Process for the solubilization of membrane proteins

Abstract

The present invention pertains to biochemistry protocols, in particular methods for the solubilization of membrane proteins in a biological sample. The method according to the present invention is based on treating a biological sample with a combination of three specific reagents: a detergent, an amphipol and a lysis agent. This method can be directly applied to samples obtained from patients, such as blood or PBMC samples, without requiring the isolation and purification of membrane proteins. Once solubilized, membrane proteins can be detected and quantified by standard protein analysis techniques for diagnostic purposes. The present invention therefore further pertains to an in vitro method for the diagnosis of a disease such as an infectious disease in a patient, wherein said method comprises a step of solubilizing membrane proteins in a sample obtained from said patient as disclosed in the present application, detecting the membrane proteins solubilized in the sample and providing a diagnosis based on the presence and / or quantity of said solubilized proteins.

Description

PROCESS FOR THE SOLUBILIZATION OF MEMBRANE PROTEINSTechnical Field

[0001] The present invention pertains to biochemistry protocols, in particular methods for the solubilization of membrane proteins in a biological sample. The present invention also pertains to the analysis of said solubilized membrane proteins for diagnostic purposes.Background Art

[0002] Although cancers and cardio-neurovascular diseases remain the most frequent causes of death in France (each representing more than 25% of French mortality), infections, especially those of the lower respiratory tract, are in 6th position in the ranking of the most frequent deaths. Early and accurate diagnosis of infections is essential to improve the quality of care in hospitals and the timeliness of patient care. In addition, administering antibiotics to people without bacterial infections increases morbidity and antimicrobial resistance rates. The rate of inappropriate antibiotic prescribing in hospitals is estimated at between 30 and 50% in France. For example, approximately 95% of patients who are given antibiotics for suspected enteric fever have negative bacterial cultures (see “Antibiotic Resistance” report published by Sante Publique France in 2022).

[0003] The WHO (World Health Organization) estimates that in 2019, 1.27 million deaths could directly be attributed to antibiotic resistance (AMR) worldwide (Murray CJL, et al. The Lancet. 2022; 399:629-655). In 2017, the WHO proposed a list of resistant bacteria by declining an urgent, medium or critical need for new antibiotics. This list includes, but is not limited to, Acinetobacter, Pseudomonas and various enterobacteriaceae (including Klebsiella, E. coll, Serratia and Proteus) which can cause serious and often fatal infections, such as sepsis and pneumonia.

[0004] According to the European Centre for Disease Prevention and Control (ECDC), France is the fourth largest consumer of antibiotics in Europe with 125,000 cases of multidrug-resistant bacterial infections, including 5,500 deaths in 2015. Its antibiotic consumption is 40% higher than the average for other European countries. In addition, it is estimated that 63.5% of multidrug-resistant bacterial infections are healthcare-associated infections and one in twenty patients acquires at least one nosocomial infection (according to Sante Publique France).

[0005] According to the annual epidemiological report of the European Union agency and of the ECDC, considering climate change and the emergence of new diseases, antibiotic resistance could become the leading cause of death worldwide, ahead of cancer, diabetes, diarrhoea, and road accidents by 2050.

[0006] According to the ECDC, between 2012 and 2021 , France has decreased its consumption of beta-lactam and penicillin (-1 .5%) quinolone (-4.4%) in hospitals. However, it has sharply increased its consumption of tetracyclines (22.7%) and carbapenems (6.2%). This shows the struggle of health services in the urge to treat patients despite recommendations of the various authorities.

[0007] Meanwhile, the ECDC also reveals that France has a tendency to increase its total consumption of antivirals: +6.9% since 2012. Indeed, antivirals targeting e.g. SARS-CoV-2 have appeared on the market, as well as anti-herpes drugs.

[0008] With the emergence of arboviruses in Europe, partly due to global warming, it is considered that new treatments will take their place in the health landscape, and inevitably lead to new resistances.

[0009] For example, in 2016, Spain saw the first case of Crimean-Congo haemorrhagic fever (CCHF), a tick-borne disease that manifests with flu-like symptoms and can lead to organ failure. In France, an increasing number of viral infections that were considered as specific to tropical or sub-tropical regions were detected in the last few years, including dengue, zika or Usutu virus infections.

[0010] Given the above, the development of new diagnostic tools must be a priority for allowing the efficient selection of appropriate treatments and prevention of drug resistance.

[0011] The detection of specific viral / bacterial biomarkers proved to be a very promising way for the detection of viral / bacterial infections. Biomarkers are used daily by all medical practitioners in the diagnosis and treatment of patients as routine tests.

[0012] For instance, tests such as complete blood count (CBC), C-reactive protein (CRP) assay, procalcitonin (PCT), microbiological cultures, molecular biology tests (PCR, NGS), are used daily for the diagnosis of various diseases including infections in hospitals. Unfortunately, these tests have limitations.

[0013] CBC is always performed when an infection is suspected as hyperleukocytosis and neutrophilia can be a sign of an infection. However, acute trauma, burns, seizures and medication can also impact the number of leukocytes. Thus, CBC, while being an indicator, has only a low specificity for detecting infections.

[0014] C-reactive protein is a protein that is secreted during the acute phase by liver cells after stimulation by inflammatory mediators such as interleukin, IL-6 and IL-8. However, CRP is moderately specific (it is increased in all inflammations and infections), its release is long (>24h) and it is not correlated to the severity of the infection.

[0015] CD64 is the type I receptor of the Fc fragment of immunoglobulin G. It plays a role in phagocytosis and macrophage activation. This parameter can be used as a biomarker for early diagnosis of bacterial infection and sepsis with relative sensitivity and specificity (Cid J et al., Journal of Infection. 2010; 60:313-319). However, this marker is also be impacted by many factors, including stress.

[0016] Procalcitonin (PCT) is the pro-hormonal form of calcitonin. It is synthesized by many tissues of the body in response to inflammatory stimuli, especially bacterial stimuli. PCT is considered as one of the best biomarkers for the diagnosis of bacterial infections in hospitals. Yet, its clinical performance remains imperfect, and it does not allow discriminating the type of infection involved.

[0017] Membrane proteins play a crucial role in immune responses and can be used as diagnostic biomarkers not only for infectious diseases but also during inflammatory diseases and cancers. However, their analysis requires the use of a flow cytometer. Although extremely useful, this technology is expensive due to the instrumentation itself and requires an expert technician capable of calibration, parameterization, acquisition, and analysis of the data. Unfortunately, during pandemics, the time allocated for collecting data is extremely limited. Detection of membrane proteins by means of a flow cytometer is therefore not compatible with a rapid and accurate detection of infections.

[0018] In addition, analysis of membrane proteins first requires isolating and purifying the protein. Such a procedure is time-consuming, and, to date, no method allows for the solubilization and analysis of multiple membrane protein at the same time in a sample.

[0019] There is therefore a need for better diagnostic tools to improve the management of infections and of diseases in general. To be effective, a diagnostic test must be sensitive, specific, inexpensive, and as fast as possible (i.e. allow establishing a diagnosis within an hour after sample collection).Summary

[0020] The invention is defined by the claims.

[0021] The present inventors have developed a method that meets all the above requirements. It allows for the simultaneous solubilization of multiple membrane proteins in a sample.

[0022] The method according to the present invention is based on treating a biological sample with a combination of three specific reagents: a detergent, an amphipol and a lysis agent. It is simple, cost efficient and does not require using complex technologies. The present method is less expensive than flow cytometry and the results can be analysed with high-throughput immunoassay automatons.

