WO2020120778A1 - Method for identifying hiv-infected cells - Google Patents

Abstract

The invention concerns a method for the in vitro identification of reservoir cells infected with a retrovirus responsible for immunodeficiency in mammals, from a biological sample from the mammals having been treated with at least one antiretroviral agent, the method comprising: - a step of determining the presence or the quantity of ASP or its transcript, - a step of comparing with a reference, and - a step of identifying the reservoir cells.

Description

[Title established by ISA under rule 37.2] METHOD OF IDENTIFYING HIV-INFECTED CELLS

The invention relates to a method for identifying infected cells, in particular by retroviruses.

Thanks to antiretrovirals, patients infected with the human immunodeficiency virus (HIV) keep their viral load (amount of virus in the blood) below the detection threshold by current methods. However, despite their effectiveness, even the most powerful antiretrovirals cannot completely eliminate the virus, because it remains latent in certain cells and thus escapes the effects of antiretrovirals which target the virus in replication or when it enters the virus. target cells. These persistent cells are called the viral reservoir.

Targeting HIV reservoir cells in patients on antiretroviral therapy has been the strategy favored by the scientific community since the end of the last century. Indeed, the eradication of these cells would completely eliminate the virus from patient cells, and thus finally offer an effective and definitive treatment of the infection.

There is therefore a real need to characterize the entire viral reservoir in order to offer appropriate therapy.

Several markers suspected of being markers of HIV reservoir cells are known from the prior art. In particular, patent applications FR 3056990 and WO 2018060979 are known for the markers CD32a and CD89. The inventors cited in these patent applications identified these cellular markers as being expressed specifically by the cells of seropositive patients under treatment and capable of producing active viruses when the antiretroviral treatment is stopped.

However, such markers are probably not the only ones which make it possible to differentiate healthy cells from reservoir cells.

Also, there is still a real need to provide means to characterize reservoir cells, and to identify new markers.

The invention particularly aims to overcome this lack.

One of the aims of the invention is therefore to propose a method for identifying HIV reservoir cells.

Another object of the invention is to propose a method making it possible to ensure that a patient is in complete remission of the infection and that there is therefore no longer any virus, that is to say that there is no there is no more tank.

Yet another object of the invention is to provide a simple means for the clinician or doctor to identify whether an HIV-positive patient has a viral reservoir.

To this end, the invention relates to a method of identifying, in particular in vitro , reservoir cells of a retrovirus responsible for immunodeficiency in mammals, from a biological sample of said mammals having been treated with at least one anti retroviral agent, said method comprising

- a step of determining the presence or absence, or even of measuring the amount of a compound chosen from:

* the antisense transcript encoding the antisense protein or asp,

* the ASP protein, and

* antibodies specific for the ASP protein,

in said biological sample of said mammals having been treated with at least one anti retroviral agent,

a step of comparing said presence or absence of said compound, or again of comparing the amount of said compound measured in the previous step, with reference samples, in particular with reference samples from non-infected patients for the reference of negativity and from uninfected patients supplemented with the synthetic reference standard corresponding to the method used for the positivity reference, and

a step of determining the presence of reservoir cells in said biological sample if i) said compound is present while it is absent in the reference sample, or ii) the amount of said compound is at least equal to the threshold defined by the statistical analysis of the negative samples for each of the methods times the quantity observed, in particular at least equal to 1.2 times the quantity measured in said reference samples.

The invention is based on the surprising observation made by the inventors that the quantity of ASP antisense transcripts, or of the ASP protein or of antibodies directed against the ASP protein, is an excellent marker of the viral reservoir of infection by HIV, and more generally retroviruses causing immunodeficiency in mammals.

In the invention, the term “reservoir cells” means the cells constituting the functional reservoir of a retrovirus inducing an immunodeficiency. These functional reservoir cells are cells that have been infected with the virus and which for some reason linked to the host cell or to the virus itself have stopped producing infectious functional virus. However, these cells are able to return to fully active cells, especially after stopping antiretroviral therapy, leading to a viral rebound.

Also, in the invention it is well considered the cells of the functional reservoir and not the cells in viral latency.

ASP (or anti-sense protein, in English AntiSense protein), is a protein encoded by the anti-sense strand of the genome of the HIV virus, and related viruses. This open reading frame is entirely overlapping with the open reading frame of the sense strand encoding the env gene. The asp gene encoding said ASP protein is found in particular in the HIV1 genome of group M.

Also, the method according to the invention consists, from a sample of a patient infected with HIV, and previously treated with at least one antiretroviral agent, in particular a multi-therapy, in detecting the presence or absence i ) of the asp antisense transcript, ii) the presence of the ASP protein itself, or iii) the presence of antibodies directed specifically against the ASP protein.

In the context of the detection of the asp transcript, conventional molecular biology techniques can be used, such as the polymerase chain reaction (PCR), quantitative or not, hybridization methods with probes labeled with radioactivity or still fluorescent markers, direct sequencing (RNAseq) or any other technique known to those skilled in the art accustomed to searching for the presence or absence of transcripts.

As part of the detection of the presence or absence of the ASP protein, immunological techniques well known to those skilled in the art can be used. For example, an ELISA test using an antibody control command for an anti ASP antibody can be used. A second antibody directed against ASP, labeled or not, will be used to detect an immobilization of the ASP protein. The use of a third antibody recognizing the constant part of the second antibody, and coupled to a molecule allowing a detection of light, chemical or fluorescent type could also be implemented.

In the context of the detection of anti ASP antibodies, the immunological techniques known to those skilled in the art will also be used. Here, the ASP protein will serve as a bait for the antibodies to be detected, and a second antibody recognizing the constant part of the antibodies, and coupled to a molecule allowing detection of the light, chemical, radioactive or fluorescent type, will be used for the detection.

Also, the method according to the invention consists, from a sample of a patient infected with HIV, and previously treated with at least one antiretroviral agent, in particular a multi-therapy, in detecting the quantity or i) of the transcript anti-sense asp , ii) the presence of the ASP protein itself, or iii) the presence of antibodies directed specifically against the ASP protein. This means that it is important to know "the relative number" of molecules (transcripts, proteins or antibodies).

The aforementioned methods for detecting presence or absence can be used, but in their "quantitative" version, that is to say allowing to give a quantified value of each of the molecules. For example, for transcripts, the quantitative PCR technique will be favored.

The second step of the aforementioned method consists of a step of comparison with one or more control samples making it possible to define the presence or absence, or the quantity of each of the molecules (transcripts, proteins or antibodies).

