Patent Application
20210254165
The present invention relates to a method for determining in vitro or ex vivo the immune status of an individual, preferably a patient, comprising a step of detecting and/or quantifying the expression, in a biological sample of said individual, of one or several sequences of HERV/MaLR, as well as the tools for implementing it and the uses thereof.
The immune system is a system for defending the body against what is recognized as non-self, such as pathogens (e.g. viruses, bacteria, parasites). In mammals, there are two main types of mechanisms: a non-specific defense mechanism, also called «innate» or «natural» immunity, and a specific defense mechanism, also called «acquired» or «adaptive» immunity.
These immune responses require very fine regulation. In a healthy individual, the immune response will be qualified as «normal» (we can also speak of immune status of immunocompetence). However, the immune response can sometimes be impaired. We will talk about inflammation status or hyperactive immune status when the immune system is more active than normal, as in the case of inflammatory or autoimmune diseases. In autoimmune diseases, the immune system of the body triggers an inflammatory response with characterized immunization against antigens of the self. Conversely, we will talk about immunosuppression status (or immunodepression or immunodeficiency or hypoactive immune status or immune paralysis), when the immune system is less active than normal.
The immunosuppression can have various origins, take many forms, and affect innate immunity and/or adaptive immunity. Particularly, the sepsis is a major public health problem, which is the leading cause of death in intensive care units. It is estimated that 28 million people develop sepsis each year worldwide, among which 8 million will die of the pathology (Fleischmann et al. (2016) American journal of respiratory and critical care medicine; 193(3): 259-72). In a patient with sepsis (also known as in a septic state), the immune response is deregulated, following an infection, leading to multiple and potentially fatal organ failure and dysfunction. This immune response is complex and evolves over time, with excessive pro-inflammatory and anti-inflammatory phenomena which may be concomitant. All of these immune system disorders lead to organ failure, immune system paralysis, and secondary infections. The septic shock is a subtype of sepsis, in which hypotension persists despite adequate vascular filling (Singer et al (2016) JAMA; 315 (8): 801-810). In the initial stage of sepsis, an inflammatory or even hyper-inflammatory response seems to predominate, which is the cause of tissue damage and organ failure, in particular in the kidney. This is why clinical trials in the field of sepsis have long focused on anti-inflammatory treatments, but with inconclusive results. More recent studies on the pathophysiology of sepsis have shown that an anti-inflammatory or immunosuppression response occurs in some patients in septic state, either concomitantly with the initial inflammation or later. The patient may then be in a state of immunosuppression, which may potentially be severe, depending on the respective degrees of pro-inflammatory and anti-inflammatory responses. These immunodepressed patients present a high risk of developing nosocomial infections (or HAI, Health-care Associated Infections), and could advantageously benefit from immunostimulatory treatments.
It therefore appears important to be able to determine the immune status of an individual, and particularly to be able to identify an immunosuppression status, in order to be able to adapt the therapeutic management. Yet, individuals with immune system disorders do not present specific clinical signs. There is therefore a significant need for the identification of biomarkers, which make it possible to determine the immune status of an individual.
Currently, the reference test for monitoring immune alterations in intensive care patients (e.g. patients with sepsis, trauma, major surgery, burns, or patients with pancreatitis) is the decreased expression of the HLA-DR (human leukocyte antigen—D related) on the surface of monocytes (mHLA-DR), measured by flow cytometry. Indeed, this marker provides valuable information in terms of prediction of mortality or the assessment of the secondary infections risk in these patients. The HLA-DR is a surface receptor belonging to the MHC (major histocompatibility complex) class II. In particular, the measurement of the mHLA-DR expression represents the gold standard for identifying whether a patient with sepsis is immunodepressed or not (Monneret and Venet (2016) Cytometry Part B (Clinical Cytometry) 90B: 376-386).
However, this approach requires extensive pre-analytical sample manipulation (Monneret and Venet (2014) HLA-DR monocyte in sepsis: shall we stop following the flow? Crit Care 18: 102). Moreover, the accessibility to a flow cytometer is not always possible in all hospitals, and the measurement is difficult to standardize from one hospital to another, or even from one operator to another.
To overcome these drawbacks, other biomarkers, using molecular biology tools, have been proposed, such as for example a biomarker based on the ratof the expression level, at the mRNA level, of CD74 on day D3 (following admission of the patient within a medical facility) on the CD74 expression level on day D1. The CD74 represents the invariant chain γ of HLA-DR. The CD74 D3/D1 expression ratio has been demonstrated to be associated with the onset of secondary infections acquired in intensive care (Peronnet et al (2017) Intensive Care Medicine; 43(7): 1013-20). The patent application W02012/101387 describes a method for determining the immune status of an individual, from the analysis of the expression of at least two genes selected from several groups of genes. It has also been proposed, in the patent application W02013/156627, a method for determining the immunodepressed or non-immunodepressed status, from the determination of the anellovirus load, in a biological sample. However, none of these biomarkers have yet come to replace the use of mHLA-DR. These biomarkers have the particular drawback of not making it possible to identify in which phase the patient is (i.e. inflammatory phase versus immunosuppressive phase), their objective being primarily to be able to identify immunodepressed patients, for whom it would be relevant to administer immunostimulant treatments.
At the date of the present invention, it therefore remains necessary to find new biomarkers, which make it possible to determine the immune status of an individual.