[0023] Therefore, according to a first embodiment, the present invention pertains to a method for the solubilization of membrane proteins, wherein said method comprises a step of treating a biological sample with a combination of a detergent, an amphipol and a lysis agent.

[0024] Surprisingly, the present inventors have shown that this method can be directly performed on a biological sample obtained from a patient, such as a whole blood sample. The proteins thereby solubilized can then be easily analysed in the liquid phase as done for many other markers such as CRP and PCT. The method according to the present invention is fast and efficient and allows maintaining the tertiary conformation of the proteins so that they can be easily detected by antibodies.

[0025] The inventors have shown that by analysing the solubilized proteins obtained by means of the method according to the present invention, it is possible to identify a wide range of membrane markers that constitute markers of cellular activation and markers of infection or inflammation. The method according to the present invention therefore allows detecting infections, determining their severity and their origin (viral or bacterial, or even fungal and parasitic).

[0026] Therefore, according to a second embodiment, the present invention further pertains to an in vitro method for the diagnosis of a disease, particularly an infectious disease, in a patient, wherein said method comprises a step of solubilizing membrane proteins in a sample obtained from said patient as described above, detecting the membrane proteins solubilized in the sample and providing a diagnosis based on the presence and / or quantity of said solubilized proteins.

[0027] The inventors have notably shown that the method according to the present invention allows identifying protein biomarkers such as: SIGLEC-1 (sialo-adhesin / CD169), CD55 (DAF), TREM-1 (Triggering receptor expressed on myeloid cells 1), CD35 (CR1), CD38 (ectoenzyme), TRAIL (tumor- necrosis-factor related apoptosis inducing ligand), HLA-DR and CD86. The process according to the present invention further allowed for the detection of cytosolic markers such as MxA. Detecting and quantifying these biomarkers therefore allows diagnosing diseases to which they are known to be correlated, such as infectious diseases.Brief Description of Drawings

[0028] Figure 1. Expression of CD169 on the surface of monocytes during COVID-19: Patients with COVID are represented as “COVID +”, healthy controls as “HC”, and “COVID - “ are patients who were hospitalized, presenting infectious symptoms but tested negative for COVID and were not affected by acute viral infections

[0029] Figure 2. CD169 is associated with the plasma concentration of IFNa: CD169 expressed in peripheral blood monocytes is moderately associated with the plasma concentration of the antiviral cytokine IFNa in patients with severe (black) and moderate (open circle) COVID ; R2 = 0,48 ; p<0,05 .

[0030] Figure 3. Protein expression window during COVID-19 infection: CD169 is an early marker of acute viral infection, decreasing over time. The figure shows monocytes expression of CD169 at Day 0 (DO) and day 7 (D7) of hospitalization in subjects with confirmed COVID. CD169 expression rapidly decreases until it becomes negative. Data analyzed by flow cytometry.

[0031] Figure 4. Plasma CD169 versus Membrane CD169: A. Plasma CD169 was assessed during the COVID-19 pandemic, both in the first wave (burgundy color and vermilion) and the third wave (in black). While it is significantly higher in the plasma of COVID-positive patients compared to that of healthy donors (in blue), it does not discriminate with plasma CD169 of hospitalized patients (COVID-negative, in green) who do not have another viral infection. Therefore, plasma CD169 does not show diagnostic value compared to membrane CD169. B. Additionally, CD169 expressed on the surface of circulating monocytes from COVID-positive patients shows a sensitivity of 93.7% compared to non-COVID patients hospitalized for reasons other than acute viral infection. Thus, diagnostic performance is superior with membrane CD169 compared to plasma CD169.

[0032] Figure 5. In vitro model of viral infection: Viral infection triggers, in the early hours postinfection, secretion of interferon a and p (primarily), allowing the secretion of numerous pro- inflammatory cytokines as well as the production of proteins involved in the immune response at the cell membrane, including CD169. As described in the literature, we have developed in our laboratoryan experimental model enabling the production of CD169 following activation by IFNa of cells (PBMC and whole blood) from blood donors at the French Blood Establishment (EFS), in order to simulate an acute viral infection in vitro

[0033] Figure 6. CD169 molecule per monocytes at different stimulation times: In the experimental model developed in the laboratory, we observe that the expression of CD169 on the membrane of monocytes is detected starting from 10 hours after the beginning of cell stimulation, and that the antigen density is then approximately 1000 pg / ml.

[0034] Figure 7. Comparison of the amount of CD169 molecules expressed on the surface of monocytes compared with extraction by the innovation process: In the experimental model developed in the laboratory, the expression of CD169 on the membrane of monocytes is correlated with the quantification of CD169 obtained through the solubilization process.

[0035] Figure 8. Results of protein solubilization and comparison to conventional flow cytometry methods: PBMCs from 5 healthy donors were stimulated with IFNa for 5h, 10h, 16h, and 24h. We assessed CD169 expression on CD64+ monocytes by flow cytometry, as well as the antigen density of CD169 molecules per monocyte, and correlated it with the concentration of CD169 obtained after cell lysis using our method. The correlation line below illustrates the results obtained with a positive correlation coefficient of R2= 0.78.

[0036] Figure 9. Comparison of amount CD169 obtained after cell solubilization from whole blood or PBMCs: With the solubilization process on PBMCs, larger quantities of receptors were obtained compared to whole blood. However, the amount of marker obtained after solubilization from whole blood exceeds the negative control value by two-fold. Millions of PBMCs were stimulated and extracted for all of these analyses and stimulated for 24 hours with interferon-a. (NS = Not stimulated; Stim = Stimulated).

[0037] Figure 10. Membrane Biomarkers for Diagnostic of Bacterial Infections Usable with the Solubilization Method: Other membrane proteins serve as promising markers for assays using the described method. This figure illustrates some of the results obtained in the laboratory after stimulation by activators mimicking bacterial versus viral infection. The combination of markers and their multiplex assay will yield high diagnostic performance. (NS = Not stimulated ; IFNa = Cell stimulation by interferon-a ; LPS = Cell stimulation by Lipopolysaccharide mimicking gram-negative infection ; PAM3 = Cell stimulation by Pam3Cys-Ser-(Lys)4, Hydrochloride mimicking primarily grampositive bacterial infection).

[0038] Figure 11. Solubilization of CD169 on 12 samples from healthy donors using the innovative method: Twelve samples from healthy donors were tested using the solubilization method without stimulation (No cell stimulation, on the left) and compared after stimulation with interferon-a for 24 hours. The production of CD169 obtained from a dry pellet of 150 pl of whole blood shows quantification of at least 1000pg / ml of protein.

[0039] Figure 12. Linear regression obtained for detection of markers CD169 (A), CD86 (B) and MxA (C).Detailed description of the invention

[0040] The present invention pertains to a method for solubilizing membrane proteins in a sample, and to methods for the diagnosis of diseases based on analyzing the membrane proteins thereby solubilized.

[0041] Therefore, according to a first embodiment, the present invention pertains to a method for the solubilization of membrane proteins, wherein said method comprises a step of treating a biological sample with a combination of a detergent, an amphipol and a lysis agent.