The control samples are multiple, and may correspond to i) one or more samples from healthy individuals who have never been infected with an immunodeficiency virus (negative controls), ii) to one or more samples of the individual tested, said sample having been taken before infection by said virus, iii) from one or more samples of individuals infected by said virus and who have not been treated with an antiretroviral agent, iv) from one or more samples of the individual tested, said sample having been taken near his infection with said virus, but before his treatment with an antiretroviral agent, or v) standards, that is to say compositions of which the quantity of molecule ( asp transcripts, ASP proteins or anti ASP antibodies) are known precisely. Obviously, within the framework of the method, it is possible to use one or more of the above-mentioned reference samples, or any other reference sample which the person skilled in the art deems useful.

The comparison steps are methods conventionally used by a person skilled in the art, consisting in carrying out differences, ratios, or any other mathematical operation making it possible to define whether there is a qualitative or quantitative difference in molecule ( asp transcripts, ASP proteins or anti ASP) between the test sample and the control or control sample (s).

In the third stage, it is determined whether or not the sample tested comes from an individual with a viral reservoir, or even to quantify said reservoir if it exists.

In the context of the presence or absence of molecules (( asp transcripts, ASP proteins or anti ASP antibodies), if detection of said molecules is possible, the patient from whom the tested sample is taken will be considered to have a viral reservoir , and therefore likely after stopping antiretroviral therapy, to undergo a viral rebound and therefore to produce active virus again. Conversely, if no molecule is detected or detectable, the individual from which the sample tested will be considered as having no viral reservoir, within the limit of detection permitted by the techniques used.

It may be particularly advantageous rather than detecting the presence or absence of the molecules ( asp transcripts, ASP proteins or anti ASP antibodies), or in addition to this detection of presence or absence, of detecting their quantity. Indeed, in order to limit false negatives (absence of detection because the method used is not sensitive enough), a quantitative method can be used. This makes it possible in particular to quantify the viral reservoir and to determine if it is weak (few cells constitute it) and therefore that the patient would be likely to have a weak viral rebound (little production of active virus when stopping the treatment), or if the reservoir is large, and in this case that the viral rebound is likely to be very significant (large production of virus at the end of the treatment).

The positivity threshold of a sample is defined as being at least equal to the threshold corresponding to the quantity of molecules ( asp transcripts, ASP proteins or anti ASP antibodies) defined by the statistical analysis of negative samples for each of the methods times the quantity observed. In particular, if the quantity measured in the sample is at least equal to 1.2 times the quantity measured in the so-called negative control sample, the individual will be considered to have a viral reservoir.

In order to determine the detection threshold, the precision and the specificity of each of the methods considered, a measurement is carried out on a panel of samples from non-infected subjects (negative) and infected subjects (positive). The results are subjected to a statistical analysis making it possible to determine the rates of specificities and sensitivity such as for example an ROC analysis (Delacour, H., et al. La Courbe ROC ( receiver operating characteristic ): principles and main applications in clinical biology , Annales de biologie clinical, 2005; 63 (2): 145-54) or any other equivalent statistical method .

In most HIV-infected patients on antiviral triple therapy, viremia (presence of virion in the plasma) is extremely low, most often undetectable. Despite the success of these triple therapies, treatment must be taken continuously, a lack of adherence leading to a rapid rebound in viremia.

If the treatments allow a control of the viremia, they do not lower the proviral load (the number of infected cells containing the proviral genome). These residual infected cells, also known as the viral reservoir, in fact group together a complex set of infected cells whose virological status and dangerousness are very variable.

Current antiviral treatments target the infectivity of the viral particle (inhibition of maturation, reverse transcription, integration, etc.) and have no direct impact on the infected cell and more particularly on those constituting the viral reservoir functional.

These drugs in themselves cannot therefore lead to modify the viral production by these cells. The impact that is observed on the CD4 + T cell population is due to the cytotoxic effect of viral production when it takes place in a significantly activated T lymphocyte.

On the other hand, certain cells, which produce little virus (macrophages, T lymphocytes activated to a lesser extent), are resistant to the cytotoxic effect of the virus and will therefore be able to persist even when infected with a productively active virus, which will be undetectable at peripheral level ( blood), as long as adherence to treatment blocks the infectious power of the particles produced and thus prevents its amplification.

1. contenant le génome viral, (qui dans le cas d’un rétrovirus comme le VIH peut être intégré au génome de la cellule hôte ou être sous forme circulaire non intégré où le virus peut produire certaines protéines virales, mais peut surtout persister dans certain type cellulaire jusqu’à ce que l’intégration soit rendue possible), et
2. ne produisant pas de virus.

The viral reservoir, in the context of an infection, is often defined as the set of infected cells, i.e.

1. containing the viral genome, (which in the case of a retrovirus like HIV can be integrated into the genome of the host cell or be in non-integrated circular form where the virus can produce certain viral proteins, but can especially persist in certain cell type until integration is made possible), and
2. not producing viruses.

A very large number of studies have demonstrated that, in the aviremic patient under treatment, the reservoir thus defined (presence of the viral genome) is in the vast majority (98%) a non-functional reservoir, that is to say unable to produce an infectious virus particle again.

Indeed, it has been well established that approximately 90% of these viruses present one or more mutations leading to the absence of production of any viral particle, or of viral particles defective for one of its components (genomes, envelope, activity of reverse transcriptase, integrase, etc.). Out of the remaining 10%, for which analyzes of the proviral genome do not allow the identification of mutations, extrinsic causes are proposed: integration site, cell state, etc. which will result in only less than 1 / 8 of these cells will be able to produce an infectious virus.

Faced with this complexity, the term functional viral reservoir has been defined, that is to say a set of infected cells capable, after single or repeated reactivation, of producing infectious particles.

To date, this functional reservoir is poorly defined and could have several aspects if we consider for example the different cellular markers which have been independently (ie exclusively) associated with this reservoir: CD2, CD30, CD32, CD89, …. The functional reservoir therefore corresponds to the viruses present, in a latent state or not, in a small fraction of the infected cells and which will be capable of producing infectious particles which will be responsible for example of the viral rebound in the event of treatment being stopped.

There is often an association that is made between reservoir and latency but it is a reducing shortcut especially in the context of a treatment which has led to an aviremia, that is to say the absence of viral particles. detectable in the plasma of the subject treated. We must first differentiate latency from quiescence.

Latency is about the virus, while quiescence is the state of cell activation. Confusion is often made because maximum viral production is associated with a significant activation state in the CD4 + T lymphocyte (which produces little or no quiescent virus). It is known that even in the case of infected cells belonging to the functional viral reservoir, an activation of this cell may not lead to the production of the virus at a given time.