The endogenous retroviruses, or ERVs (for Endogenous Retro Virus) designate stable sequences of the genome of an organism and having structural analogies with certain infectious exogenous retroviruses (including the presence of two LTRs, or Long Terminal Repeats, which surround the genes encoding for putative proteins). Their origin is uncertain, but the most likely hypothesis is that of germ cell infection with a retrovirus. Following mutations in the retrovirus which would have made it defective, the infected germ cells could have survived, and the genome of the retrovirus, integrated into the genome of the organism, could have been transmitted to the next generation, and persist in the offspring within the genome of the organism.
In humans, HERVs (Human Endogenous Retro Viruses) have only been demonstrated since the sequencing of the human genome. Together with MaLRs (Mammalian apparent LTR-Retrotransposons), which have a similar structure to HERVs, they represent 8.3% of the human genome, with a number of over 400,000 elements. By comparison, the 30,000 to 40,000 genes encoding proteins represent only 2% of human DNA. HERVs are subdivided into three major classes (I, II and III) and several groups (sometimes called «families» in the present patent application). HERVs are retroelements that are transposed only by a copy and paste mode, through an RNA intermediate and reverse transcriptase. They have long been considered as «junk DNA». While they may be inactive due to mutations or through epigenetic mechanisms, their role begins to appear, both in physiological and pathological contexts. Thus, it has been shown that HERV-W participates in one of the mechanisms ensuring the formation of the placenta. The HERV-K superfamily is the most studied in relation to carcinogenesis. The expression of certain HERVs has also been described in certain autoimmune diseases, such as multiple sclerosis or lupus erythematosus, and in interferonopathies, without any suggestion of a possible link between reactivation of HERVs and the immune status.
Thus, it has never been described or suggested that analyzing the expression of HERV in humans could be useful to determine the immune status of an individual. Furthermore, the reactivation of HERV has never been described in the pathology of sepsis.
Yet, it was discovered that, completely unexpectedly, among the approximately 420,000 existing HERV/MaLR, the analysis of the expression of some of them made it possible to determine the immune status of an individual.
Thus, the present invention relates to a method for determining in vitro or ex vivo the immune status of an individual, preferably a patient, comprising a step of detecting and/or quantifying the expression, in a biological sample of said individual (or biological test sample), of at least a part, preferably of a size of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 nucleotides, of at least one HERV/MaLR sequence selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99%, preferably at least 99.1%, preferably at least 99.2%, preferably at least 99.3%, preferably at least 99.4%, preferably at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9% of identity with one of the sequences identified in SEQ Nos: 1 to 34, from the following lists:
In the context of the present invention:
To the knowledge of the inventors, it has never been described or suggested that the detection and/or the quantification of the expression of HERV/MaLR could make it possible to determine the immune status of an individual. Particularly, the involvement of HERV/MaLR has never been described in the field of sepsis.
All of the HERV/MaLRs of SEQ ID NO: 1 to 34 have been identified among the approximately 420,000 HERV/MaLRs in the genome. These are HERV/MaLR that we can target with the HERV-V3 chip (Becker et al, BMC Genomics. 2017; 18: 286). More particularly, these HERV/MaLR are expressed in the data sets used in the Examples. Even more particularly, these expressed HERV/MaLR are modulated between the conditions of interest retained in the Examples.
The detection and/or the quantification of the expression of the HERVs according to the present invention, to determine the immune status of an individual, can be carried out by means of molecular tools which have advantages relative to the use of flow cytometry for the measurement of mHLA-DR, in terms of accessibility within hospitals and standardization. Little sample manipulation is required and the results are easy to interpret. Moreover, the expression of HERVs according to the present invention can be detected and/or quantified on several types of platforms, such as DNA chips or by PCR, in order to determine the immune status. Some of the HERVs according to the present invention can be detected early, from D1, while the measurement of mHLA-DR is carried out on D3 (Monneret and Venet (2014) Monocyte HLA-DR in sepsis: shall we stop following the flow? Crit Care 18: 102).
Preferably, the present invention relates to a method for determining in vitro or ex vivo the immune status of an individual, preferably a patient, comprising:
The reference biological sample can be of various natures, but it is preferably identical in nature, or at least similar in nature, to the nature of the test biological sample. For example, if the test biological sample is a whole blood sample, the reference biological sample will preferably be a whole blood sample, or possibly a plasma or serum sample. The biological reference sample can be a «natural» sample, that is to say from an individual whose immune status is known or determined according to a reference method (for example, by the mHLA-DR method). For example, it can be from an individual with an immune status known to be an immunocompetent status, an inflammation status, or an immunosuppression status. Preferably, if the test biological sample is from a human, the reference biological sample is also from a human. Even more preferably, the reference biological sample comes from the same individual as that from which the test biological sample comes. The reference biological sample can also be a «synthetic» sample, that is to say a sample containing a calibrated amount of at least one of the sequences SEQ ID NO: 1 to 34.
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the expression of at least 2 different sequences selected from the sequences identified in SEQ. Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34 is detected and/or quantified. The lists 1 to 4 being complementary to each other, these at least two different sequences are preferably selected in two different lists.
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the expression of at least 3 different sequences selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34 is detected and/or quantified. These at least three different sequences are preferably selected from two different lists, more preferably from three different lists.