[0042] “Membrane proteins” are a class of proteins that are embedded in or associated with biological membranes. Biological membranes are lipid bilayers that form the boundaries of cells and cellular organelles, such as the plasma membrane and the membranes of the endoplasmic reticulum, Golgi apparatus or mitochondria. Membrane proteins play a crucial role in various cellular processes, including ionic and molecular transport, signal transduction, enzymatic reactions and intercellular communication. They can be categorized into two main categories: integral membrane proteins (also referred to as transmembrane proteins) and peripheral membrane proteins (Boes, D. M., Godoy- Hernandez, A., & McMillan, D. G.. Membranes 11.5 (2021): 346).

[0043] “Integral membrane” proteins are embedded within the lipid bilayer and completely spans the phospholipid bilayer membrane, with portions of the protein extending into both the extracellular and intracellular environments. Integral membrane proteins are amphipathic: they comprise hydrophobic regions that interact with the hydrophobic core of the lipid bilayer, and hydrophilic regions that protrude outwards the membrane. Most integral membrane proteins are made of alpha helices (monotopic integral proteins comprise one single alpha helix whereas polytopic integral proteins comprise 2 or more alpha helices), but some integral proteins can also be made of p-barrel sheets.

[0044] “Peripheral membrane proteins” are extrinsic proteins that are not embedded within the lipid bilayer but are instead associated with the membrane surface through interactions with integral membrane proteins or through interactions with lipid molecules (via e.g. electrostatic and hydrogen bond interactions).

[0045] According to a preferred embodiment, the membrane proteins solubilized in the context of the present invention are integral membrane proteins.

[0046] The method according to the present invention allows for the “solubilization” of membrane proteins, i.e. allows for the membrane proteins to be extracted / separated from the biological membrane. Solubilization results in the release of the membrane proteins in an aqueous phase. Solubilization allows eliminating plasma membranes in the biological sample.

[0047] As explained above, the present inventors have shown that the method according to the present invention can be directly applied to samples obtained from a patient, without requiring any pretreatment. The “biological sample” can therefore be any sample in which membrane proteins are present. The sample according to the present invention is a cell-containing sample. Examples of such samples include fluids (including blood, lymph, cerebrospinal fluid, peritoneal fluid, synovialfluid, etc.), tissues (collected e.g. via biopsies or surgical procedures), cell samples (such as peripheral blood mononuclear cells or “PBMC”), etc. According to a preferred embodiment, the biological sample is a sample comprising leucocytes. Such samples include blood, lymph and synovial fluid samples, but also lymph nodes and spleen tissue samples. According to a more preferred embodiment, the biological sample according to the present invention is a blood sample, in particular a whole blood sample from which the plasma has been removed or a PBMC sample.

[0048] In the context of the present invention, the biological sample is treated with a combination of a detergent, an amphipol and a lysis agent.

[0049] “Detergents” are a group of compounds with an amphiphilic structure, where each molecule has a hydrophilic (polar) head and a long hydrophobic (non-polar) tail. The hydrophobic portion of these molecules may be straight- or branched-chain hydrocarbons, or it may have a steroid structure. Detergents can be classified into three categories: ionic, non-ionic, and zwitterionic. Detergents are already used for the isolation and purification of integral membrane proteins (Linke, Dirk. Methods in enzymology 463 (2009): 603-617). As known by the skilled person, the type of detergent used mainly depends on the hydrophobic or hydrophilic nature of the target protein, the sensitivity of the protein to denaturation, the preservation of the required biological activity, and the subsequent analytical techniques planned to be used. In the context of the present invention, the detergent used has been chosen to have a fairly low CMC and a small aggregation number and not to denature the protein so that it can be easily used in immunoassay techniques. According to a specific embodiment, the detergent according to the present invention can be a non-ionic detergent or a zwitterionic detergent.

[0050] According to a preferred embodiment, the detergent used in the context of the present invention is selected from the group consisting of 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton™ X-100), octylphenoxypolyethoxyethanol (IGEPAL CA-630™), TERGITOL™ 15-S-15, DDM (n-Dodecyl p-D-maltoside), DM (n-Decyl p-D-maltoside), NG (n-Nonyl p-D-glucopyranoside), OG (n- Octyl p-D-glucopyranoside), LDAO (n-Dodecyl-N,N-dimethylamine-N-oxide), 3-{Dimethyl[3- (3a,7a,12a-trihydroxy-5p-cholan-24-amido)propyl]azaniumyl}propane-1 -sulfonate (CHAPS), 3-([3- Cholamidopropyl]dimethylammonio)-2-hydroxy-1 -propanesulfonate (CHAPSO) and mixtures thereof. All these compounds can be easily obtained from various specialized manufacturers.

[0051] According to a more preferred embodiment, the detergent is selected from the group consisting of 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, CHAPS, CHAPSO and mixtures thereof.

[0052] In the context of the present invention, the detergent is typically used at a concentration above the critical micelle concentration (CMC), preferably at a concentration between 1 and 5 times the CMC. The detergent is typically introduced at a concentration comprised between 0.1 and 0.4% by weight. The skilled person will know the concentration of detergent to be used based on the type of membrane protein studied.

[0053] “Amphipol” (Apol) reagents are synthetic molecules used to stabilize and solubilize membrane proteins in the aqueous phase (see for review Popot, J-L., et al. Annual review ofbiophysics 40 (2011): 379-408). They are primarily used to study proteins that are naturally embedded in biological membranes, such as membrane proteins or ion channels. Amphipols are copolymers consisting of a hydrophobic chain and a hydrophilic chain. The hydrophobic part inserts into the lipid bilayer, mimicking the protein's natural membrane environment, while the hydrophilic part surrounds the protein, stabilizing it and keeping it in solution. The process of forming an amphipol protein-reagent complex usually involves several steps. The amphipol reagent is typically quickly added at a high concentration to the solution containing the protein. Amphipols associate with the hydrophobic parts of the protein, forming a protein-reactive complex. Once formed, the protein- reactive complex amphipol confers several benefits. It stabilizes the protein by maintaining its three- dimensional structure and protecting its exposed hydrophobic domains, allowing it to sit in an aqueous solution and be preserved in three-dimensional form. Amphipols can e.g. be selected from polyacrylate-based APol, phosphorylcholine-based APol, glucose-based APol and sulfonated APol.

[0054] In the context of the present invention, the amphipol is preferably selected from the group consisting of the polyacrylate-based amphipol A8-35, the phosphorylcholine-based amphipol C22- 43, amphipol PMAL-C8, amphipol PMAL-C9, amphipol PMAL-C12, amphipol PMAL-C16, amphipol A17, and mixtures thereof. All these compounds can be easily obtained from various specialized manufacturers. According to a preferred embodiment, the amphipol used in the context of the present invention is amphipol A8-35 or A17.

[0055] In the context of the present invention, the amphipol is introduced at high concentrations. It is typically used at a concentration between 0,5 and 5 times the CMC. The amphipol is typically introduced at a concentration comprised between 3 and 10mg / mL, preferably 4 and 7 mg / mL, more preferably of about 5 mg / mL. The skilled person will know the concentration of protein to be used based on the type of membrane protein studied. The sample is typically incubated with the amphipol at a temperature of between 25 to 30°C, preferably between 26 and 29°C, more preferably between 27,5 and 28,5°C. This incubation is typically carried out during 5 to 20 minutes, preferably between 8 and 12 minutes.