In the context of HIV, latency has several levels of understanding. Originally, this notion essentially covered clinical latency, i.e. the clinically asymptomatic phase specific to certain retroviruses between the primary infection and the phase of appearance of clinical signs (AIDS for HIV, ATL for HTLV,…). However, in these viruses there is a viral latency which results in the presence in the infected patient of cells carrying the viral genome but not producing an infectious viral particle at a given time.

By viral latency is therefore meant the absence of production of infectious viral particles but also viral transcriptional latency (that is to say the absence of transcription of the integrated proviral genome).

In reality, viral latency brings together a fairly broad spectrum of viral states. A transcriptionally active provirus could for example produce multi and / or mono-spliced transcripts allowing the production of viral helper, regulatory and envelope proteins which will not lead to the production of infectious particles in the absence of production of non-spliced viral transcripts which allow the production of the Gag and Gag-Pol poly-proteins as well as the viral genome.

The mechanisms leading to latency are still very little understood and the part of extrinsic (coming from the cell) and intrinsic (coming from the virus) factors in the induction of latency is not known.

Besides the latency induction, its maintenance but especially the mechanisms of latency reversion, which will lead a virus from a latent state to a productive state, are still unknown. It is precisely they who are going to make a latent virus be considered as part of the functional reservoir and not of a non-reactivable reservoir. In fact, only around 20% of intact viruses integrated into cells will lead to the production of viruses when the infected cells in which they are found are reactivated.

This demonstrates 1) that the possibility of activating the infected cell is not a sufficient criterion to "wake up" a virus and 2) that an intact virus, transcriptionally inactive, is present in a cell does not make it a virus. constituent of the functional tank.

There is therefore a dissociation between viral latency and the functional reservoir, since the phenomena involved in the induction of viral latency will not visibly lead systematically to the constitution of a functional reservoir.

Also, for example, the document Juan C. Zapata et al. - Virology - 2017 - pp 34-44 describes the mechanisms of viral latency, but does not give any clues to the characterization of the functional viral reservoir.

In addition to the previously mentioned aspects, a difference between viral latency and functional reservoir is the biological phenomena involved.

Indeed, a biological mechanism constituting the induction of latency is not per se a mechanism present in the functional reservoir and vice versa. For example, it is known that the virus has a mechanism allowing it to maintain latency once it is implemented. A mechanism which will intervene during the phenomenon of latency of the virus will not necessarily be present in the infected cell several years after the latency. The HIV virus indeed presents a sequence called TAR within the promoter which allows the production of the viral particles. ART corresponds to a structure of RNA that blocks transcriptional elongation in the absence of the viral protein Tat. Once a biological process has led to viral latency, and consequently that the production of the Tat protein will be inhibited, the ART will prevent viral transcription and therefore the initial process allowing the induction of latency will be necessary to induce latency but not to maintain it. The existence of the TAR structure in itself therefore clearly indicates that there are separate mechanisms for the two processes.

This is also valid for a cellular phenomenon which would be associated with the functional reservoir but which may not have a role in the establishment of latency. In illustration, a marker expressed on the surface of a cell harboring a functional reservoir such as CD32a (or other proposed markers) does not imply that this marker is expressed specifically in the cell initially, during the setting in virus latency. In other words, this marker will not make it possible to differentiate the cells in which the virus will become latent that those where it will not enter in latency: they are markers a posteriori . In addition, some of the markers associated with the functional reservoirs identify proviruses exhibiting a certain level of transcriptional activity and therefore which are not strictly speaking latent.

In summary, there is a dissociation between viral latency and functional reservoir on the one hand in terms of the cell population concerned which are only partially overlapping and on the other hand in terms of mechanistics.

In the invention, it is indeed the cells of the functional reservoir which are sought.

Advantageously, said ASP protein is the ASP protein of the human immunodeficiency virus, in particular of the human immunodeficiency virus -1 of group M.

There are two types of human immunodeficiency virus (HIV): HIV-1 and HIV-2. HIV-1 is made up of four groups (M, N, O and P), each with its own origin which has resulted from transmission from monkey to man on at least four occasions. While the simian origin of groups M and N, in fact Cameroon chimpanzees (in particular the subspecies Pan troglodytes troglodytes ), had been identified several years ago, the origin of groups O and P seems rather to be the gorilla.

The M group of HIV-1, the most common strain, is responsible for the AIDS pandemic (99% of infections out of a total of 75 million). While group P has only been detected in two individuals so far, group O has been able to spread to humans in several countries in West and Central Africa. It is estimated that it has infected nearly 100,000 people.

Advantageously, the biological sample is a blood, serum or plasma sample, and in which said compound is an antibody specific for the ASP protein.

From a simple blood, plasma or serum sample, it is possible to measure the quantity of ASP protein, or its transcript, in order to implement the method according to the invention.

Advantageously, the biological sample is a sample of cells, and in which said compound is the transcript encoding the ASP protein or the ASP protein,

It is advantageous to implement the method of the invention from a sample of patient cells, in particular taken from a simple blood test, and to search for, or quantify, the quantity of transcripts encoding the protein. ASP.

Also, this advantageous embodiment relates to a method of identifying, in particular in vitro , reservoir cells of a retrovirus responsible for immunodeficiency in mammals, from a sample of cells, in particular hematopoietic of said mammals having been treated with at least one anti retroviral agent, said method comprising

a step of determining the presence or absence, or of measuring the amount of the antisense transcript encoding the antisense protein or asp ,

in said biological sample of said mammals having been treated with at least one anti retroviral agent,

a step of comparing said presence or absence of said compound, or even comparing the amount of said compound measured in the previous step, with reference samples in particular from non-infected patients, and

a step of determining the presence of reservoir cells in said biological sample if i) said compound is present while it is absent in the reference sample, or ii) the amount of said compound is at least equal to the threshold defined by l statistical analysis of the negative samples for each of the methods times the quantity observed, in particular at least equal to 1.2 times the quantity measured in said reference samples.

Advantageously, in the aforementioned method, the ASP gene corresponds to the gene whose identifier is Gene ID: 19424028, in particular having the reference nucleic sequence (NC_001802.1) SEQ ID NO: 22 as follows: ATGCCCCAGA CTGTGAGTTG CAACAGATGC TGTTGCGCCT CAATAGCCCT CAGCAAATTG TTCTGCTGCT GCACTATACC AGACAATAAT TGTCTGGCCT GTACCGTCAG CGTCATTGAG GCTGCGCCCA TAGTGCTTCC TGCTGCTCCC AAGAACCCAA GGAACAAAGC TCCTATTCCC ACTGCTCTTT TTTCTCTCTG CACCACTCTT CTCTTTGCCT TGGTGGGTGC TACTCCTAAT GGTTCAATTT TTACTACTTT ATATTTATAT AATTCACTTC TCCAATTGTC CCTCATATCT CCTCCTCCAG GTCTGAAGAT CTCGGACTCA TTGTTGCTAT TACCACCATC TCTTGTTAAT AGCAGCCCTG TAATATTTGA TGAACATCTA ATTTGTCCAC TGATGGGAGG GGCATACATT GCTTTTCCTA CTTTCTGCCA CATGTTTATA ATTTGTTTTA TTCTGCATGG GAGGGTGATT GTGTCACTTC CTTCAGTGTT ATTTGACCCT TCAGTACTCC AAGTACTATT AAACCAAGTA CTATTAAACA GTTGTGTTGA ATTACAGTAG.

