A novel complex formed between flavivirus nonstructural NS1 protein and plasma lipoproteins

Abstract

The present invention relates to a method for the early detection, monitoring and prognosis of flavivirus-induced infections, comprising detecting complexes formed in biological samples by the flavivirus nonstructural glycoprotein NS1 and plasma lipoprotein particles during the clinical phase of infection.

Description

[Technical Field]

[0001] The present invention relates to a method for the diagnosis, monitoring and prognosis of flavivirus infection, in particular dengue virus infection, comprising the step of detecting and / or measuring the level of a complex formed by the flavivirus nonstructural glycoprotein NS1 and plasma lipoprotein particles. [Background technology]

[0002] Dengue is a global mosquito-borne viral disease affecting 3.9 billion people in 128 countries and is a leading cause of hospitalization and death in tropical and subtropical regions of the world. Dengue virus is transmitted primarily by female mosquitoes of the species Aedes aegypti and, to a lesser extent, Aedes albopictus. Dengue is caused by viruses of the Flaviviridae family, and there are four distinct but closely related serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4). The four dengue virus serotypes are estimated to infect 390 million people annually (95% confidence interval: 284–528 million), of whom 96 million (67–136 million) develop clinical signs (of any disease severity) (Bhatt et al. 2013).

[0003] Dengue is characterized by a wide range of clinical symptoms, from mild to life-threatening. More rarely, atypical manifestations of dengue virus infection may be accompanied by fulminant hepatitis, cardiomyopathy, acute renal failure, and encephalopathy. The course of the disease can be divided into three stages: (i) an acute febrile phase lasting 2–7 days with nonspecific clinical signs and the possibility of moderate hemorrhagic symptoms (petechial and mucosal bleeding); (ii) a critical phase, generally occurring once fever subsides, during which complications including severe bleeding, plasma leakage with shock, and organ damage may manifest in some patients; and (iii) a recovery phase. Early and appropriate management of severely symptomatic dengue cases is a critical step in reducing mortality.

[0004] The WHO proposed its first clinical classification guidelines for dengue in 1974, which were revised in 1997. The 1997 WHO classification system, which includes three categories—dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS)—is based on clinical and / or biological signs that are essential for correctly classifying the severity of the disease. Furthermore, as dengue case incidence increased in Asia and the Americas and the disease became widespread, many clinicians dealing with the full range of dengue cases encountered difficulties applying the 1997 WHO case definition to triage and clinical management. Following prospective clinical trials conducted across Latin American and Asian countries aimed at improving dengue case management, the WHO launched new case management guidelines in 2009 (WHO 2009), providing a cross-sectional classification of dengue cases that establishes criteria for identifying dengue cases with dangerous symptoms or severe dengue (SVD) instead of a longitudinal classification (where the previous step is required before moving on to the next step).

[0005] DENV is a small, enveloped, single-stranded RNA that encodes three structural proteins (envelope, membrane, and capsid) and seven nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) (Guzman et al. 2010). Nonstructural protein 1 (NS1) is involved in viral replication in DENV-infected cells and can be shed in large amounts into the bloodstream of patients experiencing various clinical grades of dengue disease (Alcon-LePoder et al. 2006). NS1 has been shown to bind complement and coagulation factors and trigger the production of antibodies that cross-react with surface antigens on platelets and endothelial cells, and is therefore proposed to play a role in the development of thrombocytopenia and hemorrhage in dengue disease (Rastogi et al. 2016). More recently, the secreted form of NS1 has been shown to promote endothelial permeability and vascular leakage in vitro and in vivo, as well as to critically contribute to the inflammatory cytokine storm observed in severe dengue cases (de Silva et al. 2018).

[0006] Currently, there are commercially available vaccines against dengue virus that have side effects and have proven not to be specific treatments. In the absence of vaccination, monitoring and sero-mapping of dengue virus outbreaks are crucial for controlling and containing the infection. Because the clinical symptoms of dengue virus infection are highly nonspecific, it is difficult to confirm the diagnosis without laboratory testing. Programs have been launched by the WHO to actively monitor vectors and fever cases, as well as to conduct serological and virological screening of individuals suspected of being infected with dengue virus. Therefore, the development of diagnostic assays for dengue infection is crucial.

[0007] Early diagnosis is essential for appropriate and timely treatment of patients and access to appropriate medical care, reducing the mortality rate to less than 1%. Currently available tests for dengue include RT-PCR for viral RNA and immunological tests for dengue-specific antibodies or viral proteins. However, many of these tests have significant drawbacks. For example, RT-PCR for viral RNA requires expensive laboratory equipment and skilled personnel, making it difficult to use on a large scale or in rural areas. Although some dengue-specific enzyme-linked immunosorbent assays (ELISAs) can detect IgM or IgG, which appear late during the course of infection, diagnosis as early as day 2 of infection is preferable.

[0008] A comparative analysis of four diagnostic methods for dengue infection—virus isolation, viral RNA detection, dengue-specific IgM detection, and NS1 antigen detection—revealed that NS1 antigen detection had the highest sensitivity rate compared with the other three methods (Kumarasamy et al. 2007). Alcon et al. (2002) described an ELISA for NS1 detection and demonstrated that NS1 is present at high levels in the sera of patients during primary and secondary infections. NS1 is detectable throughout the clinical phase of the disease and can be detected within the first few days of infection (as early as the first day of fever). Falconar and Young (1991) described the production of dimer-specific and dengue virus group-crossreactive mouse monoclonal antibodies against DEN2 virus NS1 and the use of these antibodies specifically in an ELISA for NS1 (Young et al. 2000). High levels of NS1 were found in acute-phase sera but not in convalescent-phase sera from some patients with serologically confirmed secondary DEN2 virus infection. PCT Patent Application No. WO 00 / 75665 describes methods for detecting the NS1 protein in its hexameric form and the selection of antibodies raised against the secreted hexameric form of NS1, along with the use of such antibodies in the early detection of flavivirus infection. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] PCT Patent Application No. WO 00 / 75665 Summary of the Invention [Problem to be solved by the invention]