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the expression of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34 different sequences selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34 is detected and/or quantified. These at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32 , at least 33, at least 34 different sequences are preferably selected from two different lists, more preferably from three different lists, even more preferably from four different lists.
The HERVs of SEQ ID NO: 1, 3 and 8 were identified from two different strategies, as described in the Examples, and are thus found in two Lists respectively. Moreover, the inventors assigned a rating for the different sequences of HERV, as explained in Examples 4 to 6. In Example 7, the HERVs were ranked in order of importance. Also, quite preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 different sequences selected from the sequences identified in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 28, or from the sequences which exhibit at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity with one of the sequences SEQ ID NO: 1, 3, 8, 11, 12, 13 and 28 is detected and/or quantified. Preferred combinations of at least 2 corresponding HERVs are listed in Table 5 below.
The method for determining in vitro or ex vivo the immune status of an individual, as described above, according to any embodiment, can also comprise a step of detecting and/or quantifying the expression, in the test biological sample, of at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 , at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49 , at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56 , at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95 gene(s) selected from the following genes:
CD74, CX3CR1, IL-10, S100A8, S100A9, MERTK, CLEC7A, CD36, TIMP2, CCL13, PTGS2, IL-12B, IL-6, IL-1A, CCL20, MX1, OAS-1, CCL15, OAS-3, EIF2AK2, IFNγ, NEFH, MMP10, SERPINB2, THBD, STAT1, CCR4, HLA-DRB1/B3, TCF7, EOMES, BCL11B, ITGA7, IL-18R1, NLRC4, CYP1B1, HGF, IL-SRA, CCLP4, CD3G, CD4OLG, CD3D, CD127, ICOS, IL-1R2, IL-1RN, IL-18, IL-18RAP, OX4OL, PD-1, PD-L1, Zonulin (HP), BTLA, C3AR1, CD154, GM-CSF, IFIH1 , IL-15, MCP1, PCSK9, STAT4, LTR82B, CIITA, LILRB2, CD177, ADGRE3, FLT-1, CD64, TREM-1, TNF-α, IL-1β, ALOX5, IL-17A, NFκB, TBX21, HIF1a , RORgT, OAS-2, GNLY, CTLA-4, TIM 3, CD274, IL-2, IL-7R, GATA3, CXCL10, FAS, GSN, MDC1, DYRK2, TDRD9, CNB1 IP1, ZAP70 and ARL14EP.
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the expression is detected and/or quantified at the RNA transcript level or messenger RNA (mRNA). The detection and/or quantification at the RNA transcript or mRNA level can be carried out by any means known to one skilled in the art. Particularly, it can be cited as examples:
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the immune status is determined as being an immunosuppression status (or immunodepression or immunodeficiency status or hypoactive immune status or immune paralysis), a normal immune status (or immunocompetence status), or inflammation status (or hyperactive immune status). The inflammation status includes the hyper-inflammation status.
Preferably, the invention relates to a method for determining the immune status of an individual, as described above, in which the individual is a patient admitted within a medical facility, preferably in an intensive care unit, in emergency department or in resuscitation. Also preferably, the individual is a trauma patient, a patient with burns, a surgical patient or a patient with sepsis, preferably a patient with septic shock. Even more preferably, the test biological sample is obtained by sampling made within 10 days, preferably within 9 days, preferably within 8 days, preferably within 7 days, preferably within 6 days, preferably within 5 days, preferably within 4 days, preferably within 3 days, preferably within 2 days, preferably within 24 hours, following admission to the medical facility.
Preferentially, the invention relates to a method for determining the immune status of an individual, as described above, in which the reference biological sample is a biological sample obtained from a healthy individual, preferably a biological sample obtained from the same individual from which the test biological sample was obtained but collected before infection or aggression, or a biological sample from an individual of known immune status, preferably with inflammation status, normal immune status, or an immunosuppression status.
Preferably, the test biological sample and/or the reference biological sample, as used in the method for determining the immune status of an individual, according to the invention, as described above, is a blood sample, preferably a sample of whole blood, plasma or serum, or a sample of peripheral blood mononuclear cells, extracted from a blood sample.
Preferably, the method for determining the immune status according to the invention, as described above, comprises a step of administering a treatment, preferably an immunomodulatory treatment, adapted to the immune status of the individual. Preferably, the immunomodulatory treatment is an immunostimulatory treatment, if it is determined that the individual has an immunosuppression status, or an anti-inflammatory treatment, if it is determined that the individual has an inflammatory status. Among the immunostimulant treatments which can be selected, mention may be made, by way of example, of the group of interleukins, in particular IL-7, IL-15 or IL-3, growth factors, in particular GM-CSF, interferons, in particular IFNγ, Toll agonists, antibodies, in particular anti-PD1, anti-PDL1, anti-LAG3, anti-TIM3, anti-IL-10 or anti-CTLA4 antibodies, transferrins and inhibitory molecules of apoptosis, FLT3L, Thymosin al , adrenergic antagonists. Among the anti-inflammatory treatments, mention may in particular be made of the group of glucocorticoids, cytostatic agents, molecules acting on immunophilins and cytokines, molecules which block the IL-1 receptor and anti-TNF treatments.
The present invention also relates to the use of at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34 sequence(s) selected from the sequences identified in SEQ Nos: 1 to 34, as appearing in Lists 1 to 4, or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34, to determine in vitro or ex vivo the immune status of an individual, preferably a patient.