The ASP protein corresponds to the protein comprising or consisting of the sequence SEQ ID NO: 1, or a protein having an identity of at least 75% at the protein level calculated outside the hypervariable zones underlined in the reference protein sequence, excluding also the deletion of the first 26 residues specific for example to viruses of subtype A of group M of HIV-1 (in italics in the sequence SEQ ID NO: 1).

The ASP protein of the HXB2 reference virus has the following sequence (SEQ ID No 1:

MPQTVSCNRC CCASIALSKL FCCCTI PDNN CLACTVSVIE AAPIVLPAAP KNPRNKAPIP TALFSLCTTL LFALVGATPN GSIFTTLYLY NSLLQLSLIS PPPGLKIS DS LLLLPP SLVN SSPVIFDEHL ICPLMGGAYI AFPTFCHMFI ICFILHGR VI VSLPSVLFDP SVLQVLL NQV LLNSCVELQ.

By “protein having at least 75% identity with said protein of sequence SEQ ID NO: 1” is meant a protein which has in amino acids 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the protein of sequence SEQ ID NO: 1.

Advantageously, these proteins comprising at least 75% identity with the protein of sequence SEQ ID NO: 1 all have the following sequence SEQ ID NO: 2: Nter- PDNNCLACTVSVIEAAPIVLPAAPKNPRNKAPIPTALFSLCTTLLFALVGATPNGSIFTTLYLYNSLLQLSLI-Cter. . In particular, this concerns the ASP proteins of the following sequence:

SEQ ID NO: 3: MPQTVSCNRC CCASIALSKL FCCCTIPDNN CLACTVSVID RAPIVLPAAP KNPRNKAPIP TALFSLCTTL LFALVGATPN GSIFTTLYLY NSLLQLSLIS PPPGLKISDP LLLLPPSLVN SSPVIFDEHL ICPLMGGAYIFPLVLFLVLFVLFLVLFLVLFLVLFVLFLVLFLVLSLVLFLVLFLVLFLVLFLVLFLVLQLVLFVLQLVLFLVLQFLVLQLVLQLVLQLVLVLVLQFLVLQLVLVLQFLVLVLQFLVLQFLVLQVLQVLQVLQVLQVLQVLQVLQVLQVLVQQ

SEQ ID NO: 4: MPQTVSCNRC CCASIALSKL FCCCTISDNN CLACTVSVID AAPIVLPAAP KNPRNKAPIP TALFSLCTTL LFALVGATPN GSIFTTLYLY NSLLQLSLIS PPPGLKISDP LLLLPPSLVN SSPVIFDEHLI CPLMGGAYI AFPTSCHMFI NCFILHGSVI VSLPSVLFDP SVLQVLLNQV LLNSCVELQ, and

SEQ ID NO: 5: MPQAVSCNIC CCASIALSKL FCCCTIPDNN CLACTVSVID AAPIVLPAAP KNPRNITPIT PTXLFSLXTT LLXALVGATP NGSIFTTLYL YNXLLQLSLI SPPPGLKISV VSLLFPPSLV NSSPVIFDEF LICPSLFMFLFMFLFMFLFMFLFMFLFMFLFMFLFMFLFMFLFMFLFMFLQFLFQFGFQFLF

Advantageously, in the aforementioned method, the ASP protein corresponds to the protein comprising or consisting of the sequence SEQ ID NO: 1, or a protein having at least 75% identity with said protein provided that it retains the sequence SEQ ID NO: 2.

Advantageously, in the aforementioned method, the ASP protein corresponds to the protein comprising or consisting of the sequence SEQ ID NO: 1, or a protein having at least 75% identity with said protein, said protein being chosen from proteins of sequence SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

The invention also relates to a method for determining, in particular in vitro , the efficacy in a biological sample of an anti retroviral therapy on the viral reservoir of mammals infected with a retrovirus responsible for human immunodeficiency, said method comprising:

a step of determining the presence or absence, or even of measuring the quantity, of a compound chosen from:

o the antisense transcript encoding the anti sense protein or ASP, in particular represented by the sequence SEQ ID NO: 22,

o of ASP protein, in particular represented by the sequence SEQ ID NO: 1, or one of its variants, in particular represented by any of the sequences SEQ ID NO: 3 to 5, or

o antibodies specific for the ASP protein,

in said biological sample,

a step of comparing said presence or absence of said compound, or again of comparing the measured quantity of said compound in the previous step, with reference samples from non-infected patients, for the reference of negativity, and from patients uninfected complemented by the synthetic reference standard corresponding to the method used for the positivity reference, and

- a step of determining the effectiveness of said antiretroviral therapy if

o said compound is absent after anti retroviral therapy while it was present in the reference sample, or

o the quantity of said compound varies by at least 1.2 times the statistically significant precision limit of the method considered.

The above method makes it possible to determine whether a therapy directed against reservoir cells is effective or not, by measuring the quantity of the viral reservoir, that is to say by measuring the quantity of markers as described above (antisense transcripts , ASP protein or antibody).

If at the end of the aforementioned method and after comparison with one or more reference samples, the markers (antisense transcripts, ASP protein or antibody) are no longer detectable, whereas they were before the administration of the treatment targeting the reservoir cells, it will be considered that the treatment has been effective. In the same way if the quantity of markers (antisense transcripts, ASP protein or antibody) is significantly reduced compared to the quantity of markers in the patient before treatment, it will also be considered that the treatment is effective.

Obviously, conversely, if the level of markers does not vary or even increase, it will then be considered that the anti-reservoir cell therapy has been ineffective.

Such a method is a major advantage in the development of therapy targeting reservoir cells, and provides a therapeutic arsenal, in combination with multi-therapies, to eradicate the virus in affected individuals.

Advantageously, said reference sample is a sample of said mammal before treatment with said antiretroviral therapy.

Indeed, the best benchmark to determine if the treatment is effective is to compare the level of markers (anti sense transcripts, ASP protein or antibody) before treatment and after treatment.