[0010] The problem underlying the present invention is therefore to provide new markers that have diagnostic and prognostic added value compared to the commonly used NS1 marker, in particular to enable better medical care of patients at risk of developing severe dengue and to reduce mortality. [Means for solving the problem]

[0011] The problem is solved by the present invention as defined in the claims. [Brief explanation of the drawings]

[0012] [Figure 1-1] Dengue virus nonstructural NS1 binds to human HDL and LDL lipoprotein particles. (A) Size-exclusion chromatography (SEC) profiles from an NS1 pull-down experiment show a clear shift to a lower elution volume after incubation with human serum (black line) compared with NS1 protein alone (blue line). The NS1 protein partners were identified by SDS-PAGE and N-terminal sequencing as apolipoprotein B-48, a scaffold for low-density lipoprotein (LDL), in the first SEC elution peak, and ApoA-1, a scaffold for high-density lipoprotein (HDL), in the second elution peak. (B) Biolayer Interferometry signals for titrations of NS1 binding to HDL (left panel) and LDL (middle panel) particles, respectively. Steady-state single-molecule models of binding fitted to the NS1 binding signals for HDL (black dots) and LDL (white dots) are shown in the right panel. [Figure 1-2]Dengue virus nonstructural NS1 binds to human HDL and LDL lipoprotein particles. (A) Size-exclusion chromatography (SEC) profiles from an NS1 pull-down experiment show a clear shift to a lower elution volume after incubation with human serum (black line) compared with NS1 protein alone (blue line). The NS1 protein partners were identified by SDS-PAGE and N-terminal sequencing as apolipoprotein B-48, a scaffold for low-density lipoprotein (LDL), in the first SEC elution peak, and ApoA-1, a scaffold for high-density lipoprotein (HDL), in the second elution peak. (B) Biolayer Interferometry signals for titrations of NS1 binding to HDL (left panel) and LDL (middle panel) particles, respectively. Steady-state single-molecule models of binding fitted to the NS1 binding signals for HDL (black dots) and LDL (white dots) are shown in the right panel. [Figure 2-1] Visualization of NS1-HDL and NS1-LDL complexes by electron microscopy. Representative fractions of NS1-LDL (A), NS1-HDL (B), and purified NS1 input protein (C) were negatively stained with 2% uranyl formate and analyzed with a Tecnai G2 Bio-Twin electron microscope. Images were acquired with an Eagle camera and recorded in low-dose mode on a Falcon II direct electron detector. [Figure 2-2] Visualization of NS1-HDL and NS1-LDL complexes by electron microscopy. Representative fractions of NS1-LDL (A), NS1-HDL (B), and purified NS1 input protein (C) were negatively stained with 2% uranyl formate and analyzed with a Tecnai G2 Bio-Twin electron microscope. Images were acquired with an Eagle camera and recorded in low-dose mode on a Falcon II direct electron detector. [Figure 3]Image processing of electron micrographs reveals the presence of NS1 dimers on the surface of HDL particles. (A, B) Representative electron micrographs are shown from left to right: purified HDL particles (A) and representative NS1-HDL complexes (B) from the three most representative classes of NS1-HDL complexes. (C) Fit of the dimeric NS1 3D structure to the most abundant class of NS1-HDL complexes. [Figure 4-1] Figure 1 shows that NS1-HDL complexes trigger the production of proinflammatory cytokines in human primary macrophages. Macrophages were collected from four different donors and incubated with various effectors as indicated for 24 hours. LPS stimulation was used as a positive control. At the end of the incubation period, cell culture supernatants were collected, and the concentrations of TNFα, IL-1β, IL-6, and IL-10 were quantified using Luminex assays. Data represent the mean ± SEM. Mean cytokine levels were compared using two-way analysis of variance (Anova). [Figure 4-2] Figure 1 shows that NS1-HDL complexes trigger the production of proinflammatory cytokines in human primary macrophages. Macrophages were collected from four different donors and incubated with various effectors as indicated for 24 hours. LPS stimulation was used as a positive control. At the end of the incubation period, cell culture supernatants were collected, and the concentrations of TNFα, IL-1β, IL-6, and IL-10 were quantified using Luminex assays. Data represent the mean ± SEM. Mean cytokine levels were compared using two-way analysis of variance (Anova). [Figure 5-1]Figure 1 shows standard ELISAs for quantification of (A) NS1 antigen, (B) NS1-ApoA1 / HDL complex, or (C) NS1-ApoE-positive lipoprotein particles. A schematic diagram of the principles of the three different ELISAs is shown on the left side of the figure. (A) Standard curves of in vitro reconstituted dengue NS1. Purified DENV-NS1 was incubated at known concentrations in normal plasma obtained from human donors at 37°C for 1 hour and 30 minutes. DENV-NS1 was captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound NS1-mAb17A12 complex was further detected with peroxidase-labeled anti-dengue NS1 MAb (8G6). (B) Standard curves of in vitro reconstituted dengue NS1-HDL complex for DENV-NS1. NS1-HDL complexes were captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound complexes were further detected with a commercially available anti-ApoA1 polyclonal antibody followed by a species-specific peroxidase-conjugated secondary antibody. (C) An anti-ApoE polyclonal antibody was instead used as the secondary antibody to detect NS1 complexes formed with ApoE. Concentration values ​​reported on the x-axis are shown as NS1 equivalent concentrations, an estimate based on our observation that all NS1 molecules bind to HDL, which is present in large excess in this experimental setting. All curves are representative of at least three sets of experiments. [Figure 5-2]Figure 1 shows standard ELISAs for quantification of (A) NS1 antigen, (B) NS1-ApoA1 / HDL complex, or (C) NS1-ApoE-positive lipoprotein particles. A schematic diagram of the principles of the three different ELISAs is shown on the left side of the figure. (A) Standard curves of in vitro reconstituted dengue NS1. Purified DENV-NS1 was incubated at known concentrations in normal plasma obtained from human donors at 37°C for 1 hour and 30 minutes. DENV-NS1 was captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound NS1-mAb17A12 complex was further detected with peroxidase-labeled anti-dengue NS1 MAb (8G6). (B) Standard curves of in vitro reconstituted dengue NS1-HDL complex for