The present invention also relates to a method for identifying or selecting a treatment, preferably an immunomodulatory treatment, more particularly an immunostimulating treatment or an anti-inflammatory treatment, suitable for treating an individual, preferably a patient, comprising the following steps of:
a. determining the immune status of said individual by a method as previously described
b. identifying an appropriate treatment from the immune status determined in step a)
Preferably, the immunomodulatory treatment is an immunostimulatory treatment, if it is determined that the individual has an immunosuppression status, or an anti-inflammatory treatment, if it is determined that the individual has an inflammation status.
The present invention further relates to a method for evaluating the effectiveness of a treatment, preferably an immunomodulatory treatment, more particularly an immunostimulating treatment or an anti-inflammatory treatment, on an individual, preferably a patient, comprising the following steps of:
a. detecting and/or quantifying the expression of at least part of a sequence selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the identified sequences in SEQ Nos: 1 to 34, in a first biological sample of said individual, collected before the treatment, and in a second biological sample of said individual, collected after the treatment
b. comparing the expression obtained for the 2 biological samples in step a)
c. evaluating the effectiveness of the treatment from the comparison of step b)
Another subject of the present invention concerns an amplification primer comprising, or consisting of, a nucleotide sequence complementary to at least part of a sequence selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34, and the sequences complementary to these sequences. Preferably, the amplification primer according to the invention is selected from the primers presented in Table 6.
The present invention also concerns a pair of amplification primers, consisting of two amplification primers selected from the primers as described above, and making it possible to amplify, preferably making it possible to specifically amplify, at least part of a sequence selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34, and the sequences complementary to these sequences. Preferably, the pair of amplification primers according to the invention is selected from the pairs of primers presented in Table 7.
Another subject of the present invention concerns a hybridization probe, whose nucleotide sequence comprises, or consists of, a nucleotide sequence complementary to at least part of a sequence selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which have at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34, and the sequences complementary to these sequences. Preferably, the hybridization probe according to the invention is selected from the hybridization probes presented in Table 8.
Another subject of the invention is the use of at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least 25, preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32, preferably at least 33, preferably at least 34 pairs of primers according to the invention, as described above,
and/or at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8 , preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, of preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least less 25, preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32, preferably at least 33, preferably at least 34 hybridization probes according to the invention, as described above,
to determine in vitro or ex vivo the immune status of an individual, preferably a patient.
Another subject of the invention is a method for determining in vitro or ex vivo the immune status of an individual, as described above, in which at least one, preferably at least 2, preferably at least 3, of preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least 25, preferably at least 26, preferably at least 27, preferably at least 28, of preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32, preferably at least 33, preferably at least 34 pairs of primers as described above;
and/or at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, of preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least 25 , preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32, preferably at least 33, of preferably at least 34 hybridization probes as described above, are used.
Another subject of the invention is a kit comprising means for amplifying and/or detecting at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5 , preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, of preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least 25, preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30 , preferably at least 31, preferably at least 32, preferably at least 33, preferably at least 34 sequences selected from the sequences identified in SEQ Nos: 1 to 34 or from the sequences which exhibit at least 99% identity with one of the sequences identified in SEQ Nos: 1 to 34.
Preferably, in the kit according to the invention:
the amplification means comprise, preferably consist of at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least at least 24, preferably at least 25, preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32 , preferably at least 33, preferably at least 34 pairs of primers as described above;
and/or the detection means comprise, preferably consist of, at least one, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably at least 13, preferably at least 14, preferably at least 15, preferably at least 16, preferably at least 17, preferably at least 18, preferably at least 19, preferably at least 20, preferably at least 21, preferably at least 22, preferably at least 23, preferably at least 24, preferably at least 25, preferably at least 26, preferably at least 27, preferably at least 28, preferably at least 29, preferably at least 30, preferably at least 31, preferably at least 32, preferably at least 33, preferably at least 34 hybridization probes such as described above.
Also preferably, the kit according to the present invention further comprises means for amplifying and/or detecting other biomarkers, particularly endogenous biomarkers (or loci), such as other HERV/MaLR and/or genes, preferably genes involved in the inflammation and/or the immunity, and/or housekeeping genes, and/or exogenous biomarkers, such as viruses. Among the genes involved in the immunity, the following genes may be mentioned in particular, of particular interest:
CD74, CX3CR1, IL-10, S100A8, S100A9, MERTK, CLEC7A, CD36, TIMP2, CCL13, PTGS2, IL-12B, IL-6, IL-1A, CCL20, MX1, OAS-1, CCL15, OAS-3, EIF2AK2, IFNγ, NEFH, MMP10, SERPINB2, THBD, STAT1, CCR4, HLA-DRB1/B3, TCF7, EOMES, BCL11B, ITGA7, IL-18R1, NLRC4, CYP1B1, HGF, IL-SRA, CCLP4, CD3G, CD4OLG, CD3D, CD127, ICOS IL-1R2, IL-1RN, IL-18, IL-18RAP, OX4OL, PD-1, PD-L1, Zonulin (HP), BTLA, C3AR1, CD154, GM-CSF, IFIH1, IL-15, MCP1, PCSK9, STAT4, LTR82B CIITA, LILRB2, CD177, ADGRE3, FLT-1, CD64, TREM-1, TNF-α, IL-1β, ALOXS, IL-17A, NFκB, TBX21, HIF1a, RORgT , OAS-2, GNLY, CTLA-4, TIM 3, CD274, IL-2, IL-7R, GATA3, CXCL10, FAS, GSN, MDC1, DYRK2, TDRD9, CNB1 IP1, ZAP70 and ARL14EP.
