cells or of an organ. They also
`
`include for example treatments that induce immune function deficiency as a side
`
`effect, such as chemotherapy or radiotherapy. Immunosuppressive treatments usually
`
`include e.g. glucocorticoids, antiproliferative and antimetabolic drugs (rapamycin,
`
`everolimus, mycophenolate mofetil, mycophenolic acid), calcineurin inhibitors
`
`(cyclosporine, FK506, voclosporin), S1P-R agonists
`
`(FI'Y720), malononitrilamides
`
`(FK778),
`
`and
`
`antibodies
`
`(e.g.
`
`antithymocyte
`
`globulin)
`
`including monoclonal
`
`antibodies (e.g. muromonab-CD3, daclizumab, basiliximab, rituximab, alemtuzumab,
`
`infliximab, adalimumab, efalizumab).
`
`An “immunostimulating treatment”, according to the present invention, is any type
`
`of treatment intended to induce or enhance immune function. Immunostimulating
`
`treatments include treatments that stimulate specific immune response, treatments
`
`that stimulate non-specific immune response and treatments that stimulate both
`
`specific and non-specific immune responses. Such immunostimulating treatments are
`
`usually given to treat conditions associated with non-deliberate immunosuppression.
`
`Immunostimulating treatments that stimulate the immune response have been
`
`described in the literature,
`
`ranging from small synthetic molecules (poly |:C,
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`levamisole,
`
`inosine pranobex) to living microorganisms (Corynebacterium parvum),
`
`and including complex mixtures of bacterial components with mineral oils (Freund's
`
`adjuvant) or inorganic salts (aluminum and magnesium hydroxide/phosphate) and,
`
`more
`
`recently,
`
`recombinant proteins modulating immunity
`
`(e.g.
`
`cytokines,
`
`antibodies against cellular receptors). According to the present invention anti-viral
`
`treatments are also considered immunostimulating treatments. Anti-viral treatments
`
`include for example oseltamivir (Tamiflu), zanamivir (Relenza),
`
`interferon, which
`
`inhibit viral synthesis in infected cells, particularly alpha-interferon, used in the
`
`treatment of hepatitis B and C.
`
`Thus,
`
`in one embodiment of the method of the invention,
`
`the immunomodulation
`
`treatment
`
`is an immunosuppressive treatment.
`
`In another embodiment of
`
`the
`
`method of the invention, the immunomodulation treatment is an immunostimulating
`
`treatment.
`
`The said adaptation of the immunomodulation treatment may be either a reduction
`
`or suppression of the said immunomodulation treatment or the continuation of the
`
`said immunomodulation treatment at the same or an increased dose. The skilled
`
`person will appreciate that the treatment will be continued if the desired effect on
`
`the subject’s immune system is not achieved. For example, when the treatment is
`
`administered for stimulating the immune system function in order to compensate for
`
`a deficit
`
`thereof,
`
`the
`
`treatment
`
`is
`
`continued
`
`if
`
`the patient
`
`shows
`
`an
`
`immunosuppressed phenotype. Likewise, an immunosuppressive treatment will be
`
`continued if the subject displays a non-immunosuppressed status. On the other hand,
`
`the treatment will be reduced or suppressed if the desired effect on the subject’s
`
`immune system has been attained. This is the case for example when the treatment
`
`seeks to obtain immunosuppression in order to perform e.g. organ transplantation,
`
`while the subject shows an i mmunosuppression status.
`
`In a preferred embodiment,
`
`the condition associated with immunodeficiency is
`
`transplant rejection.
`
`Thus,
`
`the invention relates to a method for treating or preventing transplant
`
`rejection in a transplanted subject, said method comprising:
`
`a) determining the immunosuppressed or non-immunosuppressed status of the said
`
`transplanted subject according to the methods of the invention, and
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`b) adapting an immunosuppressive treatment to the said transplanted subject.
`
`The present invention thus provides an immunosuppressive treatment for use in
`
`treating or preventing transplant rejection in a transplanted subject, wherein the
`
`said use comprises the steps of:
`
`a) determining the immunosuppressed or non-immunosuppressed status of the said
`
`transplanted subject according to the methods of the invention, and
`
`b) adapting the said immunosuppressive treatment to the said transplanted subject.