DENV-NS1. NS1-HDL complexes were captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound complexes were further detected with a commercially available anti-ApoA1 polyclonal antibody followed by a species-specific peroxidase-conjugated secondary antibody. (C) An anti-ApoE polyclonal antibody was instead used as the secondary antibody to detect NS1 complexes formed with ApoE. Concentration values ​​reported on the x-axis are shown as NS1 equivalent concentrations, an estimate based on our observation that all NS1 molecules bind to HDL, which is present in large excess in this experimental setting. All curves are representative of at least three sets of experiments. [Figure 5-3]Figure 1 shows standard ELISAs for quantification of (A) NS1 antigen, (B) NS1-ApoA1 / HDL complex, or (C) NS1-ApoE-positive lipoprotein particles. A schematic diagram of the principles of the three different ELISAs is shown on the left side of the figure. (A) Standard curves of in vitro reconstituted dengue NS1. Purified DENV-NS1 was incubated at known concentrations in normal plasma obtained from human donors at 37°C for 1 hour and 30 minutes. DENV-NS1 was captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound NS1-mAb17A12 complex was further detected with peroxidase-labeled anti-dengue NS1 MAb (8G6). (B) Standard curves of in vitro reconstituted dengue NS1-HDL complex for DENV-NS1. NS1-HDL complexes were captured using immobilized anti-NS1 monoclonal antibody (17A12), and the bound complexes were further detected with a commercially available anti-ApoA1 polyclonal antibody followed by a species-specific peroxidase-conjugated secondary antibody. (C) An anti-ApoE polyclonal antibody was instead used as the secondary antibody to detect NS1 complexes formed with ApoE. Concentration values ​​reported on the x-axis are shown as NS1 equivalent concentrations, an estimate based on our observation that all NS1 molecules bind to HDL, which is present in large excess in this experimental setting. All curves are representative of at least three sets of experiments. [Figure 6]This figure shows the detection of NS1-ApoA1 complexes in the plasma of hospitalized dengue virus-infected patients. Apolipoprotein A1 is a scaffolding protein for HDL. The concentration of NS1-ApoA1 complexes represents the abundance of viral lipoprotein NS1-HDL particles in plasma samples. NS1-ApoA1 complexes were quantified by ELISA in two groups of hospitalized patients classified as exhibiting dengue with dangerous symptoms (DWWS, n = 36; Panels A and B) or severe dengue (SVD, n = 19; Panels C and D). The two groups were tested after hospital admission (ADM) or during follow-up visits (F-VIS) (Panels A and C), or at the time of admission and, for patients who recovered during their hospital stay, at the time of discharge (Panels B and D). On average, two blood samples were collected from each patient, 3–4 days apart. The dotted line in each graph indicates the threshold for positivity. [Figure 7] Figure 6 shows the detection of NS1-ApoE complexes in the plasma of hospitalized dengue virus-infected patients. The samples described in Figure 6 were tested using an ApoE-specific ELISA. The assay described in Figures 5 and 6 was slightly modified by replacing the secondary anti-ApoA1 polyclonal antibody with a commercially available anti-ApoE polyclonal antibody. The legends for the four panels A, B, C, and D are the same as in Figure 6. [Figure 8] Figure 1 shows correlation tests between NS1-ApoA1 and NS1-ApoE concentrations and disease severity in dengue virus-infected patients showing either DWWS or SVD. The concentrations of NS1-ApoA1 (Panel A) or NS1-ApoE (Panel B) complexes are plotted separately for two clinical groups of patients who developed either DWWS or SVD during DENV infection. The mean values ​​observed for the different parameters are indicated by flat bars and compared using two-way analysis of variance (Anova). [Figure 9]Figure 5 shows that the ability of NS1 protein to bind to HDL is shared among flaviviruses. ELISA detection of NS1-ApoA1 complexes with NS1 from different flaviviruses. Purified NS1 from different flaviviruses (yellow fever, YF; Zika; West Nile, WN; Japanese encephalitis, JE; and tick-borne encephalitis, TBE) was spiked into normal plasma for 1 hour and 30 minutes at 37°C, and NS1-HDL complexes were further detected by capture ELISA. A calibration curve was obtained using in vitro reconstituted dengue NS1-HDL complexes. Different flavivirus NS1-HDL complexes were captured using the immobilized anti-dengue NS1 monoclonal antibody (17A12) depicted in Figure 5, and the bound complexes were further detected with a commercially available anti-ApoA1 polyclonal antibody followed by a species-specific peroxidase-labeled secondary antibody. Concentration values ​​reported on the x-axis are shown as NS1 equivalent concentrations, an estimate based on our observation that all NS1 molecules bind to HDL, which is present in large excess in this experimental setting. [Figure 10] Figure 1 shows a correlation test between NS1-ApoA1 and NS1-ApoE concentrations and plasma leakage intensity in dengue virus-infected patients upon admission to the hospital. The concentration of NS1-ApoA1 (A) or NS1-ApoE (B) complexes is plotted against the intensity of pleural fluid observed by ultrasound and scored as 0 (no detectable pleural fluid), 1 (moderate effusion), and 2 (massive effusion) for dengue-risk patients (DWWS). All severe dengue patients (SVD) were grade 2. A statistical difference was observed between the DWWS grade 0 and SVD groups of patients for NS1-ApoA1 complexes (p = 0.005), but not for NS1-ApoE complexes (p = 0.42; ns, not significant). [Figure 11] Figure 1 shows the detection of NS1-ApoB complexes in the plasma of hospitalized dengue virus-infected patients. Apolipoprotein B is a scaffolding protein for LDL. NS1-ApoB complexes were quantified by ELISA (A) (Materials and Methods) in dengue virus-infected patients (n=7) or non-dengue hospitalized patients (n=3). The two groups were tested for the presence of NS1-ApoB complexes at the time of admission to the hospital (ADM). [Figure 12] Figure 1 shows that binding of NS1-HDL lipoprotein complexes can be blocked by NS1-specific MAbs. Several anti-dengue NS1 MAbs were incubated with purified NS1, and the resulting immune complexes were exposed to immobilized HDL. The residual binding capacity of NS1 was measured by BLI as in Figure 1B. Purified NS1 alone was used as a positive control. Notably, none of the MAbs tested in the absence of NS1 bound to HDL particles. DETAILED DESCRIPTION OF THE INVENTION