Even more preferably, the kit according to the present invention comprises means for amplifying and/or detecting at most 100, preferably at most 90, preferably at most 80, preferably at most 70, preferably at most. plus 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10 biomarkers, in total.
The present invention also concerns the use of a kit as described above, for determining in vitro or ex vivo the immune status of an individual, preferably a patient.
SEQ ID NO 6 (F), by PBMCs, taken from 5 healthy volunteers, stimulated by LPS and quantified by biochips after the completion of the endotoxin tolerance model. The y-axis represents the fluorescence intensity of each of the hybridization probes: (A) 207113_s_at for the TNF alpha, (B) 207433_at for the IL-10, (C) 190665001-HERV0376uL_at for SEQ ID NO 1, (D) 121601801-HERV0492uL_at for SEQ ID NO 4, (E) 011052702-MALR1044uL_at for SEQ ID NO 5 and (F) 011052202-HERV1033uL_at for SEQ ID NO 6. The three conditions are represented, NS for the negative controls (without stimulation), LPS for cells stimulated once with 100 ng/ml of LPS and ET for cells subjected to two stimulations with LPS (2 ng/ml then 100 ng/ml).
The present invention is illustrated in a non-limiting manner by the following examples.
The endotoxin tolerance corresponds to a temporary state of the inability of a cell or organism to respond to endotoxin stimulation, resulting from a first endotoxin stimulation.
The endotoxin tolerance model was set up to mimic on the one hand an inflammatory context induced by a stimulation by lipopolysaccharide (LPS) and on the other hand the monocytic anergy which represents a state of «no response» of the cells. These components of immunity are found in different types of patients, such as patients suffering from sepsis, trauma, burns or who have undergone major surgery.
Materials and Methods
PBMC Stimulation
The endotoxin tolerance model was established from 5 bags of citrated blood from healthy volunteers collected at the EFS in accordance with standard procedures for blood donation, and used immediately after receipt. PBMCs, peripheral blood mononuclear cells, are isolated using a density gradient adjusted to 2 million cells per mL and cultured in X-Vivo medium (Lonza) at 37° C. and 5% CO2. The endotoxin used in this model is the lipopolysaccharide (LPS), which is a major component of the outer membrane of Gram negative bacteria. The LPS is obtained from a mixture of three strains of Escherichia coli: 0111: B4, 055: B5 and 0127: B8 (Sigma). All of the conditions are carried out in biological triplicates. In this ex-vivo model of endotoxin tolerance, the PBMCs are first cultured for 15 hours without (NS control cells and LPS cells mimicking the inflammatory condition) or with a dose of 2 ng/ml of LPS (ET cells mimicking monocytic anergy, immunosuppression condition). After a washing step, the PBMCs are incubated a second time for 6 hours without (NS control cells) or with a dose of 100 ng/ml of LPS (LPS and ET cells).
At the end of the experiments, the culture supernatants are harvested and stored at −80° C. The cells are also harvested, lysed and stored at −80° C. before performing RNA extraction (Qiagen commercial kit) for the transcriptomic study. To validate the efficiency of the model, the concentrations of the pro-inflammatory cytokine TNF-α («tolerizable gene») and of the anti-inflammatory cytokine IL10 («non-tolerizable gene») are determined from the culture supernatants of PBMC by ELISA (commercial R&D System kits).
RNA Extraction and Amplification
The mRNAs are extracted using commercial kits (RNeasy Mini Plus kit, QIAGEN) from the ex vivo cultures of PBMCs described above.
The total mRNAs are then assayed and characterized. The characterization of the mRNA is carried out by capillary electrophoresis using the Bioanalyser 2100. The quality of the mRNA samples is evaluated by the calculation of the RIN (RNA Integrity Number). This value is based on the detection of 18S and 28S ribosomal RNAs, if the RIN tends towards 10 it means that the mRNA is intact (a RIN 7 is accepted).
The synthesis of the complementary DNA (cDNA) and the amplification steps are carried out by a linear and isothermal method described in 2005 by the company NuGEN Technologies (Kum N et al. Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications. Clinical chemistry. 2005; 51 (10): 1973-81). The Ribo-SPIA amplification process is used (Watson JD et al. Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system. BMC genomics. 2008;9:84) from 16 ng of Total RNA (WTO pico commercial kit, Nugen) and consists of three steps. The first step is the production of the first strand of cDNA by reverse transcription from an mRNA template, this by using a mixture of random primers and oligo-dT. The second step, consists in adding DNA polymerase to the reaction, which induces the production of the second strand of cDNA. The third step, involves the SPIA amplification by strand displacement. Hybrid DNA/RNA primers are degraded by the RNAse H activity of DNA polymerase when complexed with the cDNA template. Single-stranded DNA synthesis (complementary to the mRNA template) is initiated and continues, authorizing new SPIA primers to be fixed to the cDNA template, thus sustaining the repetitive process of strand synthesis. The cDNAs are then fragmented into fragments of 50-200 bp using a DNase from 5 μg of purified and amplified DNA (commercial kit, Nugen) and are also labeled in 3′ (Nugen commercial kit). The amplification and fragmentation of the cDNA are verified on the Bioanalyser. The amplification profile spans a cDNA size range from 25 to 4000 nucleotides, with a peak around 1500 pb. The fragmentation profile should be centered on a nucleic acid population around 100 nucleotides in size, which is recommended for hybridization on an Affymetrix microarray.