[0056] The method according to the present invention also includes treating the biological sample with a lysis agent. The “lysis agent” is a compound that causes the breakdown of the cell membranes comprised in the sample, leading to the release of the contents of the cell. Lysis agents are known to one of skill in the art (see for review Shehadul Islam, M., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). Micromachines, 8(3), 83). Lysis agents can be chemical, biological or mechanical lysis agents. Chemical lysis agents typically comprise a lysis buffer (which will induce an alkaline lysis of the cell membranes) that can be combined with a detergent. Biological lysis agents generally include enzymes such as lysozyme, lysostaphin, zymolase, cellulose, proteases or glycanase. The type of enzyme selected depends on the nature of the cells to be lysed (bacteria, yeast, plant cells, etc...). According to a preferred embodiment, the lysis agent is a mechanical lysis agent, i.e. an agent that physically induces the breakdown of cell membranes by using shear force. Mechanical lysis agents typically include beads such as glass beads, zirconium beads, stainless steel beads and ceramic beads. According to a preferred embodiment, the lysis agent according tothe present invention comprises or consists in zirconium beads. Beads are typically used in combination with a centrifugation of the sample. Centrifugation typically allows enhancing the lysing effect of the beads and also allows separating the supernatant, containing the solubilized membrane proteins, from the cell residues.

[0057] The skilled person will know how (amounts and conditions) to use the lysis agent in the context of the method of the present invention.

[0058] In the context of the present invention, the detergent, amphipol and lysis agent are used in “combination”, that means that the sample can be treated with each of these 3 compounds simultaneously (treating the sample with the detergent, the amphipol and the lysis agent at the same time), sequentially (by treating the sample with one compound after the other, in any order), or partially sequentially (by first treating the sample with two compounds simultaneously and then with the third compound, or by first treating the sample with one compound and then with the two other compounds simultaneously).

[0059] According to a preferred embodiment, the method according to the present invention comprises the following steps: a. treating a biological sample with a detergent; b. adding an amphipol; and c. treating the mixture obtained after step b. with a lysis agent.

[0060] Typically, step a. is performed at a temperature between 1 and 10°C, preferably between 3 and 5°C. The sample is typically treated with the detergent over a period of between 30 seconds and 15 minutes, preferably of less than 6 minutes, prior to step b.

[0061] As mentioned above, step b is typically performed by incubating the mixture obtained after step a. with the amphipol at a temperature of between 25 to 30°C, preferably between 26 and 29°C, more preferably between 27,5 and 28,5°C (typically 28°C). This incubation is typically carried out during 5 to 20 minutes, preferably between 8 and 12 minutes.

[0062] Typically, step c. is performed at a temperature between 1 and 10°C, preferably between 2 and 4°C. The mixture is typically treated with the lysis agent over a period of less than 2 minutes, preferably between 5 and 30 seconds more preferably between 12 and 17 seconds.

[0063] The conditions under which the combination of the detergent and amphipol are used (concentration, temperature, and ionic strength) enable minimal solubilization, but sufficient to obtain a detection threshold for the proteins of interest, avoiding the risks associated with the use of high concentrations of detergents (which could lead to poor protein conformation, loss of stability and / or activity). The membrane proteins can then be detected and quantified by immunoassay.

[0064] As mentioned above, the method according to the present invention does not require a step wherein membrane proteins are purified. Therefore, according to a specific embodiment, the present invention does not comprise an additional step wherein the membrane proteins are purified.

[0065] “Purifying” the membrane proteins refers to a step wherein the membrane proteins are isolated, in other words, it is not necessary to enrich the sample is said protein to further analyze it.

[0066] According to a further embodiment, the method can also comprise adding at least one proteinase inhibitor, typically simultaneously with the detergent, or after having added the detergent but prior having added the amphipol. Proteinases are enzymes that can cleave proteins and degrade them, which can lead to a loss of integrity and function of the proteins of interest. During the solubilization of membrane proteins, endogenous proteinases can be released from membranes and potentially affect the stability of target proteins. The addition of proteinase inhibitors during solubilization can help prevent this unwanted degradation. The skilled person knows several proteinase inhibitors that can be used in the context of the present invention. Mention can be made of the proteinase inhibitor cocktail with or without EDTA commercialized by Roche under the reference complete™.

[0067] The detergent can also be added simultaneously with a buffer such as a Good’s buffer (i.e. a buffer selected from HEPES, MES, ADA, PIPES, ACES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Tris, Glycinamide, Glycylglycine, Bicine and TAPS, preferably Tris and HEPES). The detergent can also be added simultaneously with a salt such as magnesium chloride (MgCI2) or sodium chloride, N- Acetylglucosamine (GIcNAc).

[0068] The detergent can therefore be LDAO alone or administered simultaneously with Tris and proteinase inhibitors, or with HEPES and GIcNAc, or be CHAPSO and be administered simultaneously with HEPES, or be Triton™ X-100 and be administered simultaneously with Tris and sodium chloride, or be CHAPS alone.

[0069] According to another embodiment, the method according to the present invention can also comprise steps of preparing the sample with techniques well known by the person of ordinary skilled in the art. For instance, the method can comprise, prior to treating the sample with the combination of the detergent, amphipol and lysis buffer, a washing step aiming at treating the sample with a high- stringency solution (referred to as the washing buffer) that limits non-specific interactions. The method can also comprise, prior to treating the sample with the combination of the detergent, amphipol and lysis buffer, a permeabilization step wherein the sample, either firstly submitted to a washing step or not, is treated with a permeabilization buffer. However, as shown in the examples below, such additional steps, and in particular a permeabilization step are not necessary in the context of the present invention.

[0070] According to a specific embodiment, the method according to the present invention can therefore comprise the following steps: a’, treating a biological sample with a washing buffer and / or a permeabilization buffer; a. treating the washed and / or permeabilized biological sample thereby obtained with a detergent b. adding an amphipol; andc. treating the mixture obtained after step b. with a lysis agent.

[0071] The washing buffer according to the present invention typically comprises a compound selected from the group consisting of a surfactant such as polysorbate and / or poloxamer, a Good’s buffer, preferably HEPES or Tris, and phosphate-buffered saline (PBS). The washing buffer can further comprise a detergent or sodium chloride. The washing buffer can e.g. comprise HEPES and a detergent, or alternatively Tris, sodium chloride and a polysorbate, or alternatively PBS, sodium chloride, BSA and FCS.

[0072] The sample is typically incubated with the washing buffer over a period of time comprised between 1 and 30 minutes, preferably between 2 and 10 minutes, more preferably around 5 minutes, and at a temperature comprised between 15 and 25°C, more preferably between 20 and 23°C.

[0073] Treatment with the washing buffer can advantageously be combined with centrifugation in conditions well-known by the skilled person. After incubation, the supernatant it typically eliminated prior to treating the sample with the permeabilization buffer or directly with the combination of a detergent, amphipol and lysis buffer.

[0074] The permeabilization buffer according to the present invention typically comprises a compound selected from sodium chloride and Good’s buffers such as Tris. The permeabilization buffer can further comprise a detergent such as Triton™ X-100. The permeabilization buffer can e.g. comprise sodium chloride and Triton™ X-100, or alternatively Tris, or alternatively Tris, sodium chloride and Triton™ X-100.

[0075] The sample is typically incubated with the permeabilization buffer over a period of time comprised between 1 and 30 minutes, preferably between 2 and 10 minutes, more preferably around 5 minutes, and at a temperature comprised between 1 and 10°C, more preferably between 2 and 6°C.