[0013] We found that dengue virus NS1, secreted by infected cells as atypical lipoprotein particles, binds with strong affinity to serum high-density lipoprotein particles (HDL), which play a major role in vascular homeostasis, inflammation, and thrombosis, and, to a lesser extent, low-density lipoprotein particles (LDL). The NS1-HDL complex (but not the NS1 protein alone) induces cytokine production in primary human macrophages, suggesting that this complex represents a previously unreported active form of the viral protein. Concurrently, we developed three ELISAs based on the detection of NS1-ApoA1, NS1-ApoB, and NS1-ApoE in DENV-infected patients to assess the concentrations of complexes formed between NS1 and HDL, between NS1 and LDL, or between NS1 and a larger panel of lipoprotein particles (optionally HDL, IDL, LDL, VLDL, and chylomicrons). NS1-ApoA1 complexes were detected in the majority of acute-phase blood samples and appeared to be inversely correlated with the number of days of fever. In contrast, NS1-ApoE complex concentrations increased over time during the 12-day observation period. Biostatistical analysis of various virological and clinical markers revealed that NS1-HDL complex concentrations were inversely correlated with the degree of disease severity, particularly the intensity of plasma leakage. NS1-HDL complexes therefore represent a novel diagnostic marker with prognostic added value that is more relevant than NS1 protein itself and has yet to be more fully evaluated in larger clinical trials.

[0014] The present invention therefore relates to a complex formed by flavivirus nonstructural protein 1 (NS1) and endogenous lipoprotein particles present in plasma selected from among high density lipoprotein particles (HDL), low density lipoprotein particles (LDL), intermediate density lipoprotein particles (IDL), very low density lipoprotein particles (VLDL) and chylomicrons.

[0015] According to certain embodiments, the complex has a strong affinity (equilibrium dissociation constant K D In a more particular embodiment, the complex is formed by flavivirus NS1 and an apolipoprotein A1 (ApoA1)-positive lipoprotein particle.

[0016] According to a particular embodiment, the complex has a lower affinity (equilibrium dissociation constant K D In a more particular embodiment, the complex is formed by flavivirus NS1 and an apolipoprotein B (ApoB)-positive lipoprotein particle, or flavivirus NS1 and an apolipoprotein E (ApoE)-positive lipoprotein particle.

[0017] The present invention also relates to a biomarker for the diagnosis, prognosis or monitoring of flavivirus infection or an associated disease, preferably dengue virus infection or an associated disease, comprising a complex formed by flavivirus nonstructural protein 1 (NS1) and an endogenous lipoprotein particle, in particular a plasma lipoprotein particle according to the present disclosure.

[0018] The present invention also relates to the use of the presence or level of a complex formed by flavivirus nonstructural protein 1 (NS1) and endogenous lipoprotein particles, particularly plasma lipoprotein particles, as defined above, as a biomarker for the diagnosis, prognosis, or monitoring of flavivirus infection or related diseases, preferably dengue virus infection or related diseases, according to the present disclosure. In some preferred embodiments, the NS1-HDL particle complex, preferably the NS1-ApoA1-positive lipoprotein particle complex, is used as a biomarker for the prognosis of flavivirus infection or related diseases. In some other preferred embodiments, the NS1-plasma lipoprotein particle complex, preferably one or more of the NS1-ApoA1, NSI-ApoE, and NS1-ApoB-positive lipoprotein particle complex, is used as a biomarker for the diagnosis of flavivirus infection or related diseases. In some other preferred embodiments, the NS1-plasma lipoprotein particle complex, preferably one or more of the NS1-ApoA1 and NSI-ApoE-positive lipoprotein particle complex, is used as a biomarker for the monitoring of flavivirus infection or related diseases.

[0019] The present invention also relates to an in vitro method for diagnosing, prognosing or monitoring a flavivirus infection or associated disease, comprising detecting the presence or level of a complex formed by flavivirus nonstructural protein 1 (NS1) as defined above and plasma lipoprotein particles in a biological sample obtained from a subject.

[0020] The complexes can be detected by any suitable means, using standard techniques, e.g., chemical, physical, or other techniques, hi some embodiments, the complexes are detected using immunochemical methods.

[0021] For the purposes of the present invention, the term "nonstructural protein 1" is used interchangeably with "nonstructural glycoprotein 1" and "NS1" and encompasses the native protein obtained from the culture supernatant of mammalian cells infected with a flavivirus or transformed and purified using an expression system containing the gene for the NS1 protein of said flavivirus. NS1 may be in a hexameric, monomeric, or dimeric form.

[0022] The present invention also provides an in vitro method for quantifying the complex formed by flavivirus nonstructural protein 1 (NS1) and endogenous lipoprotein particles, in particular plasma lipoprotein particles, in a biological sample obtained from a subject, comprising: a. contacting a biological sample with an antibody specific for flavivirus NS1 and / or an antibody specific for an endogenous lipoprotein, particularly a plasma lipoprotein, to form an immune reaction product; b. detecting the presence of an immune reaction product; c. Quantifying the complex formed by NS1 and endogenous lipoprotein particles, particularly plasma lipoprotein particles; The present invention also relates to the method comprising:

[0023] In some embodiments, the method comprises: a. contacting a biological sample with an antibody specific for flavivirus NS1 to form a first immune reaction product; b. contacting the first immune reaction product with an antibody specific for a plasma lipoprotein to form a second immune reaction product; c. detecting the presence of a second immune reaction product; d. Quantifying the complex formed by NS1 and plasma lipoprotein particles; Includes.

[0024] Flaviviral and flavivirus according to the present invention refer in particular to and include dengue virus, West Nile virus, Japanese encephalitis virus, Zika virus and yellow fever virus. According to an advantageous embodiment of the invention, the flavivirus is dengue virus.