Analysis by Biochip
The identification of sequences exhibiting an expression differential is based on the design and the use of a high density DNA chip in GeneChip format, called
HERV-V3, designed by the inventors and whose manufacture has been subcontracted to the Affymetrix company. This chip contains probes that are hybridized to distinct HERV sequences within the human genome. These sequences are extracted from a database specific to the inventors, already published (Becker et al. A comprehensive hybridization model allows whole HERV transcriptome profiling using high density microarray, BMC Genomics 2017 18: 286).
The HERV-V3 chip targets 353,994 HERV/MaLR elements, and over 1,500 immunity genes.
Once the cDNAs have been amplified and fragmented, they can be hybridized on the HERV-V3 chip, in an oven at 50° C. for 18 hours with constant stirring at 60 rpm for hybridization. A fluidics system allows automating the washing and coloring steps, and finally after all of these steps, the chip is read using a fluorometric scanner.
The raw dataset is created from the aggregation of the CEL files of each chip by traditional Affymetrix methods.
After a first quality control of the raw data, several steps are carried out: correcting the background noise by the Robust Multi-array Average (RMA) method, standardizing the data of each chip by quantiles, grouping the probe data into sets of probes (probes in probesets) and smoothing of the median. A second step of quality control is carried out. All these steps thus make it possible to obtain a matrix containing the normalized data.
The pre-treatment of the chips as well as the statistical analysis are carried out by using R/Bioconductor.
A pre-analysis step consists on evaluating the quality of the chips, before and after normalization. For this, several criteria must be taken into account: the quality of the RNA, the amplification and fragmentation controls of the cDNA, the image of the chips produced after the scan, the hybridization of the Affymetrix controls, the signal intensity (before and after normalization), probeset homogeneity (RLE and NUSE plots), chips correlation (before and after normalization) and principal component analysis. For all of these criteria, a statistical analysis allows identifying the extreme values for each chip and the data are then grouped together. Chips that pass less than 5 quality controls are removed from the analysis. For each data set, a decision table is then produced to summarize all the quality criteria, and quickly identify the chips to be removed from the analysis.
Sometimes it is necessary to have a corrective method for the analysis of data. This so-called COMBAT correction (for Combining BATches) makes it possible to correct the technical variability of the dataset and thus bring out its biological variability.
Finally, a data filter step was performed to reduce the data set and gain statistical power for the analyses. The intensity threshold was defined as the minimum intensity value for which the 75th percentile of the distribution of the coefficients of variation is below 10%. In this way, the intensity threshold is 25.5. The probesets below the intensity threshold in more than 68% of all samples (31 of 45 samples) are eliminated.
Among the 71,063 probesets targeting HERVs/MaLRs and the 42,560 probesets targeting genes selected in the previous step, a differential expression analysis was performed.
Studying the differential expression between two conditions is like calculating the expression rator Fold Change (FC). For example an expression value equal to 10 under condition A, and a value equal to 5 under condition B, the FC of NB is equal to 2. The FC data will be represented in log2 FC. To determine that a gene or a HERV/MaLR sequence is differentially expressed between two conditions, the Limma method was used (Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 2004, 3:Article3). Statistical tests and their associated p-value are calculated to evaluate the significance of the observed expression changes. The p-values were adjusted by controlling the false discovery rate (FDR, due to multiple tests) according to the method of Benjamini and Hochberg (Hochberg et al. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 1995, Vol. 57, No. 1 (1995), pp. 289-300). A probeset is considered to be significantly differentially expressed when the absolute value of FC in log2 is greater than 1 and the fitted p-value is below 0.05 (see
The sequences which were identified as being the most differentially expressed by the biochip were validated by RT-qPCR from the same samples as those which made it possible to produce the HERV-V3 chips.
Results
As indicated above, to validate the efficiency of the model, the concentrations of the pro-inflammatory cytokine TNF-α («tolerizable gene») and of the anti-inflammatory cytokine IL10 («non-tolerizable gene») are determined from the supernatants of PBMC culture by ELISA (commercial R&D System kits).
As shown in
These results allow validating the effectiveness of this model.
An analysis of the expression of HERV/MaIR and genes in unstimulated PBMC (NS, control), stimulated once with LPS (LPS, inflammatory condition) or twice stimulated with LPS (ET, immunosuppression condition) was made on the HERV-V3 biochip, designed by the inventors.
The processing of the data generated by the analysis of the HERV-V3 chips using this method made it possible to identify a set of 4 sets of probes (or «probesets»). These «probesets» are the most statistically differentially expressed among the sequences showing a statistically significant difference in expression between the different conditions (healthy patients, inflammatory condition and immunosuppression condition). As indicated previously, for the expression differential to be statistically significant, the absolute value of log2 Fold Change must be greater than or equal to 1 and the adjusted p-value must be less than or equal to 0.05. These criteria will apply for all examples. These 4 «probesets» are associated with HERV sequences identified by SEQ ID NO 1 and 4 to 6. The chromosomal location of each sequence is given in the GRCh38 reference. In Table 9, below, the list of identified sequences.