`
`In other words, the invention relates to the use of an immunosuppressive treatment
`
`in the preparation of a medicament for treating or preventing transplant rejection in
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`a transplanted subject, wherein:
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`a) the immunosuppressed or non-immunosuppressed status of the transplanted said
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`subject is determined according to the methods of the invention, and
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`b) the said immunosuppressive treatment
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`is adapted to the said transplanted
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`subject.
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`In another preferred embodiment, the condition associated with immunodeficiency is
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`an infection.
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`Thus this embodiment relates to a method of treating an infection in an infected
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`subject, said method comprising:
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`a) determining the immunosuppressed or non-immunosuppressed status of the said
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`infected subject according to the methods of the invention, and
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`b) adapting an immunostimulating treatment to the said infected subject.
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`The present invention thus provides an immunostimulating treatment for use in
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`treating an infection in an infected subject, wherein the use comprises the steps of :
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`a) determining the immunosuppressed or non-immunosuppressed status of the said
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`transplanted subject according to the methods of the invention, and
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`b) adapting the said immunostimulating treatment to the said subject.
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`In other words, the invention relates to the use of an immunostimulating treatment
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`in the preparation of a medicament for treating an infection in an infected subject,
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`wherein :
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`a) the immunosuppressed or non-immunosuppressed status of the transplanted said
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`subject is determined according to the methods of the invention, and
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`b) the said immunostimulating treatment is adapted to the said transplanted subject.
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`An anellovirus according to the invention is a non-enveloped virus, with a small
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`circular single-stranded DNA genome which is replicated through double-stranded
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`intermediates, and which may contain up to 4 open reading frames: ORF1, ORF2,
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`ORF3 and ORF4. Open reading frames (ORF)
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`1
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`(long) and 2 (short) are partially
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`overlapping. ORF3 and 0RF4 are smaller. Anelloviruses are subgrouped into torque
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`teno virus ('l'l'V), torque teno mini virus ('l'l'MV), and torque teno midi virus ("l—I'MDV),
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`with known hosts including humans, non-human primates and domestic animals
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`(Biagini et al., J Gen Virol, 88: 2696—2701, 2007; Hino & Miyata, Rev Med Virol, 17:
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`45—57, 2007; Leary et al., J Gen Virol, 80: 2115—2120., 1999).
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`By “anellovirus”, it is herein referred to any virus belonging to the Anelloviridae
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`family of viruses, including, but not limited to, the Torque Teno viruses ("l—I'Vs), the
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`Torque Teno midiviruses ('I'I'MDVs), and the Torque Teno miniviruses, also formerly
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`known as the Torque Teno-like miniviruses (Tl'MVs). The prototype strain of Torque
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`Teno Virus ('lTV-1a) has a genome size of 3853 nucleotides. The prototype strain of
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`Torque Teno Minivirus, formerly known as 'l'l'V-like minivirus ('l'l'MV-NLC030), has a
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`genome size of 2915 nucleotides. Finally,
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`the Torque Teno Midivirus has been
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`described, with a genome of 3242 nucleotides for the prototype strain ('lTMDV-MD1-
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`073). Anelloviruses are highly variable in sequence. For example, nucleotide
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`sequences of full-length genomes of TN can vary by 40 %, and those of 'I'I'MDV by 33
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`%.
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`The “viral load” according to the invention is the number of nucleic acid sequences
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`of a virus present in a biological sample. The viral load reflects the severity of a viral
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`infection. Preferably,
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`the viral
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`load refers to the proportion of a nucleic acid
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`sequences in a biological sample which belong to the said virus. More preferably, the
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`viral load represents the number of copies of the said virus in a biological sample.
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`The viral load can for example be determined by estimating the amount of the virus
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`in a biological sample from a subject.
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`As used herein, the term “subject” refers to a vertebrate, preferably a mammal, and
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`most preferably a human.
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`By “biological sample”,
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`it is herein referred to any sample that is taken from a
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`subject, which includes but is not limited to, for example, blood, serum, plasma,
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`sputum, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen,
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`seminal fluid, tears, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample,
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`a sacral ganglion sample, adipose tissue,
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`lymphoid tissue, placental tissue, upper
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`reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal
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`central nervous system tissue. Preferably, the biological sample is blood or is derived
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`from blood, such as plasma or serum.