[0025] The term "lipoprotein particle" is used interchangeably with "lipoprotein" in this description and includes high-density lipoprotein (HDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very-low-density lipoprotein (VLDL), and chylomicrons. The terms "HDL," "LDL," "VLDL," and "IDL" are used interchangeably with "HDL particle," "LDL particle," "VLDL particle," and "IDL particle," respectively. "NS1-ApoA1 complex," "NS1-ApoE complex," and "NS1-ApoB complex" refer to complexes of NS1 with ApoA1-, ApoE-, and ApoB-positive apolipoprotein particles, respectively.

[0026] In certain embodiments of the invention, the antibodies used may be synthetic, monoclonal, or polyclonal, and may be produced by techniques well known in the art. Such antibodies bind specifically (as opposed to non-specifically) via the antigen-binding site of the antibody. Monoclonal antibodies include antigen-binding fragments and chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.

[0027] According to a particular embodiment of the method, an antibody specific for flavivirus NS1 is used as a capture antibody and an antibody specific for plasma lipoprotein particles is used as a detection antibody.

[0028] According to an advantageous embodiment of said method, the antibody specific for flavivirus NS1 is coated on a solid support and the antibody specific for plasma lipoprotein is a detection or revelation antibody, which is optionally conjugated to a suitable label.

[0029] According to another advantageous embodiment of the method, if the antibody specific for plasma lipoproteins is not conjugated to a label, a third antibody raised against this antibody and conjugated to a suitable label is used to detect the presence of the second immune reaction product. The third antibody is a conventionally used antibody, such as an IgG raised against this antibody, in particular raised in goats, pigs or donkeys. Among the labels used, mention may be made of fluorescent labels, biotin / streptavidin systems, non-isotopic labels or enzymes, such as horseradish peroxidase or alkaline phosphatase.

[0030] According to another advantageous embodiment of said method, the antibody specific for a plasma lipoprotein is an antibody specific for ApoE.

[0031] According to another advantageous embodiment of the method, the plasma lipoprotein particles are high density lipoprotein (HDL) particles, in which case the plasma lipoprotein-specific antibody may be an antibody specific for apolipoprotein A1 (ApoA1).

[0032] According to another advantageous embodiment of said method, the plasma lipoprotein particles are low density lipoprotein (LDL) particles, in which case the plasma lipoprotein-specific antibody may be an antibody specific for apolipoprotein B.

[0033] In some embodiments, an in vitro method for quantifying complexes formed by flavivirus nonstructural protein 1 (NS1) and endogenous lipoprotein particles comprises first contacting a biological sample with an antibody specific for a plasma lipoprotein, and then contacting the biological sample with an antibody specific for flavivirus NS1.

[0034] In some other embodiments, an in vitro method for quantifying complexes formed by flavivirus nonstructural protein 1 (NS1) and endogenous lipoprotein particles comprises simultaneously contacting a biological sample with an antibody specific for a plasma lipoprotein and an antibody specific for flavivirus NS1.

[0035] The present invention also relates to an in vitro method for prognosing severe flavivirus infection in a flavivirus-infected subject, comprising carrying out the in vitro method for quantifying complex NS1-plasma lipoprotein particles described above in a biological sample obtained from the subject after infection, and preferably during primary or acute infection, wherein the higher the level of the complex, the lower the risk of developing severe flavivirus infection. In a preferred embodiment of the method, the complex is formed by flavivirus NS1 and HDL, preferably flavivirus NS1 and ApoA1-positive lipoprotein particles.

[0036] The present invention also relates to an in vitro method for monitoring flavivirus disease in a subject infected with a flavivirus, comprising carrying out the method for quantifying complex NS1-plasma lipoprotein particles described above on biological samples obtained from the subject at different time points during the flavivirus disease.

[0037] The present invention also relates to an in vitro method for diagnosing flavivirus infection in a subject, comprising carrying out the above-described method for quantifying complex NS1-plasma lipoprotein particles on a biological sample obtained from the subject, wherein the presence of a complex formed by flavivirus NS1 and plasma lipoprotein particles in the sample indicates flavivirus infection.

[0038] According to a particular embodiment of the in vitro method for diagnosing flavivirus infection, the complex formed by flavivirus NS1 and HDL particles, preferably the complex formed by flavivirus NS1 and ApoA1-positive lipoprotein particles, is quantified.

[0039] According to another particular embodiment of the in vitro method for diagnosing flavivirus infection, the method for quantifying complex NS1-plasma lipoprotein particles described above is repeatedly performed on a biological sample obtained from the subject to quantify flavivirus NS1-HDL particle complexes and / or flavivirus NS1-LDL particle complexes and / or flavivirus NS1-other plasma lipoprotein particle complexes, preferably one or more of flavivirus NS1-Apo1-positive lipoprotein particle complexes, flavivirus NS1-ApoB-positive lipoprotein particle complexes, and flavivirus NS1-ApoE-positive lipoprotein particle complexes; in particular flavivirus NS1-Apo1-positive lipoprotein particle complexes and flavivirus NS1-ApoE-positive lipoprotein particle complexes.

[0040] The present invention also provides a kit for detecting a complex formed by flavivirus NS1 and plasma lipoprotein particles in a biological sample obtained from a subject, comprising: a.An antibody specific for flavivirus NS1; b. an antibody specific for a plasma lipoprotein, preferably specific for HDL, more preferably specific for ApoA1; c. a means for detecting the production of an immune reaction product between the two antibodies and a complex formed by flavivirus NS1 and plasma lipoprotein particles; The present invention also relates to a kit as described above, comprising:

[0041] Like the antibodies specific for plasma lipoproteins used in the kits according to the invention, the antibodies specific for flavivirus NS1 may be polyclonal antibodies, monoclonal antibodies, or antigen-binding portions thereof.

[0042] According to a particular embodiment, the antibody specific for a plasma lipoprotein is specific for HDL, preferably for the ApoA1 protein carried by HDL.

[0043] According to another particular embodiment, the antibody specific for plasma lipoproteins is specific for LDL, preferably for the ApoB protein carried by LDL.

[0044] According to another particular embodiment, the plasma lipoprotein-specific antibody recognizes several populations of plasma lipoproteins, and is preferably specific for the ApoE protein.