As observed in
The sequence SEQ ID NO 6, for its part, has greater expression under the NS and LPS conditions compared to the ET condition, with statistically significant differences between NS and ET, and between LPS and ET (
These results show a modulation of the expression of these sequences in the endotoxin tolerance model and thus their ability to be used as a marker of immune status.
The results also show that the sequences SEQ ID NO 4 to 6 have a «tolerizable» profile (inflammatory condition) in an endotoxin tolerance model while the sequence SEQ ID NO 1 has a «non-tolerizable» profile (immunosuppression condition) in an endotoxin tolerance model.
The results show that the same profile as that obtained on the biochips is observed for each identified sequence. Thus, the data obtained by the HERV-V3 biochip are confirmed by RT-qPCR (
Materials and Methods
Patients and Biological Samples
This retrospective observational study was conducted in patients aged 37 to 77 years (13 men, 7 women, median age: 59 years) admitted to intensive care following septic shock.
Whole blood samples were collected in PAXgene tubes (PreAnalytix) from these 20 patients in septic shock on days 1 (D1) and 3 or 4 (D3) after admission to intensive care, and were then stored (retrospective cohort).
Patients in this cohort were stratified according to the expression level of HLA-DR at the surface of monocytes (mHLA-DR). The expression of mHLA-DR was measured on D3 (days 3 or 4) by flow cytometry.
20 patients from this cohort were selected, 10 patients (50%) had high expression of HLA-DR on day 3 or 4 (more than 30% of expression), and 10 patients (50%) had a low expression of HLA-DR on day 3 or 4 (less than 30% of expression).
Patients with high expression of HLA-DR are considered to have immunocompetent status (DR+) and patients with low expression of HLA-DR are considered to have immunodepressed status (DR-).
5 healthy volunteers are also included in this study.
RNA Extraction
RNA extraction was performed using the PAXgene Blood RNA Kit (PreAnalytix) following the manufacturer recommendations. Before the RNA elution step, residual genomic DNA was removed by the action of DNAse. The RNA concentration was determined by fluorimetry (RNA assay kit on Qubit, Life
Technologies). The quality of the RNA was then controlled using the RNA 6000 Nano kit on a Bioanalyzer (Agilent Technologies), the collections with a RIN (RNA Integrity Number) greater than 6 being considered to be of good quality.
The steps of RNA amplification, biochip analysis and validations of sequences by RT-qPCR which were identified as being differentially expressed by the biochip are carried out as described in Example 1.
Results
The processing of the data generated by the analysis of the HERV-V3 chips using this method made it possible to identify a set of 6 sets of probes (or «probesets»). These «probesets» are the most statistically differentially expressed among the sequences showing a statistically significant difference in expression between the two conditions (immunocompetent condition and immunosuppression condition). These 6 «probesets» are associated with HERV sequences identified by SEQ ID NO 2, 3, 7, 8, 11 and 12. The chromosomal location of each sequence is given in the GRCh38 reference. In Table 10, below, the list of the 6 identified sequences.
As observed in
Thus, these results show the usefulness of the sequences SEQ ID NO 2 and 7 as markers, from D1, of immunosuppression. The sequences SEQ ID NO 3, 8, 11 and 12, for their part, are markers of immunosuppression at D3.
These sequences, identified as being differentially expressed by the biochip, were validated by RT-qPCR. The results are illustrated in
Profiles similar to those obtained on the biochips are observed for each identified sequence. Thus, the data obtained by the HERV-V3 biochip are confirmed by RT-qPCR.
Materials and Methods
Patients and Biological Samples
This retrospective observational study was conducted in patients admitted to resuscitation in 6 French hospitals from 2009 to 2011. The inclusion criteria were as follows:
The exclusion criteria were as follows:
Among all patients, 102 patients met the following criteria:
Then, the patients of this cohort were stratified according to the ratof the level of expression of CD74 on D3 to the level of expression of CD74 on D1, carried out by RT-qPCR.
The cohort was divided into 2 categories. People with a CD74 D3/D1 ratio greater than 1.23 are called «high» and are considered to have an immunocompetent status. Those with a CD74 D3/D1 ratof less than 1.23 are called «low» and are considered to have an immunodepressed status.
Among the 102 patients in this cohort, 52 patients (51° A) are considered to have an immunodepressed status («low» CD74 D3/D1 ratio) and 50 patients (49%) are considered to have an immunocompetent status («high» CD74 D3/D1 ratio).
The RNA extraction step is implemented as described in Example 2.
The steps of amplifying the RNA, analyzing by biochip and validating by RT-qPCR (on D1 and D3) the sequences which have been identified as being differentially expressed by the biochip are implemented as described in Example 1.
Results
The processing of the data generated by the analysis of the HERV-V3 chips using this method made it possible to identify a set of 2 sets of probes (or «probesets»). These «probesets» are the most statistically differentially expressed among the sequences showing a statistically significant difference in expression between the two conditions (immunocompetent condition and immunosuppression condition). These 2 «probesets» are associated with HERV sequences identified by SEQ ID NO 9 and 10. The chromosomal location of each sequence is given in the GRCh38 reference. In Table 11, below, the 2 identified sequences.