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`According to the invention, the anelloviral load in a subject means the viral load of
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`any virus of the Anelloviridae family hosted by said subject. Thus determining the
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`anelloviral
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`load in a subject according to the invention comprises estimating the
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`number of sequences of any virus of the Anelloviridae family in a biological sample
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`from the said subject. In particular, there is no selection, according to the invention,
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`of specific anellovirus species to be measured in the said biological sample.
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`Preferably, determining the anelloviral load comprises determining the amount of
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`active and/or
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`inactive viral copies.
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`It comprises determining the amount of
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`circulating, integrated or latent viral copies. More preferably, the anelloviral load
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`corresponds to circulating copies of anellovirus.
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`The levels of anellovirus may be determined by measuring levels of anellovirus DNA,
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`anellovirus RNA, or anellovirus proteins. The method according to the invention may
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`thus comprise another preliminary step, between the taking of the sample from the
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`patient and step a) as defined above, corresponding to the transformation of the
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`biological sample into a double-stranded DNA (dsDNA) sample, or into an mRNA (or
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`corresponding cDNA) sample, or into a protein sample, which is then ready to use for
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`in vitro detection of anellovirus in step a). The said dsDNA may correspond either to
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`the whole anellovirus genome or only to a portion of it. Once a ready-to-use dsDNA,
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`mRNA (or corresponding cDNA) or protein sample is available, the detection of the
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`anellovirus may be performed, depending on the type of transformation and the
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`available ready-to-use sample, either at
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`the genomic DNA (i.e. based on the
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`presence of at least one sequence consisting of at least a part of the anellovirus
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`genome), mRNA (i.e. based on the mRNA content of the sample) or at the protein
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`level (i.e. based on the protein content of the sample).
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`Preferably,
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`the levels of anellovirus are determined by measuring levels of
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`anellovirus DNA.
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`Methods for detecting a nucleic acid in a biological sample include inter alia
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`hybridization with a labelled probe, amplification,
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`including PCR amplification,
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`sequencing, and all other methods known to the person of skills in the art. The
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`amount of nucleic acid transcripts can be measured by any technology known by the
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`skilled person. In particular, the measure may be carried out directly on an extracted
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`messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA)
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`prepared from extracted mRNA by technologies well-known in the art. From the
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`mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using
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`any technology known by a person skilled in the art, including nucleic microarrays,
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`quantitative PCR, and hybridization with a labelled probe.
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`In a first embodiment of the invention, the levels of anellovirus DNA are measured by
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`sequencing. As used herein, the term "sequencing" is used in a broad sense and refers
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`to any technique known by the skilled person including but not limited to Sanger
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`dideoxy
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`termination
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`sequencing, whole-genome
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`sequencing,
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`sequencing
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`by
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`hybridization, pyrosequencing, capillary electrophoresis, cycle sequencing, single-
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`base extension sequencing, solid- phase sequencing, high-throughput sequencing,
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`massively parallel
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`signature sequencing (MPSS),
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`sequencing by reversible dye
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`terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing,
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`sequencing
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`by
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`ligation,
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`short-read
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`sequencing,
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`single-molecule
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`sequencing,
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`sequencing-by-synthesis,
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`real-time
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`sequencing,
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`reverse-terminator
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`sequencing,
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`nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD(R)
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`sequencing, MS-PET sequencing, mass spectrometry, and combinations thereof.
`
`Optionally, DNA is fragmented randomly, generally by physical methods,, prior to
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`sequencing. The anellovirus DNA may be sequenced by any technique known in the
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`art,
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`including sequencing by ligation, pyrosequencing, sequencing—by-synthesis or
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`single-molecule sequencing. Sequencing also includes PCR-based techniques, such as
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`for example quantitative PCR or emulsion PCR.
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`Sequencing is performed on the entire DNA contained in the biological sample, or on
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`portions of the DNA contained in the biological sample. It will be immediately clear
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`to the skilled person that the said sample contains at least a mixture of anellovirus
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`DNA and of DNA from the host subject. Moreover, the anellovirus DNA is likely to
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`represent only a minor fraction of the total DNA present in the sample.
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`A first approach that addresses these challenges is
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`to sequence and quantify
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`sequences which are known to be specific of the anellovirus genome. Indeed, the
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`inventors have identified two consensus sequences, represented by SEQ ID No. 4 and
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`SEQ ID No. 5, based on the comparison between all the anelloviral sequences. Thes