[0045] According to a particular embodiment, the kit comprises several antibodies specific for plasma lipoproteins, each of these antibodies being specific for one population of plasma lipoproteins.

[0046] In some embodiments, the kit comprises an antibody specific for ApoB; preferably, the kit further comprises an antibody specific for ApoA1 and / or ApoE.

[0047] According to a particular embodiment, the antibody specific for said plasma lipoprotein carries a detectable label.

[0048] According to a particular embodiment, said means for detecting the production of an immune reaction product comprises a third antibody directed against an antibody specific for a plasma lipoprotein and conjugated to a suitable label.

[0049] According to certain embodiments, the kit further comprises instructions for use of said kit.

[0050] According to certain embodiments, the kit further comprises at least one standard sample, which may be a plasma sample from a healthy donor, a patient whose lipid markers have returned to normal at the time of hospital discharge and who has essentially recovered from the disease, or a patient who was hospitalized without delay after fever (i.e., had had a fever for 3 days or less at the time of admission) spiked with a known amount of purified flavivirus NS1.

[0051] This standard allows a more reliable definition of the positivity threshold that should be considered for the assay.

[0052] For the NS1-ApoA1 and NS1-ApoB assays, the calculation of the positivity threshold was based on the principle that patients whose lipid markers had returned to normal at the time of discharge (i.e., Total cholesterol >3 mmol / mL, LDL cholesterol >1.5 mmol / mL, HDL cholesterol >0.4 mmol / mL, triglycerides <2.7 mmol / mL; n = 9) had essentially recovered from the disease and had become negative for the NS1-ApoA1 complex (Figure 5). The positivity threshold was then considered to be twice this mean value. The strategy for estimating the positivity threshold for the NS1-ApoE assay differs due to the fact that the trend of NS1-ApoE complex formation / accumulation is opposite to that of the NS1-ApoA1 complex, since NS1-ApoE concentrations increase overall over time. The calculation of the mean value was based on the group of patients who were admitted without delay after fever (i.e., fever ≤3 days at admission; n = 11).

[0053] The kit according to the invention is used in the in vitro method according to the invention.

[0054] The present invention also provides a method of treating a flavivirus infection or an associated disease in a subject in need thereof, comprising: - diagnosing flavivirus infection in a subject by detecting the presence or level of a complex formed by flavivirus nonstructural protein 1 (NS1) and plasma lipoprotein particles in a biological sample obtained from the subject, wherein the presence or level of the complex is indicative of flavivirus infection; - administering appropriate treatment if the subject is diagnosed with a flavivirus infection; The present invention also relates to a method, including:

[0055] The NS1-plasma lipoprotein particle complexes are detected as disclosed herein, preferably using a method for quantifying the complexes according to the present disclosure.

[0056] The treatment may be any suitable therapy known in the art for treating flavivirus infection, such as antiviral therapy, immunotherapy, and combinations thereof. The treatment may include administration of an antibody that blocks the formation of a complex between NS1 and endogenous lipoproteins. The treatment may also include administration of exogenous lipoprotein particles.

[0057] In some embodiments of the treatment method, the severity of flavivirus infection is determined by detecting the level of NS1-HDL complexes, with a higher level of NS1-HDL complexes indicating a lower risk of developing a severe form of flavivirus infection. The complexes are preferably NS1-ApoA1-positive lipoprotein particle complexes. Detection is preferably performed during primary or acute infection, as disclosed above. Detection advantageously allows for tailoring of an individual's treatment depending on the severity of the disease.

[0058] In some embodiments, the treatment method includes monitoring flavivirus disease in a subject infected with a flavivirus, by performing the method for quantifying complex NS1-plasma lipoprotein particles described above on biological samples obtained from the subject at different times during the flavivirus disease.

[0059] In certain embodiments of the invention, the flavivirus NS1 is NS1 from a dengue virus and / or the flavivirus infection is a dengue virus infection.

[0060] In certain embodiments of the invention, the biological sample is blood, plasma or serum.

[0061] In a particular embodiment of the invention, detection of NS1-lipoprotein complexes is an early detection during the clinical phase of flavivirus infection, particularly during primary or acute flavivirus infection.

[0062] In certain embodiments of the invention, the subject is a human.

[0063] The features described herein above, as well as other features of the invention, will become clear on reading the examples and figures illustrating experiments carried out by the inventors that complement the features and definitions set out in the present description, but the examples are not limiting with respect to the described invention. [Example]

[0064] Materials and Methods DENV or flavivirus NS1 spikes in human plasma DENV2 recombinant NS1 protein (400 μg) was incubated in 1 mL of serum or plasma obtained from healthy donors (provided by the Pasteur Institute IcareB Biological Facility) for 1 h at 37°C. All human samples complied with ethical regulations.

[0065] NS1 serum pulldown The mixture was then purified through a Strep-tactin column (Iba) and washed twice with PBS MgCa (Gibco), followed by 14 column volumes of PBS 0.3 M NaCl and a further 5 column volumes of PBS MgCa. Elution was performed using 2.5 mM D-desthiobiotin (Iba) in PBS Mg / Ca.

[0066] Size Exclusion Chromatography (SEC) Gel filtration of recombinant NS1, HDL, or NS1-HDL solutions was performed on a Superdex 200 10 / 300 column equilibrated with PBS MgCa++ at 0.4 ml / min, collecting 0.5 ml fractions. Elution profiles were compared with those of Bio-Rad standards.

[0067] SDS-PAGE of NS1 and NS1-HDL SEC fractions All protein samples were denatured with 5x Laemmli Sample Buffer (Bio-Rad) containing β-mercaptoethanol and boiled at 95°C for 5 min. They were separated by discontinuous sodium dodecyl sulfate (SDS) 10% polyacrylamide gel electrophoresis (SDS-PAGE precast gel, Bio-Rad). SDS-PAGE gels were stained with Coomassie Blue solution (Bio-Rad).

[0068] Biolayer Interference We titrated DENV2 NS1 binding to HDL or LDL using the Octet Red (ForteBio) Biolayer Interference Assay (BLI). Experiments were performed in 96-well plates at 25°C with a shaking speed of 1000 rpm.