As observed in
Identically for the expression of the sequence SEQ ID NO 10 (
Thus, these results show the usefulness of these SEQ ID NOs 9 and 10 sequences as a marker of the immunosuppression state on D3.
These sequences, identified as being differentially expressed by the biochip, were validated by RT-qPCR on D1 and D3 from 30 patients out of the 102 of the cohort. The results are illustrated in
Profiles similar to those obtained on the biochips are observed for each identified sequence. Thus, the data obtained by the HERV-V3 biochip are confirmed by RT-qPCR from 30 patients.
For each of the sequences identified in Examples 1 to 3 (SEQ ID NO 1-12), the inventors have assigned a rating. This rating is based on the expression profiles, in chips, of the sequences SEQ ID NO 1 to 12 observed on the cohort of Example 2.
The inventors have assigned ratings ranging from 1 to 4 stars. All the criteria are visual, from graphs showing the expression of the different identified sequences.
As seen previously, all these sequences have already been selected on the basis of the level of expression and the differential of expression. The method of assigning ratings is described in Table 12, below.
Here in Table 13, below, the rating obtained for each of the sequences identified in Examples 1 to 3.
For the identification of markers specific to immunosuppression, the inventors used the data generated by the analysis of the HERV-V3 chips from the samples of Example 2.
Thus, the inventors selected the HERV sequences differentially expressed on D3 between the patients considered to be immunodepressed (DR−) and those considered to be immunocompetent (DR+), but which were not differentially expressed between the healthy volunteers of the study and all the patients (whether DR+or DR−, whether on D1 or D3).
This selection allowed the identification of 17 HERV sequences. For each of the identified sequences, the inventors assigned a score according to several criteria (expression profile, fold change, level of expression, consistency of the expression profiles between the sense and antisense probes, difference in expression visible from D1 and absence of too much variability between patients). Below Table 14 describes the mode of attributing points for each criterion.
Thus, thanks to this mode of attributing a score, the inventors selected 10 candidates and excluded 7 candidates whose performances seemed insufficient. Here in Table 15, below, the 10 markers identified with their respective score.
The sequences SEQ ID 13 to 18 and 19 to 21 are newly identified sequences. As for the sequence SEQ ID 8, this is a sequence which has already been identified in Example 2.
For the identification of specific markers of inflammation, the inventors used the data generated by the analyses of the HERV-V3 chips from the samples of Examples 1 and 2.
Thus, the inventors selected the HERV sequences on the one hand differentially expressed between the LPS condition (immunosuppression condition) and the NS condition (negative controls), in Example 1, and on the other hand differentially expressed between the patients on D1 or D3 compared to healthy volunteers, of Example 2.
This selection made it possible to identify 13 HERV sequences. As in Example 5, for each of the identified sequences, the inventors assigned a score based on the same criteria. The mode of attributing points for each criterion is described in Table 16, below.
Thanks to this mode of attributing a score, the inventors selected 7 candidates and excluded 6 candidates whose performances appeared to be insufficient. Here in Table 17, below, the 7 markers identified with their respective score.
This strategy for identifying inflammation marker made it possible to identify 6 new sequences (SEQ ID 22 to 27) and to find a sequence already identified in Example 1 (SEQ ID 1). It should also be noted that 2 sets of probes (probesets) target the same HERV sequence (SEQ ID 1).
For this example, the aim was to determine a signature of markers making it possible to best discriminate between patients considered to be immunodepressed and those considered to be immunocompetent. To do this, the inventors used the data generated by the analyses of the HERV-V3 chips from the samples of Examples 2 and 3.
The inventors selected the HERV sequences which were differentially expressed on D3 between the patients considered to be immunodepressed (DR-) and those considered to be immunocompetent (DR+). This selection allowed identifying a list of 193 HERV sequences.
In order to obtain a reduced signature still making it possible to best discriminate between patients considered to be immunodepressed and those considered to be immunocompetent from these 193 HERV sequences, the inventors applied the «Random Forests» method (Tin Kam Ho, Random Decision Forests, AT&T Bell Laboratories). This method makes it possible to classify each sequence according to its discriminating power between the two studied conditions (immunosuppressed and immunocompetent).
Then, to identify the optimal number of sequences in the signature, the inventors calculated the prediction performances on the cohort of example 3, by using the discriminating power for signatures of size ranging from 2 to 30 markers between the patients having a low CD74 ratbetween D3 and D1 and those with this same high ratio. Thus they determined that the signature of a size of 10 sequences had the best performances (best area under the curve: AUC) (cf.
In Table 18, below, the list of markers making up the signature making it possible to best discriminate between patients considered to be immunodepressed and patients considered to be immunocompetent.
It will be noted that this signature is composed of 8 sequences using 10 different sets of probes. Indeed, 3 sets of probes target the same sequence (SEQ ID NO 28). In addition, in this signature we find the sequence SEQ ID 3 already identified in Example 2. The HERV sequences appear in the table above in an order reflecting their «importance» in the stratification of patients according to their immune status, based on a score attributed to them by the Random forests classification algorithm.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18180991.4 | Jun 2018 | EP | regional |
| 18/58167 | Sep 2018 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/067330 | 6/28/2019 | WO | 00 |