Even more advantageously, reference is made to the aforementioned method in which said ASP protein is the ASP protein of the human immunodeficiency virus, in particular of the human immunodeficiency virus -1 of group M.

In another aspect, the invention relates to kit for the detection of mammalian retrovirus reservoir cells comprising:

- means for detecting a compound chosen from

o an anti sense transcript encoding the anti sense protein or ASP

o the ASP protein, in particular represented by the sequence SEQ ID NO: 1, or one of its variants, in particular represented by any of the sequences SEQ ID NO: 3 to 5, or

one or more antibodies specific for the ASP protein, and

- at least one biological reference sample.

The above-mentioned kit contains the essential elements making it possible to detect the presence of at least one of the markers (antisense transcripts, ASP protein or antibody) making it possible to quantify the viral reservoir. Associated with these detection means, the kit also includes at least one biological reference sample.

Concerning the reference biological samples, it may be either a determined quantity of purified markers (RNA, proteins or antibodies) which in particular correspond to an average which can be observed either in healthy individuals or in infected patients who have not yet been treated with antiretroviral therapy. The biological sample can also correspond to extracts of cells, or even cells, originating from infected and not yet treated patients, or cells or extracts thereof originating from healthy individuals.

The detection means contained in the kit can be in particular:

- to detect the antisense transcript, specific oligonucleotides enabling quantitative PCR to be carried out, and in particular the pair of oligonucleotides as represented by the sequences SEQ ID NO: 11 and SEQ ID NO: 12,

To detect the protein, antibodies specific for the ASP protein, in particular capable of recognizing the sequence SEQ ID NO: 1, or any protein comprising this sequence, and

- to detect antibodies, peptides, in particular of sequence SEQ ID NO: 2, of ASP, or the protein ASP, in particular of sequence SEQ ID NO: 1 or of sequence SEQ ID NO: 3 to 5.

Also, advantageously, a kit is proposed comprising:

oligonucleotides making it possible to detect the anti sense ASP transcript, in particular oligonucleotides of sequence SEQ DI NO: 11 and SEQ ID NO: 12, and / or

- antibodies specifically recognizing the ASP protein, and in particular antibodies specific for the sequence SEQ ID NO: 2, and / or

peptides of the ASP protein, in particular the peptide of sequence SEQ ID NO: 2, or the protein ASP, in particular of sequence SEQ ID NO: 1 or of sequence SEQ ID NO: 3 to 5, and

- at least one reference biological sample, said reference biological sample being either a sample from a healthy individual, or a sample from a patient infected with said virus but who has not been treated with antiretroviral therapy, or them types of samples.

Brief description of the figures

The invention will be better understood on reading the following figures:

Measurement of anti-ASP antibodies by LIPS: Plasmas from patients infected (HIV +) or non-infected (HIV-) by HIV-1 are incubated with the renilla-ASP fusion protein and then the experiment is continued according to the protocol. from LIPS described in Example 2. The figure shows the Relative Light Unit (RLU) for the two groups of patients. The horizontal line indicates the positivity threshold for which the specificity (true positive rate) is equal to 100%.

Measurement of antisense viral transcription in ex-vivo CD4 + T lymphocytes: CD4 + T lymphocytes from a patient infected with HIV-1 and on anti-viral treatment were stimulated for 5 days by anti-CD3 antibodies and anti-CD28 then maintained in IL-2. At the different times indicated on the graph, part of the cells were recovered to allow analysis of the antisense viral transcription according to the protocol described in Example 1.

Infection of T lymphocytes by a mutant not expressing ASP: CD4 + T lymphocytes isolated from a healthy donor have been infected with an HIV virus expressing ASP (gray curve) or not expressing ASP (black curve). At different post-infection times (in days), the viral production was evaluated by determining the percentage of cells expressing the viral protein GAG by cytometry and intracellular labeling.

Infection of dendritic cells with a mutant not expressing ASP: Dendritic cells derived from monocytes isolated from a healthy donor have been infected with an HIV virus expressing ASP (gray curve) or not expressing ASP (black curve). At different post-infection times (in days), the viral production was evaluated by determining the percentage of cells expressing the viral protein GAG by cytometry and intracellular labeling.

represents a graph showing the Q-RTPCR analysis of the relative ratio of the Gag transcript and of ASP (Y axis) in latently infected cells (U1 - A) or productive: U1 activated by PMA (B) or TNFalpha (C). The relative ratio measured in the latent U1 line (A) is taken as a reference

represents a graph showing the Q-RTPCR analysis of the relative ratio of the ASP and Gag transcript (Y axis) in infected cells in productive (JK - A) or latent (J-Lat (B) and U1 (C)). The JK line (C) is taken as a reference. *** represents latent infection.

represents a graph showing the Q-RTPCR analysis of the relative ratio of the Gag transcript and of ASP (Y axis) in infected cells used to perform it FIG. 6 in a presentation similar to that of FIG. 5. (B ) represents the ratio measured in latently infected J-lat cells and (C) in productively infected jurkat cells. (A) represents the relative ratio in latently infected U1 cells similar to the representation in Figure 6, and serves as a reference.

Supplement to FIG. 2 showing that the detectable antisense transcript on D0 is no longer detectable after 5 days of stimulation, in two patients.

Examples

Example 1: RT- protocol qPCR specific strand for the detection of the ASP transcript in the cells of infected patients

Equipment used

1) Primers

Reverse transcription primers:
- ASP: 5'- ATC ATG AAG CAT CTA GAT TCA AAG TGC TGC GGA GCA GCA GG AAG CAC TAT- 3 '(SEQ ID NO: 6)
- Gag: 5'- TCA TCC ATC CTA TTT GTT CCT GAA G-3 '(SEQ ID NO: 7)
- Actin Beta: 5'- AAG GGA CTT CCT GTA ACA ATG CA-3 '(SEQ ID NO: 8)
- RPL19: 5'- TGC GTG CTT CCT TGG TCT TA-3 '(SEQ ID NO: 9)
- HPRT: 5'- GGC GAT GTC AAT AGG ACT CC-3 '(SEQ ID NO: 10)

QPCR primers:
- ASP: 5'-sense primers - ATG CCC CAG ACT GTG AGT TG-3 '(SEQ ID NO: 11) and 5'-anti-sense - ATC ATG AAG CAT CTA GAT TCA AAG TGC TGC-3' (SEQ ID NO: 12).

The ASP-specific transcription and qPCR reverse primers were designed to detect the ASP transcript from different M group subtypes, and target the highly conserved 5 'end of the ASP transcript between the different subtypes.

- Gag: primers sense 5 '- GTG GGG GGA CAT CAA GCA G-3' '(SEQ ID NO: 13) and antisense 5' - TGC TAT GTC ACT TCC CCT TGG -3 '(SEQ ID NO: 14).