[0076] According to a specific embodiment, the present invention pertains to a method for the solubilization of membrane proteins, said method comprising the following steps: treating a biological sample such as a whole blood sample from which the plasma has been removed or a PBMC sample with a washing buffer; treating the washed biological sample with a permeabilization buffer; treating the biological sample thereby obtained with a detergent; adding an amphipol; and treating the mixture thereby obtained with a lysis agent.

[0077] As mentioned above, once solubilized, membrane proteins can be detected and quantified by standard protein analysis techniques for diagnostic purposes. As shown in the present specification, the solubilisation method according to the present invention allows for the detection of membrane markers that constitute markers of cellular activation and markers of infection or inflammation. The method according to the present invention therefore allows detecting cellularresponses observed during specific diseases such as infections and allows determining their severity and their origin (viral or bacterial, or even fungal and parasitic).

[0078] Therefore, according to a further embodiment, the present invention pertains to an in vitro method for the diagnosis of a disease in a patient, wherein said method comprises a step of solubilizing membrane proteins in a sample obtained from said patient as described above, detecting the membrane proteins solubilized in the sample and providing a diagnosis based on the presence and / or quantity of said solubilized proteins.

[0079] The “disease” according to the present invention can be any disease that can be detected based on the presence of membrane protein markers. In the context of the present invention, the membrane protein is therefore a diagnostic marker which can be used for the diagnosis of a disease to which it is correlated.

[0080] Membrane markers are indeed known to be correlated with multiple diseases (see for review Varady, Gybrgy, et al. Biomarkers in medicine 7.5 (2013): 803-819). For instance, dysfunctions of tyrosine kinase receptors (such as growth hormone receptors) are known to be correlated with developmental diseases, diabetes, atherosclerosis and cancer development. Overexpression of HER2 is e.g. known to be correlated with aggressive forms of breast cancer (see e.g. Pupa, Serenella M., et al. Cancers 13.19 (2021): 4778). EGFR mutations are correlated with various cancers including lung cancer (see da Cunha Santos, Gilda, Frances A. Shepherd, and Ming Sound Tsao. Annual Review of Pathology: Mechanisms of Disease 6 (2011): 49-69). Cluster of differentiation (CD) markers are also commonly used as diagnostic markers in a variety of infectious and neoplastic diseases including HIV, acute and chronic leukemias (such as CD38 which is associated to chronic lymphoid leukemia) and solid cancers including colorectal cancer. (Ellmark, Peter, et al. Genomics Protocols (2008): 199-209). Membrane proteins are particularly suitable for the diagnosis of infectious diseases. Therefore, according to a preferred embodiment, the disease diagnosed in the context of the present invention is an infectious disease.

[0081] An “infectious disease” refers to a condition caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi. The European Centre for Disease Prevention and Control (ECDPC) provides the following list for infectious diseases: acquired immunodeficiency syndrome (AIDS), anthrax, arenavirus, avian influenza virus, Bordetella (pertussis), borreliosis (including Lyme disease), botulism, brucellosis, campylobacteriosis, chickenpox (varicella), chikungunya virus disease, chlamydia infection, cholera, ciguatera fish poisoning (CFP), clostridioides difficile infections, congenital rubella, congenital syphilis, coronavirus, coxsackievirus, Creutzfeldt-Jakob disease (CJD), Crimean-Congo haemorrhagic fever (CCHF), cryptosporidiosis, cutaneous warts, dengue and Dengue Haemorrhagic Fever (DHF), diphtheria, Ebola virus disease, echinococcosis, enteric fever, enterohaemorrhagic Escherichia coll (EHEC) infection, enterovirus infection, Escherichia coll infection, flu, food- and waterborne diseases, German measles (rubella), giardiasis, gonorrhoea, Haemophilus infection, haemorrhagic fever, haemorrhagic fever with renal syndrome; Hantavirus infection, hepatitis such as hepatitis A, hepatitis B, hepatitis C and hepatitis E, HIV infection, human papillomavirus infection (HPV), influenza in humans, avian origin, influenza in humans, pandemic,influenza in humans, swine origin, invasive Haemophilus influenzae disease, invasive meningococcal disease, invasive pneumococcal disease, Japanese encephalitis virus, Lassa fever, legionnaires’ disease, leishmaniasis, leptospirosis, listeriosis, lymphogranuloma venereum (LGV), malaria, measles, meningococcal disease, Middle East respiratory syndrome coronavirus, Mosquito- borne diseases, Mpox (Monkeypox), mumps, nephropathia epidemica, Nipah virus disease, Norovirus infection, paratyphoid fever, pertussis, plague, pneumococcal disease, poliomyelitis, Q fever, rabies, Respiratory Syncytial Virus (RSV), Rift Valley fever, rotavirus infection, rubella, S. pneumoniae, salmonellosis, SARS-CoV-2, seasonal influenza, severe acute respiratory syndrome (SARS), sexually transmitted infections, shigellosis, sindbis fever, smallpox, streptococcus pneumoniae, swine-origin influenza, seasonal influenza, syphilis, tetanus, tick-borne diseases, tick- borne encephalitis (TBE), Toscana virus infection, toxoplasmosis, trichinellosis, tuberculosis (TB), tularaemia, typhoid and paratyphoid fever, variant Creutzfeldt- Jakob disease (vCJD), viral haemorrhagic fever, viral hepatitis, West Nile virus infection, yellow fever, yersiniosis and zika virus disease.

[0082] The present inventors have shown that the method according to the present invention allows detecting the following transmembrane proteins: SIGLEC-1 (sialo-adhesin / CD169), CD55 (DAF), TREM-1 (Triggering receptor expressed on myeloid cells 1), CD35 (CR1), CD38 (ectoenzyme), TRAIL (tumor-necrosis-factor related apoptosis inducing ligand), HLA-DR and CD86. The process according to the present invention further allows for the detection of cytosolic markers such as MxA.

[0083] CD169 (Siglec-1 Sialic acid binding Immunoglobulin-like lectins or Sialoadhesin) is a membrane protein of the sialic acid-binding immunoglobulin-like lectin family and is specifically expressed on monocytes, tissue macrophages, and dendritic cells. It facilitates cell-cell interactions through carbohydrate recognition and participates in the regulation of cellular functions of innate and acquired immune systems (Sakumura, Naoto, et al. "CD169 expression on monocytes as a marker for assessing type I interferon status in pediatric inflammatory diseases." Clinical Immunology 250 (2023): 109329). It has recently been shown that the expression of CD169 on the surface of monocyte cells (MoCD169) increases in patients with viral infection, but not in those with bacterial infection (Bourgoin et al. Immunity, inflammation and disease 8.1 (2020): 106-123). These infections involved many but not exclusively single-stranded RNA viruses (flaviviruses such as dengue, Zika, but also HIV, SARS-CoV-2, Lassa and Marburg virus, CMV, measles, influenza, EBV (in the acute phase) rhinovirus. Indeed, CD169 expression is induced on the surface of monocytes after stimulation with IFN-a, IFN-p and IFN-co, but not IFN-y (Bourgoin et al. (2020)). MoCD169 expression is also increased in patients with autoimmune and autoinflammatory diseases in which IFN-a is involved in pathogenesis, such as SLE (systemic lupus erythematosus) and systemic sclerosis (Biesen, Robert, et al. Arthritis & Rheumatism 58.4 (2008): 1136-1145; York, Michael R., et al. Arthritis & Rheumatism 56.3 (2007): 1010-1020).