[0069] HDL had a uniform diameter of 9.6 nm and was commercially available (Merck Millipore). Streptavidin A (SA) "dip and read" biosensors (ForteBio) were activated and loaded with biotinylated anti-ApoA-I or anti-ApoB antibodies, followed by the respective lipoproteins and then the NS1 protein solution. The BLI signal of NS1 bound to the lipoproteins was monitored over time, and controls of NS1 bound to the free tip and free antibody, as well as background noise from buffer interactions with the lipoproteins or antibodies, were measured. The controls were subtracted from the corresponding signal for each NS1 concentration. The binding signals were analyzed using Scrubber software for data extraction and normalization, BIAevaluation (BIACORE) for data control subtraction, and Profit (Quantumsoft) for data fitting.

[0070] Capture ELISA of NS1-ApoA1 or NS1-ApoE lipoprotein complexes Briefly, microtiter plates were coated overnight with immunoaffinity-purified mouse anti-NS1 polyclonal antibody or dengue NS1-specific monoclonal antibody 17A12 (deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) under the terms of the Budapest Treaty under number I-3186 on March 4, 2004). After saturating and washing the wells, serial dilutions of purified dengue virus type 1 NS1-spiked human serum or dengue virus-infected human serum were added to the wells for 2 hours at room temperature. The wells were washed again and incubated with anti-ApoA-I or anti-ApoE goat polyclonal antibody for 1 hour at 37°C, followed by development with a peroxidase-conjugated secondary antibody in 3,3",5,5"-tetramethylbenzidine solution. Negative controls were measured in the absence of antigen. Absorbance values ​​were corrected using the mean values ​​of the negative controls. Healthy donors were recruited through the IcareB Platform at the Institut Pasteur.

[0071] Detection of NS1-ApoB lipoprotein complexes by capture ELISA The protocol used is depicted in Figure 11(A). Microtiter plates were coated overnight with purified mouse anti-NS1 polyclonal or anti-NS1 monoclonal antibodies. After saturating and washing the wells, serial dilutions of dengue virus-infected human sera or control patients infected with other pathogens were added to the wells for 2 hours at room temperature. The wells were washed again and incubated with anti-ApoB biotinylated goat polyclonal antibodies for 1 hour at 37°C, after which peroxidase-conjugated streptavidin was developed with a 3,3",5,5"-tetramethylbenzidine solution. Quantification of NS1-ApoB complexes, reported in Figure 11(B), was based on a standard curve obtained using normal human plasma spiked with known concentrations of purified recombinant NS1 antigen (A).

[0072] Serum samples from patients infected with dengue virus Human sera were collected from patients infected with dengue virus type 1 during the 2011-2012 epidemic in Cambodia. Dengue virus diagnosis was confirmed by RT-PCR on blood samples collected on the first day of hospitalization. Each patient was also tested by MAC-ELISA and hemagglutination inhibition (HI) tests on paired sera (acute and convalescent phase). Various clinical and biochemical information was available for the included patients, allowing us to classify them into groups with different disease severity according to the 2009 WHO classification system. Classical dengue fever was not observed among hospitalized patients classified using the 2009 classification; approximately two-thirds of patients had dengue with warning signs (DWWS), and one-third developed severe dengue (SVD).

[0073] Electron Microscopy Imaging Purified HDL or NS1-HDL complexes were spotted onto glow-discharged carbon grids (CF300, EMS, USA), negatively stained with 2% uranyl formate (UFA) pH 7.4, analyzed with a Tecnai G2 Bio-Twin electron microscope (FEI, USA), and imaged with an Eagle camera (FEI, USA). Image frames were recorded in low-dose mode on a Falcon II direct electron detector (FEI, USA).

[0074] Image analysis HDL and NS1-HDL negative staining images were CTF corrected (phase flip) and sorted using XMIP software (Scheres et al. 2005). Corrected images were imported into Relion (Scheres 2012). The recommended particle picking strategy was applied as follows: manual selection of particles matching the HDL or NS1-HDL size was performed on a small number of images (approximately 15). 2D classification (40 classes) was performed, and five representative, distinct classes were selected as templates for automatic picking, resulting in approximately 30,000 particles. 2D classification (200 classes) was then performed. Classes that clearly represented artifacts were suppressed, and a new run of 2D classification (200 classes) was performed.

[0075] Primary monocyte culture and differentiation into macrophages PBMCs were isolated from whole blood using Ficoll gradient centrifugation (Eurobio). CD14+ cells were purified by magnetic bead separation of PBMCs using a CD14+ Human Positive Selection Kit (StemCell) and plated at 1 ml / ml in Teflon plates (Sarstedt) at 7 ml per plate in the following medium: RPMI-1640 (Gibco), 2 mM L-glutamine (Life Technologies), 1% penicillin-streptomycin (Life Technologies) at concentrations of 10,000 units penicillin and 10 mg streptomycin / ml, 10 mM Na-pyruvate (Life Technologies), 10 mM HEPES (Life Technologies), 1% MEM vitamins (Life Technologies) from stock, 1% NEAA (Life Technologies), 50 μM beta-mercaptoethanol (Life Technologies), and 15% human serum. Monocytes were cultured in differentiation medium for 6–8 days, after which macrophages were scraped off the Teflon plates and counted. After spinning, macrophages were resuspended at 1 μg / ml in the same medium (except containing 10% FBS instead of human serum).

[0076] Macrophage immune activation assay Macrophages were plated on a P24 plate (Corning) at a density of 0.5 × 10 6 Macrophages were seeded at 1000 cells / mL. After 2 hours of cell settling and adhesion, macrophages were incubated with plain PBS, HDL, NS1, or NS1-HDL mix for 24 hours before supernatant collection. Luminex 5-plex assays were performed on all supernatants according to the manufacturer's recommendations (R&D Systems Human 5-plex Kit). Standards were run on each plate per assay to titrate the levels of cytokines present.

[0077] statistical analysis Significant differences between groups were determined by one-way ANOVA analysis performed using GraphPad Prism software.