- Actin beta: primers sense 5’- CCA ACC GCG AGA AGA TGA -3 ’(SEQ ID NO: 15) and antisense 5’- CCA GAG GCG TAC AGG GAT AG -3’ (SEQ ID NO: 16).

- RPL19: primers 5 ′ direction - CTC TAG TGT CCT CCG CTG TG -3 ’(SEQ ID NO: 17) and antisense 5’- CTT CCG CTT ACC TAT GCC CA-3’ (SEQ ID NO: 18).

- HPRT: primers 5 ′ direction - TGA CAC TGG CAA AAC AAT GCA -3 ’(SEQ ID NO: 19) and antisense 5’- GGT CCT TTT CAC CAG CAA GCT -3’ (SEQ ID NO: 20).

2) Reagents

RNeasy Mini Kit (Qiagen)

Superscript transcriptase III (Thermofisher)

PCR Clean-up Gel extraction Kit (Macherey-Nagel)

LightCycler 480 SYBR Green I Master (Roche Life Science)

RT protocol- qPCR specific strand

1) Isolation of patient cells

The cells are isolated from blood tubes (4 to 7 mL) taken from an anticoagulant.

Cells can be isolated by different approaches including:

All white blood cells: erythrocyte lysis and centrifugation

Monocytic fraction: Isolation on ficoll cushion

Isolation of total CD4 + T cells, for example by negative selection, which prevents their activation. : RosetteSep kit (Stemcell)

The cells are either used immediately or frozen at -80 ° C. in the form of a dry pellet. The total RNAs are then subsequently extracted from these cells.

The total RNAs are extracted according to the protocol of the “RNeasy Mini Kit” kit (Qiagen). The total RNA obtained is assayed with the nanodrop.

2) Reverse transcription

An amount of RNA between 600 ng and 2 µg is used as a template for the reverse transcription step. Primers specific to ASP and Gag transcripts, but also housekeeping genes encoding the proteins Actin Beta, RPL19 and HPRT are used for reverse transcription. At the 3 ’end of the ASP primer, a non-complementary sequence called an anchor has been added. This sequence specifically amplifies the cDNAs from the reverse transcription of ASP mRNA, and not the DNAs from a possible self-priming reaction of RNA. The reverse transcription is carried out at 55 ° C. using the reverse transcriptase Superscript III. The cDNAs obtained are purified on a column in order to remove the primers used for the reverse transcription, using the "PCR Clean-up Gel extraction" kit.

3) qPCR specific strand

The qPCR reaction is carried out in a 384-well plate. For each cDNA sample, the expression of three housekeeping genes (actin beta, rpl19 and hprt) and two target genes (asp, gag) is analyzed. Each measurement of the expression of a gene for a given sample is carried out in triplicate (in three different wells).

In the same well are added:

- 2.5 µL of the "lightcycler 480 SYBR green II master mix" mix which contains DNA polymerase, SYBR green and dNTPs.

- 0.5 µL of the specific primers of the target gene to be amplified (1 µM final).

- 2 µL of cDNA. The cDNAs purified at the end of the reverse transcription step are used directly in the qPCR reaction.

The qPCR plate is centrifuged for 1 minute at 2000 g.

The fluorescence emitted during the qPCR reaction is measured by the LightCycler480 device (Roche). The cycle parameters used for qPCR are as follows:

5 min at 95 ° C

55 cycles: 10 sec denaturation at 95 ° C

10 sec hybridization at 60 ° C

10 sec elongation at 72 ° C

Measurement of the temperature at which 50% of the DNA is denatured at the end of the qPCR (Melting temperature).

4) Analysis of the qPCR

For the same sample, the expression of the target genes gag and asp is quantified relatively by the standard straight line method. Beforehand, ranges of dilutions of known concentrations were carried out for each gene (target genes and household genes). These ranges were made from amplagons of gag , asp , actin beta , rpl19 and hprt obtained from a line stably transduced by a molecular clone of HIV-1. These dilution ranges of known concentrations make it possible to determine the concentration of the target genes and of the household genes. The expression of the target genes is then normalized by the LightCycler 480 software with respect to that of the household genes. The result obtained is expressed without unit, and corresponds to the relative expression of gag and asp.

In each plate of qPCR, a reminder point of the dilution range of each gene is deposited.

The melting temperature (Tm) is analyzed for each gene in the samples and then compared to that obtained for the standard range. This step verifies the specificity of the amplified sequence.

The concentration of each gene is determined using standard lines for each sample, and the relative expression of the target genes is calculated.

FIG. 2 shows the quantification of the antisense viral transcription obtained with the procedure presented in Example 1 on cells isolated from a patient infected with HIV and receiving antiviral treatment (and therefore aviremic). It is found that in the cells of this individual, one can detect, from their isolation, a certain level of antisense transcription (J0), specific to each individual. During phase A, the cells are stimulated which leads to an awakening of the latent viruses. There is then a decrease in the antisense transcription (J5). During phase B, the cessation of cellular stimulation, and therefore of viral production, is associated with an increase in antisense transcription (D9).

FIG. 8 shows the quantification of the antisense viral transcription obtained with the procedure presented above on cells isolated from two distinct patients infected with HIV and receiving antiviral treatment (and therefore aviremic). It is found that in the cells of these individuals, it is possible to detect, as soon as they are isolated, a certain level of antisense transcription (J0), specific to each individual. In the cells of these stimulated patients (CD3 / CD28 + rhIL2) for 5 days in order to provoke the awakening of the latent viruses, the antisense viral transcription becomes undetectable.

Example 2: Protocol of the LIPS technique

Equipment used

1) Plasmid constructs

The LIPS experiments were carried out with a recombinant ASP protein produced in HEK 293T. The ASP coding sequence used is derived from the molecular clone HXB2 and is therefore of subtype B. This sequence has been optimized for the use of codons in eukaryotic cells, as described previously by Torresilla et al. (Torresilla et al., 2013). For the constructs coding for the luciferase-ASP (luci-ASP) and luciferase-p24 (luci-p24) fusion proteins, a Flag sequence (DYKDDDDK - SEQ ID NO: 21) was inserted upstream of the ORF of the luciferase. For clarity, the coding sequences Flag-luci-ASP and Flag-luci-p24 will be named luci-ASP and luci-p24 below. The luci-ASP and luci-p24 ORFs were inserted into the expression plasmid p-CAGGS, under the control of the chicken β-actin promoter and the enhancer of Cytomegalovirus (CMV) (Niwa et al., 1991).