[0084] Table 1 below discloses viruses for which a CD168 overexpression was observed in the monocytes of infected subjects:Table 1: List of viruses inducing CD169 overexpression in monocytes of infected subjects

[0085] The method according to the present can thus advantageously allow for the diagnosis of infectious diseases, including those listed in Table 1 based on the detection of CD169.

[0086] CD55 (DAF for “Decay acceleration factor”) is expressed on the surface of various cell types, including red blood cells, endothelial cells, leukocytes, and epithelial cells. Its expression is essential to protect these cells from complement attack, which is a collection of plasma proteins that act in cascades to eliminate pathogens and damaged cells. It is a membrane glycoprotein involved in regulating the immune system and protecting cells from non-specific complement activation, a key component of the innate immune system. The primary function of this protein is to inhibit non-specific complement activation by regulating the early stages of the complement cascade, more specifically the classical pathway and the alternative pathway. It works by accelerating the degradation of C3 and C5 convertases, enzymes involved in the formation of membrane attack complexes (MACs) that can damage target cells. Data show that increased CD55 levels on neutrophils and monocytes is a potent marker of bacterial infection and viral infection.

[0087] CD35 (or CR1 for “Complement Receptor 1 ”) is a membrane glycoprotein found on erythrocytes, leukocytes, glomerular podocytes, hyalocytes, and splenic follicular dendritic cells. It binds to complement activation fragments, including C3b and C4b. It acts as an important regulator of complement activity by promoting its elimination from the bloodstream and protecting immunesystem cells from overactivation. By binding to complement fragments, CD35 facilitates their degradation and eliminates them, preventing potential autoimmune damage caused by uncontrolled complement activation. CD35 is expressed on many cells such as red blood cells, myeloid cells, and lymphocytes. The amount of CD35 expressed by neutrophils can be used as a marker for infections and can further be used for discriminating viral infections from bacterial infections.

[0088] CD14 has the ability to identify multiple groups of ligands of Gram-positive and Gram-negative bacteria such as lipids, peptidoglycan, and other surface motifs. The best-studied ligand is lipopolysaccharide (LPS) from Gram-negative bacteria. To potentially be recognized, LPS requires the association of the lipoprotein-binding protein (LBP) that presents LPS to CD14. CD14 is a coreceptor that is constitutively expressed on the surface of monocytes / macrophages. It plays an important role in the presentation of LPS to TLR and actively contributes to intracellular signals and promotes the expression of genes responsible for the immune response such as the release of cytokines by effector cells. CD14 occurs in two forms: membrane CD14 (mCD14) and soluble CD14 (sCD14). SCD14 is found in plasma and is produced by cleavage of mCD14 or by cell secretion by cathepsin D and other proteases in plasma or in the phagolysosome. The 13 kDa N-terminal fragments are the subtype of sCD14 (sCD14-ST) which has been named presepsin. Presepsin is released into the systemic circulation by proteolysis and exocytosis. In the blood, the observed increase in CD14 receptors is a direct response to the action of lipopolysaccharides (LPS) on neutrophils.

[0089] TREM-1 (triggering receptor expressed on myeloid cells 1) or CD354, is an amplifier of inflammation inducing the production of cytokines, as well as the expression of many surface molecules. It is expressed on monocytes, neutrophils, and macrophages. It plays a key role in inflammation, amplifying it by activating the TREM-1 / DAP12 pathway through interaction with ligands or stimulation by bacterial lipopolysaccharides. This amplifies the production of cytokines and pro- inflammatory chemokines (Bouchon A, Dietrich J, Colonna M. J Immunol. 2000; 164:4991-4995; Arts RJW, et al., Eur Cytokine Netw. 2011 ; 22:11-14). It is involved in trans-epithelial migration of neutrophils and effector responses, such as degranulation and phagocytosis. It also contributes to macrophage survival and the development of an adaptive immune response. The TREM-1 receptor and its signaling pathways contribute to the pathology of several acute and chronic non-infectious inflammatory diseases, including atherosclerosis. During a bacterial infection, the TREM-1 protein is activated. The expression of the TREM-1 receptor increases on the surface of neutrophils and monocytes upon the recognition of specific bacterial components, such as LPS present in the wall of gram-negative bacteria (Bouchon et al, Nature 410.6832 (2001): 1103-1107; Bouchon et al, The Journal of Immunology. 2000; 164:4991-4995; Matos et al. Critical Reviews in Microbiology. 2021 ; 47:290-306; Fortin et al. International Immunology. 2006; 19:41-50). The activation of the TREM-1 protein leads to the release of inflammatory mediators and amplification of the inflammatory response, thus promoting the elimination of bacteria.

[0090] CD38 The CD38 protein is a cell surface enzyme belonging to the ADP-ribosyl cyclase family of glycoproteins. It is expressed on various cell types, including lymphocytes, plasma cells,endothelial cells, and epithelial cells. The main function of the CD38 protein is to catalyze the conversion of nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose (ADPR) and nicotinamide. This reaction also produces free calcium and cyclic ADPR (cADPR). These metabolites play an important role in many biological processes, such as cell signaling, intracellular calcium regulation, energy metabolism, and lymphocyte activation. The CD38 protein is involved in various cellular functions and physiological processes for example, the regulation of intracellular calcium by producing cADPR, which acts as a secondary messenger to release calcium from intracellular stores. This contributes to many calcium-dependent cellular processes, such as muscle contraction.

[0091] TRAIL (tumor-necrosis-factor related apoptosis inducing ligand) is a protein belonging to the tumor necrosis factor (TNF) family. It acts as a cell death ligand that can induce apoptosis (programmed death) in some cells. TRAIL is expressed in various cell types, including lymphocytes, natural killer (NK) cells, dendritic cells, endothelial cells, and tumor cells. TRAIL binds to its appropriate receptors, it activates an intracellular signaling cascade that can trigger apoptosis in susceptible cells. Tumor cells are often sensitive to the apoptotic effect of TRAIL, which has generated considerable interest in its potential use in cancer treatment. TRAIL is a marker in viral infections and was shown as a potent marker for discriminating viral and bacterial infections.

[0092] CD86 and CD80 are co-stimulators and can both interact with different receptors, resulting in opposite effects. At the onset of the immune response, CD86 is expressed on cells in primary lymphoid tissue. It is expressed constitutively on antigen-presenting cells and is rapidly upregulated in response to infection. It was therefore involved in the early phases of the immune response.

[0093] The MerTK protein, also known as Mer-tyrosine kinase, is a receptor in the receptor tyrosine kinase family. It is involved in the regulation of various cellular processes, including phagocytosis, inflammation, tissue homeostasis, and suppression of the immune response. MerTK is expressed primarily by cells of the immune system, such as macrophages, dendritic cells, and endothelial cells. Its key role is to orchestrate phagocytosis of apoptotic cells and foreign bodies, thus contributing to the resolution of inflammation and the maintenance of tissue homeostasis. Its activation can inhibit the production of pro-inflammatory cytokines and promote the secretion of anti-inflammatory cytokines, thus contributing to the resolution of inflammation. MerTK can also regulate cell proliferation and cell survival, including cancer cells. Alteration of MerTK expression or activity has been associated with several pathological conditions, including autoimmune diseases, chronic inflammation, neurodegenerative diseases, and cancers. Due to its involvement in immune regulation and phagocytosis, MerTK may represent a potential therapeutic target for the development of therapies to modulate inflammatory responses or improve apoptotic cell clearance. MerTk has also been shown in combination with HLA-DR, as a biomarker of bacterial infection by flow cytometry (Velly, Laetitia, et al. Journal of Infection 82.4 (2021): 11-21).