2) Stamps

Lysis buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl, 1% Triton X-100, 50% glycerol and 2 tablets of protease inhibitors for 50 ml of buffer

Buffer A: 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. A stock solution of Tris at 500 mM pH 7.5 is produced. Buffer A is produced extemporaneously from this mother solution. Buffer A is filtered (0.2 µm) before use and renewed every 15 days. The pH of the Tris stock solution is checked before each use.

3) Reagents

- Sepharose beads coupled to A / G chimeric proteins (Protein A / G Ultralink resin, Thermo Scientific).

- Luciferase substrate (Promega).

LIPS protocol .

1) Transfection of constructions

The day before, HEK 293 T are seeded in a 6-well plate at a concentration of 1,106 cells / ml per well.

The next day, the HEKs are transfected with the following constructs: p-CAG-luci, p-CAG-luci-ASP or p-CAG-luci-p24.

Only one construction is transfected per well.

Three wells are transfected for each construction.

3µg of each construct is transfected. The 3 μg of DNA are added with 400 μL of NaCl and 9 μg of Polyethylenimine, incubated for 20 minutes at room temperature then added to the corresponding HEK wells.

The culture medium is renewed 5 to 6 hours after the transfection.

2) Cell collection and lysis

The culture medium is removed 48 hours post-transfection.

The cells are washed with 1X PBS.

For 3 wells transfected with the same construction: 700 μL of cold lysis buffer (released from -20 ° C at the last moment) are added to a well and the cells are peeled off using a cell scraper. The 700 μL of buffer and detached cells are added to the 2nd well and the cells of the 2nd well are removed with the cell scraper. This operation is repeated for the 3rd well.

The cell lysates obtained for each condition (700 μL of lysate correspond to 3 wells transfected with the same construction) are sonicated in ice. 3 pulses of 5 seconds spaced 10 seconds at medium power (about 30-40) are performed.

The lysates are then centrifuged 2 times 4 minutes at 12,500 rpm at 4 ° C.

At the end of this step, total cell lysates enriched in recombinant protein (luciferase, luciferase-ASP or luciferase-p24) are obtained.

3) Calculation of the number of LU (light units ) per µL of lysate (LU / µL)

Serial dilutions to 1 / 10th of each total lysate are made in the lysis buffer in a 96-well plate (50 μL final per dilution).

The serial dilutions made are placed in a plate with a white background and the luciferase substrate is added. The luminescence reading obtained, expressed in LU, is read by a luminometer.

A calibration line corresponding to the luminescence obtained as a function of the volume of cell lysate present in the well (pure lysate = 1 μL; lysate diluted to 1 / 10th = 0.1 μL) is carried out. The equation of this straight line makes it possible to calculate the number of LUs obtained after adding the luciferase substrate for 1 μL of each total lysate (luciferase, luci-ASP and luci-p24).

The total lysates are aliquoted and then placed at -80 ° C.

At the end of this step, the total volume of lysate necessary to obtain a luminescence of 10 ⁷ LU per well can be calculated.

4) Preparation of patient plasmas

Different plasma samples are used:

Plasmas from HIV-1 infected patients.

Plasmas from uninfected donors provided by the French Blood Establishment.

Plasmas from uninfected donors and infected patients are diluted 1 / 10th in buffer A containing. The manipulation of the plasmas of infected patients is done in a level 3 containment laboratory until the addition of buffer A which inactivates the virus possibly present in the plasma of infected patients.

Plasmas diluted 1 / 10th can be stored for one month at 4 ° C.

5) Realization of LIPS

Each plasma sample is analyzed in triplicate (in 3 different wells). The reaction is carried out in a 96-well plate. Three 96-well plates are successively used for the LIPS:

A transparent 96-well plate with a flat bottom (plate 1).

A transparent 96-well plate with conical bottom (plate 2; for washes).

A white 96-well plate with a flat bottom (plate 3; for reading the luminescence).

Use of the plate 1. In each well are added:

40 µL of buffer A

10 µL of plasma from non-infected patients or donors is added.

50 µL of total lysate containing luciferase alone or the luci-ASP or luci-p24 proteins, diluted in buffer A so as to obtain 1.10 ⁷ LU per well.

The plasma samples previously diluted 1/10 in buffer A are again diluted 1/10 in the well. In the end, the equivalent of 1µL of undiluted plasma is used in each well for an experiment.

Plasmas from uninfected patients or donors are incubated with the total lysate containing luciferase or the luci-ASP / luci-p4 fusion protein with stirring for 1 h at room temperature.

Sepharose beads coupled to A / G chimeric proteins are added to each well. The volume of beads added (1.5 μL per well) was calculated so as to allow immunoprecipitation of all the Immunoglobulins G present in 1 μL of human plasma. The bead resin used has a binding capacity greater than 20 mg of human immunoglobulins (Ig) G / mL of beads (Protein A / G Ultralink resin, Thermo Scientific). The beads and any luci-ASP / antibody complexes formed are incubated for 1 hour at room temperature.

The beads and any luci-ASP / antibody complexes formed are transferred to plate 2 to perform the washings. The washes make it possible to eliminate the luciferase, luci-ASP or luci-p24 proteins which would not have been complexed by antibodies and immuno-precipitated by the beads. Four washes with buffer A and two washes with PBS 1X are carried out. During each wash, the plate 2 is centrifuged for 2 min at 2000 g. After the last wash, the ball / antibody complexes are taken up in 50 μL of PBS 1X.

The ball / antibody complexes taken up in 50 μL of PBS 1X are transferred to plate 3 for reading the luminescence. The luciferase substrate is added to each well. The luminescence is read at 0.1 s on the luminometer.

For each experiment, several wells are used for positive and negative controls:

Negative control: A point deposited in triplicate corresponding to a healthy donor plasma.

Positive controls:

A dilution range of an antibody recognizing the antigen is added to an uninfected donor plasma.

A point deposited in triplicate corresponding to the total lysate before immunoprecipitation (INPUT).
Figure 1 illustrates the type of results that can be obtained with the LIPS method. It represents the measurement of anti-ASP antibodies in the plasma of non-infected subjects (n = 15) and infected subjects (n = 20). By establishing a positivity threshold allowing to have the best specificity (the minimum of false positives), we note that there is indeed in the infected patient the development of an anti-ASP antibody response and that it may present a variable level depending on the individual. In addition to FIG. 2, FIGS. 3 and 4 present results obtained during an experimental infection of stimulated CD4 + T lymphocytes and of dendritic cells isolated and differentiated from the same healthy subject then infected in vitro . In activated T lymphocytes, the inventors have shown that the antisense transcription is undetectable (Laverdure et al.) And therefore that the ASP protein is not produced. Infection of these cells with a virus incapable of producing ASP, by introduction of a STOP codon, 12 amino acids after the initiator codon, shows that in this context ASP does not logically play a role. Indeed, we see that the production of the mutant virus (black curve) can be superimposed on that of the wild virus (gray curve).

In dendritic cells, the inventors have shown that antisense transcription is particularly active. When these cells are infected with the mutant virus incapable of producing ASP, there is surprisingly an increased production of virus (FIG. 4, black curve) compared to that observed with the wild virus (gray curve).

These results support a role of the ASP protein in the control of viral production by the infected cell. That is to say, when it is produced, it results in a decrease or absence of viral production. The aim of this regulation could be either the avoidance of the immune response (to avoid the production of viral antigens which would be recognized) or the avoidance of the cytopathic effects associated with viral production and which lead to the death of the producer cell ( avoidance to keep the reservoir cell alive in this case).

All of these results are therefore in favor of the invention which proposes to use ASP, the anti-ASP response and antisense transcription as parameters for the evaluation of the viral reservoir.

Figure 5 shows the analysis of sense and antisense transcription in infected cells of the U1 line.

The cells of the U1 line are a subclone of U937 cells (premonocytic cell originating from a histiocytic lymphoma) which have been chronically infected with HIV. These cells have very low constitutive production of the virus and are used as a latency model. Stimulation of cells helps induce viral production. For the experiment which made it possible to obtain the results of FIG. 5, 1.5 million cells were stimulated either with PMA (phorbol myristate acetate) at the concentration of 15 ng / ml, or rhTNF-alpha at the concentration of 2 ng / ml is unstimulated (latent U1) for 4 hours before harvesting the cells before the RNAs are analyzed according to the protocol of Example 1. After quantification of the sense RNA (Gag transcript) and antisense (ASP transcript) , the variation of the ratio of these transcripts was evaluated by taking as a reference the ratio obtained in the non-stimulated cells. This result shows that when the virus integrated into this cell line passes from a latent state to a productive state thanks to the stimulation of the PKA of the cells by PMA or to a stimulation by a pro-inflammatory cytokine, the viral transcription is strongly unbalanced to the meaning transcription. There is therefore an equilibrium state of the two latent viral transcripts and during the production of the virus the antisense transcription will be disadvantaged.

Figures 6 and 7 show the analysis of viral transcription in cell lines infected either productively or chronically with HIV.

Cells of the Jurkat lymphocyte line were infected with HIV virus strain NLAD8 and then, after monitoring the productivity of the infection, the cells were recovered. In parallel, cells chronically infected with the virus, models for studying viral latency, were recovered: J-lat cells which are cells chronically infected with an HIV virus of strain R7, less and containing GFP and U1 cells, a subclone of U937 cells (premonocytic cell originating from a histiocytic lymphoma) which have been chronically infected with HIV of the LAV strain. After extraction of the RNAs, the sense and antisense viral transcripts were analyzed as described in Example 1. If we consider the ratio of antisense / sense transcription in infected cells in a productive manner (FIG. 6: JK) we note that this ratio is increased when the virus is latent (Figure 6: J-lat and U1) compared to the situation when viral production is effective. To obtain FIG. 7, the previous results were obtained using, as for FIG. 5, the ratio obtained in the unstimulated U1 cells is taken as a reference. It can then be seen that, as the reactivation of viral production in chronically infected cells, productive infection of a lymphocyte line is associated with an increase in sense viral transcription compared to antisense transcription, the latter being more favorably present in cells where the virion is latent, or at the very least produced at a very low level.

The invention is not limited to the embodiments presented and other embodiments will be apparent to those skilled in the art.

Claims (9)

1. Method for in vitro identification of reservoir cells of a retrovirus responsible for immunodeficiency in mammals, from a biological sample of said mammals having been treated with at least one anti retroviral agent, said method comprising:
   a step of determining the presence or absence, or also of measuring the amount of a compound chosen from:
   o the anti-sense transcript encoding the anti-sense protein or asp ,
   o the ASP protein, and
   o an antibody specific for the ASP protein,
   in said biological sample of said mammals having been treated with at least one anti retroviral agent,
   a step of comparing said presence or absence of said compound, or again of comparing the measured quantity of said compound in the previous step, with reference samples from non-infected patients for the reference of negativity and from non-infected patients supplemented by the synthetic reference standard corresponding to the method used for the positivity reference, and
   a step of determining the presence of reservoir cells in said biological sample if
   o said compound is present while it is absent in the reference sample, or
   o the quantity of said compound is at least equal to the threshold defined by the statistical analysis of negative samples for each of the methods times the quantity observed.
2. The method according to claim 1, wherein said ASP protein is the ASP protein of the human immunodeficiency virus, in particular of the human immunodeficiency virus group -1.
3. A method according to claim 1 or claim 2, wherein the biological sample is a blood, serum or plasma sample, and wherein said compound is an antibody specific for the ASP protein.
4. A method according to claim 1 or claim 2, wherein the biological sample is a cell sample, and wherein said compound is the transcript encoding the ASP protein or the ASP protein.
5. Method according to any one of claims 1 to 4, in which the ASP protein corresponds to the protein, encoded by the gene whose identifier is: Gene ID: 19424028.
6. Method for determining in vitro the efficacy in a biological sample of an anti retroviral therapy on the viral reservoir of mammals infected with a retrovirus responsible for human immunodeficiency, said method comprising:
   a step of determining the presence or absence, or even of measuring the quantity, of a compound chosen from:
   o the antisense transcript encoding the anti sense protein or ASP,
   o the ASP protein, or
   o an antibody specific for the ASP protein,
   in said biological sample,
   a step of comparing said presence or absence of said compound, or again of comparing the measured quantity of said compound in the previous step, with reference samples from non-infected patients, for the reference of negativity, and from patients uninfected complemented by the synthetic reference standard corresponding to the method used for the positivity reference, and
   - a step of determining the effectiveness of said antiretroviral therapy if
   o said compound is absent after anti retroviral therapy while it was present in the reference sample, or
   o the quantity of said compound varies by at least 1.2 times the statistically significant precision limit of the method considered.
7. The method of claim 6, wherein said reference sample is a sample of said mammal before treatment with said antiretroviral therapy.
8. The method according to claim 6 or claim 7, wherein said ASP protein is the ASP protein of the human immunodeficiency virus, in particular of the human immunodeficiency virus group -1.
9. Kit for the detection of retrovirus reservoir cells comprising:
   - means for detecting a compound chosen from
   o the anti sense transcript encoding the anti sense protein or ASP
   o the ASP protein, or
   o an antibody specific for the ASP protein, and
   - at least one biological reference sample.

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