[0094] The diagnosis method according to the present invention can therefore advantageously comprises detecting and evaluating the quantity of the above membrane proteins so as to determinewhether the patient suffers from an infection (e.g. viral or bacterial) or other disorders including autoimmune diseases, chronic inflammation, neurodegenerative diseases, and cancers.

[0095] According to a further embodiment, and in order to further improve the specificity and selectivity of the method according to the present invention, it is possible to detect, in addition to the membrane proteins solubilised according to the method of the present invention, other proteins having an interesting marker value. The present inventors have e.g. shown that it is possible to combine the detection of a membrane protein as described above with the detection of e.g. the human MxA, the human myxovirus resistance protein 1 (Interferon-induced GTP-binding protein Mx1).

[0096] The human MXA protein belongs to the superfamily of large dynamin-like GTPases; characterized by their high molecular weight in the range of 80-100kDa. MxA is unique among these GTPases because it is induced by type I (IFN-a and p) and type III (IFN-A) proteins and it has intrinsic antiviral activity by inhibiting the replication of a number of viruses, including influenza, measles and bunyavirus (Haller, O., M. Frese, and G. Kochs. Revue scientifique et technique (International Office of Epizootics) 17.1 (1998): 220-230). Human myxovirus resistance protein 1 (MxA) is a key mediator of interferon-induced antiviral response against a wide range of viruses. It is located in the cytoplasm in association with the membranes of the smooth endoplasmic reticulum. The human MxA protein exhibits broad antiviral activity and is capable of inhibiting many RNA (including influenza A virus) and DNA viruses at different stages of their replication cycles. It can inhibit the formation of functional viral structures, thereby compromising the integrity and function of viral particles during viral assembly. Its expression is usually very low or absent under normal conditions or in the absence of viral infection. Thus, the detection of high levels of the MxA protein in biological samples, such as blood, serum, or tissues, indicates the presence of an active viral infection.

[0097] In the context of the present invention, the “patient” or “subject” is a mammal (e.g. a cow , a buffalo, a horse, a dog, a cat, a pig, a rodent or a primate). In a particular embodiment, the patient is a human.

[0098] In the context of the present invention, the diagnosis is provided based on the analysis of the membrane proteins, i.e. based on their presence and / or on their quantity. For instance, as shown in the experimental section of the present specification, the presence of high levels of CD169 are representatives of early viral infection. It is therefore possible to diagnose an infectious disease based on the presence and quantity of solubilized CD169 detected in a sample.

[0099] The person skilled in the art is familiar with many techniques that are used on a daily basis to determine the presence and quantity of a protein / peptide in a sample. Such methods typically involve contacting the biological sample to be analyzed with an agent capable of specifically binding the target protein. This agent is usually a polyclonal or monoclonal antibody. The presence of the protein is then typically detected by standard immunodetection methods after separation of the proteins by electrophoresis (technique also called "Western blotting") or by immunoassays by direct, indirect, competition or immunocapture methods (such techniques include e.g. "ELISA" or immunoturbidimetry or immuno-nephelometry). The formation of a complex between the protein of interestand the antibody(s) targeting said protein is usually detected and quantified by measuring an enzymatic reaction generating a colored, chemiluminescent or fluorescent product (ELISA) or by measuring the absorbance of light passing through the sample after formation of the protein / antibody complex (immuno-turbidimetry or immuno-nephelometry). Common techniques also include polyacrylamide gel electrophoresis (PAGE), mass spectrometry or methods such as Bradford assay.

[0100] The present invention will be further illustrated by means of the following examples. These examples are provided for illustrative purposes only and should not be interpreted as limiting the scope of the invention.Examples

[0101] Example 1

[0102] Protocol for the solubilization of membrane proteinsF01031 STEP-1

[0104] Wash buffer dOOPul):

[0105] High-stringency solution that limits non-specific interactions.

[0106] 50 mM HEPES (pH 7.4) + 0.1 % detergent or 10 mM Tris-HCI (pH 7.4) + 150mM NaCI + 0.1 % Tween-20 or 10 mM Tris-HCI (pH 8.0) + 150 mM NaCI + 2.5% Tween-40 or PBS + 0.7M NaCI + 1 % BSA + 5% FCS

[0107] Incubation 5 minutes at a temperature 20-23°C; centrifugation 500g 7 min at 4°C; Remove the supernatant.

[0108] STEP-2 (permeabilization and solubilization)

[0109] Permeabilization buffer: (200pl)

[0110] NaCI 150mM + Triton™ X-100 1 % or Tris-HCI (pH 8.0) 50 mM or 20mM Tris-HCI (pH 7.4) 150 mM NaCI + 1 % Triton™ X100.

[0111] Incubation 5 minutes at 4°C.

[0112] Solubilization buffer: (1 OOpI)

[0113] 10 mM Tris-HCI pH 8.0 + + protease inhibitors or HEPES buffer (pH 7.5) 20mM + MgCI2 2mM + 0.8pg / ml CHAPSO (final concentration 0.02%) or 50 mM Tris-HCI (pH 7.4) +150 mM NaCI + 0.5% Triton™ X-100 + or 0.5% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate).

[0114] The two permeabilization and solubilization buffers are added with a ratio of 2 / 3 - 1 / 3.

[0115] Inhibitory Protease Cocktail: (3pl - complete™ - Roche).

[0116] 1 ng / ml pepstatin + 2.5 mM iodoacetamide + 2.5 pg / ml aprotinin + 2.5 mM ethylene diamine tetraacetic acid + 0.1 mg / ml soybean trypsin inhibitor + 1 mM phenyl methyl sulphonyl fluoride + 0.5 ng / ml leueptin; and / or EDTA or EGTA or Benzamidine or AEBSF.

[0117] + Amphipol Reagent: A8-35: 5,4 mg / mL at 28°C during 10 minutes.[01181 STEP-3

[0119] Membrane grinding:

[0120] 1 x 25 mg Zirconium balls 0.10 or 0.15 mm diameter mixing power 2, 15 seconds, at a temperature of 2-4°C.

[0121] Centrifuge for 7 minutes from 0°C to 4°C speed 10000 g.[01221 STEP-4: Specific ELISA plate deposition of the proteins of interest and guantitative evaluation of the extracted target proteins.

[0123] Results:

[0124] As showed during the COVID-19 outbreak in 2020 (Bedin, Anne-Sophie, et al. The Journal of infectious diseases 223.4 (2021): 562-567), CD169 is highly expressed on the surface of monocytes in circulating blood, changing from a low-expressed basal state to a very strong increase detectable by flow cytometry (Figure 1). As described in the literature, CD169 is induced by interferon a; cytokine secreted by immune system cells, including myeloid and pDCs (dentritic plasmacytoid cells) in response to viral infections in the mucous membranes. During COVID, the method according to the present invention allowed to show a correlation of CD169 with IFN a (Figure 2).

[0125] The protein is greatly increased during the acute phase of the disease and decreases over time to finally become negative on average 8 days later (Figure 3). This protein is exceptionally interesting because it has the particularity of not being expressed in the case of chronic infection. Its soluble form exists, but it has major defects in sensitivity and specificity as a biomarker because its increase in plasma persists after resolution of the acute phase of infection and it is not specific for viral infections (Figure 4).

[0126] An in vitro model of interferon a stimulation was developed and allowed observing the expression kinetics over time of the CD169 protein on peripheral blood monocytes (Figure 5). This allowed to demonstrate the increase in the number of CD169 antigen sites on the surface of monocytes over time (Figure 6) and to make a correlation between the protein solubilized by the method according to the present invention and the expression of the protein at the membrane identified by flow cytometry (Figure 7 and 8).

[0127] As shown in Figure 9, thanks to the present solubilization process, it is possible to use PBMC or whole blood samples (by removing the plasma beforehand). This process allowed obtaining highly discriminating results between cells stimulated and not stimulated in vitro by interferon a after 10 hours of culture for moderate levels of cell expression.

[0128] Other markers were studied to distinguish between viral and bacterial infections (Figure 10). These markers can be solubilized with the innovative method described in the present specification.

[0129] Figure 11 shows an example of solubilization of 12 healthy controls before and after stimulation with interferon-alpha for 24 hours.

[0130] Example 2

[0131] Protocol for the solubilization of membrane proteins out of 150 000 cells

[0132] The following simplified process, wherein no permeabilization is performed, was developed:F01331 STEP-1

[0134] Wash buffer CIOOQuI):

[0135] High-stringency solution that limits non-specific interactions.

[0136] 50 mM HEPES (pH 7.4) + 0.1% detergent or 10 mM Tris-HCI (pH 7.4) + 150mM NaCI + 0.1% Tween-20 or 10 mM Tris-HCI (pH 8.0) + 150 mM NaCI + 2.5% Tween-40 or PBS + 0.7M NaCI + 1% BSA + 5% FCS or PBS

[0137] Centrifugation 500g 7 min at 4°C; Remove the supernatant.

[0138] STEP-2 (solubilization)

[0139] Solubilization buffer: (150|jl) at room temperature

[0140] 10 mM Tris-HCI pH 8.0 + protease inhibitors or HEPES buffer (pH 7.5) 20mM + MgCI22mM + 0.8|jg / ml CHAPSO (final concentration 0.02%) or 50 mM Tris-HCI (pH 7.4) +150 mM NaCI + 0.5% Triton™ X-100 + or 0.5% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate).

[0141] Inhibitory Protease Cocktail: (3pl - complete™ - Roche).

[0142] + 150 pl Amphipol Reagent: A8-35: 5,4 mg / mL at 28°C during 10 minutes.F01431 STEP-3

[0144] Membrane grinding:

[0145] 1 x 25 mg Zirconium balls 0.10 or 0.15 mm diameter (mixing power low), 5 seconds, at a temperature of 2-4°C.

[0146] Centrifuge for 7 minutes from 0°C to 4°C speed 10000 g.[01471 STEP-4: Specific ELISA plate deposition of the proteins of interest and guantitative evaluation of the extracted target proteins.

[0148] Example 3

[0149] Sample analysis

[0150] Samples

[0151] Between March 15 and July 30, 2024, 37 heparinized whole blood samples were collected. Among these samples, 15 cases of acute viral infections were biologically confirmed, including 6 women and 9 men, with an average age of 56.3 years. The diagnoses were distributed as follows:

[0152] - 11 cases of COVID-19, including 5 severe cases;

[0153] - 1 severe case of para-influenza virus type 3 infection;

[0154] - 1 moderate case of Epstein-Barr virus (EBV) infection;

[0155] - 2 cases of varicella-zoster virus (VZV) infection.

[0156] In addition, 17 bacterial infections were biologically identified in 5 women and 12 men, with an average age of 65.2 years. A distinction was made between:

[0157] - 7 severe bacterial infections (including 3 Escherichia coli, 1 Haemophilus influenzae and 2 Streptococcus pneumoniae);

[0158] - 4 polymicrobial bacterial infections, all severe;

[0159] - 6 moderate bacterial infections.

[0160] Flow cytometry

[0161] All samples were evaluated for a selection of markers as described in the present application; here we present the results for markers CD169, CD86 and MxA. (HLA-DR, TREM-1 also show a significant correlation - data not shown).

[0162] Solubilization using the extraction process of Example 2

[0163] 150 pl of whole blood were collected and solubilized using the extraction process.

[0164] Finally, the solubilized protein supernatant was collected and stored frozen at -80°c prior to ELISA analysis.

[0165] Analysis

[0166] The results obtained by Elisa (protein concentration) were compared with the mean fluorescence (MFI) obtained by flow cytometry; the R++ software (RppHIM) was used to analyze the linear regressions obtained between the two techniques. The results are shown in Figure 12.

[0167] The solubilization protocol as described above allowed easily and rapidly detecting cell markers including CD169, CD86 and MxA. The solubilization process presented herein therefore allows for the rapid and simple diagnostic of infections (viral or bacterial), and can be automated in a medical context, using clinical samples such as whole blood or PBMC. The process is based on the specific combination of three agents - a mild detergent (such as Triton X-100, CHAPS), an Amphipol (e.g. A8-35) and a mechanical lysis agent (zirconium beads) - applied to extract membrane proteins without altering their conformation, for detection by immunoassays compatible with medical biology automats.

Claims

Claims

1. A method for the solubilization of membrane proteins, wherein said method comprises a step of treating a biological sample with a combination of a detergent, an amphipol and a lysis agent.

2. The method according to claim 1 , wherein said membrane proteins are integral membrane proteins.

3. The method according to claim 1 or 2, wherein said biological sample is a sample comprising leucocytes.

4. The method according to any one of the preceding claims, wherein said biological sample is a whole blood sample from which the plasma has been removed or a peripheral blood mononuclear cells sample.

5. The method according to any one of the preceding claims, wherein said detergent is selected from the group consisting of 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, 3-{Dimethyl[3-(3a,7a,12a-trihydroxy- 5p-cholan-24-amido)propyl]azaniumyl}propane-1 -sulfonate (CHAPS), 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) and mixtures thereof.

6. The method according to any one of the preceding claims, wherein said amphipol is selected from the group consisting of amphipol A8-35, amphipol C22-43, amphipol PMAL-C8, amphipol PMAL-C9, amphipol PMAL-C12, amphipol PMAL-C16, amphipol A17 and mixtures thereof.

7. The method according to claim 6, wherein said amphipol is amphipol A8-35 or A17.

8. The method according to any one of the preceding claims, wherein said lysis agent comprises beads, preferably zirconium beads.

9. The method according to any one of the preceding claims, wherein said method comprises the following steps: a. treating said biological sample with said detergent; b. adding said amphipol; and c. treating the mixture obtained after step b. with said lysis agent.

10. The method according to any one of the preceding claims, wherein said method further comprises a step of treating the biological sample with a washing buffer and / or a permeabilization buffer prior to treatment with the combination of detergent, amphipol and lysis agent.

11. An in vitro method for the diagnosis of a disease in a patient, wherein said method comprises a step of solubilizing membrane proteins in a sample obtained from said patient as recited in any one of claims 1 to 10, detecting the membrane proteins solubilized in the sample and providing a diagnosis based on the presence and / quantity of said solubilized membrane proteins.

12. The method according to claim 11 , wherein said disease is an infectious disease.

13. The method according to claim 11 , wherein said infectious disease is a viral infectious disease.

14. The method according to any one of claims 11 to 13, wherein said membrane proteins include CD169.

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