[0078] antibody Anti-ApoA1 goat polyclonal antibody was provided by Novus Biologicals (product number NB400-147). Anti-ApoE goat polyclonal antibody is provided by Calbiochem (product number 178479). Peroxidase-conjugated secondary antibody is provided by Southern Biotech (product number 6425-05). Anti-ApoB biotinylated goat polyclonal antibody was provided by ABCAM (product number ab20898). Peroxidase-conjugated streptavidin is provided by Interchim (product number 396888). Biotinylated antibodies against ApoA1 or ApoB were provided by ABCAM (product numbers ab27630 and ab20898, respectively). Mouse anti-NS1 polyclonal antibody is obtained as described by Alcon-LePoder et al. 2006. The following murine hybridoma cell cultures secreting dengue NS1-specific monoclonal antibodies have been deposited at the Collection Nationale de Cultures de Microorganismes (Paris, France) under the following accession numbers: mAb 17A12 I-3186, March 4, 2004 mAb 8G6 I-5291, March 13, 2018 mAb 1A11 I-5290, March 13, 2018 mAb4F7 I-3185, March 4, 2004

[0079] Example 1 DENV NS1 preferentially binds to human high-density lipoproteins - Identification of a novel NS1 protein ligand We performed pull-down assays using purified preparations of a tagged version of DENV2 NS1 in serum obtained from healthy human donors and analyzed the resulting products by size-exclusion chromatography (SEC) (Fig. 1A). Compared with the NS1 protein alone, the pull-down SEC profile showed an additional peak and a large shoulder at a lower elution volume (Fig. 1A). The protein content was analyzed by SDS-PAGE, and the identity of prominent protein bands was determined by mass spectrometry. The band migrating with a molecular weight (MW) of 29 kDa corresponded to apolipoprotein A1 (ApoA1), and the one with a higher MW corresponded to apolipoprotein B-48 (>250 kDa). These two major species correspond to the major scaffolding proteins of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively (Fig. 1A).

[0080] Example 2 DENV NS1 preferentially binds to human high-density lipoproteins - Binding affinity measurement by biolayer interference We used biolayer interferometry (BLI) to assess the relative affinity of NS1 for HDL and LDL particles (Figure 1B). Purified HDL and LDL particles were immobilized on streptavidin-coated biosensors and further loaded with biotinylated antibodies against ApoA1 or ApoB, respectively. NS1 titration binding experiments on HDL- or LDL-loaded biosensors showed the true amplitude of the interferometry signal relative to the background (Figure 1B). On the right side of Figure 1B, equilibrium values ​​for NS1 titration for LDL and HDL are reported, including deviations observed in at least three experiments. We obtained K values ​​of 63.8 nM and 1.4 μM, respectively. DTo compare the equilibrium dissociation constants of NS1 with HDL and LDL, the titration values ​​were interpreted by fitting the data to a steady-state single-site binding model. This difference suggested that NS1 preferentially binds HDL. Notably, the NS1-HDL complex remained stable over a 6-day period (data not shown), suggesting that once formed, the protein remains tightly bound to lipoprotein particles.

[0081] Example 3 DENV NS1 preferentially binds to human high-density lipoproteins - Visualization of NS1-HDL complexes by electron microscopy We further analyzed the complexes by negative staining electron microscopy (Figures 2 and 3). As previously shown by others, human HDL and LDL particles appear as smooth spheres with diameters of approximately 10 and 20 nm, respectively (Zhang et al. 2015).

[0082] The NS1-HDL and NS1-LDL complexes have sizes consistent with these values ​​(Fig. 2). However, in contrast to the smooth appearance of HDL alone, the NS1-HDL complex exhibited a granular surface with protruding rod-like structures (Fig. 3). These nodules correspond well to the dimensions of the NS1 dimer. NS1 in its dimeric form is known to expose a large hydrophobic patch on one of its faces. NS1 dimers can therefore directly insert into the lipid phase of HDL, a feature that confers particularly high stability to the complex.

[0083] Example 4 DENV NS1 Preferentially Binds to Human High-Density Lipoprotein-Characterization of the Biological Activities Associated with NS1-HDL Complexes HDL is a potent modulator of inflammation, and under physiological conditions, these lipoproteins are primarily anti-inflammatory regulators. Conversely, NS1 is known to trigger the secretion of proinflammatory cytokines in macrophages (Modhiran et al. 2015). To establish whether NS1 requires binding to HDL to become functional, we compared the activation state of macrophages treated with various effectors. We used human primary macrophages differentiated from monocytes isolated from various donors and stimulated the cells with NS1 alone (10 μg / mL), HDL alone (2.5 μg / mL), NS1-HDL (a mix of 10 and 2.5 μg / mL, respectively), and LPS as a positive control. After a 24-hour incubation period, supernatants were collected, and the levels of IL-1beta, TNF-alpha, IL-6, and IL-10 were measured using a Luminex assay (Figure 4).

[0084] With NS1 and HDL alone, we observed no difference in cytokine levels compared with the negative control (Figure 4), whereas LPS consistently induced high cytokine levels (data not shown). These observations ruled out any cytotoxic effects from background contaminants in the NS1 and HDL purified samples and indicated that NS1 itself was unable to induce proinflammatory activation of human macrophages. In contrast, the NS1-HDL complex increased cytokine secretion to a significant level in different donor cells compared with HDL or NS1 alone (Figure 4). This demonstrated that the NS1-HDL complex is the bioactive form of the protein and a potent modulator of inflammation.

[0085] Example 5 Quantification of viral or host factors in plasma from DENV-infected patients - Quantification of NS1 and NS1-lipoprotein complexes in plasma Next, we evaluated the presence of NS1-HDL complexes compared with secreted forms of NS1 in DENV-infected patients. To this end, we used the classical ELISA for NS1 detection (Alcon-LePoder et al. 2006) and developed two ELISAs to specifically detect NS1 complexes in human plasma samples. In the first assay, we used an anti-NS1 monoclonal antibody to capture NS1-HDL complexes and an anti-ApoA1 polyclonal antibody to detect the bound material (Figure 5). We instead used an anti-ApoE polyclonal antibody as a secondary antibody to potentially detect NS1 complexes formed with other types of lipoprotein particles as well (IDL, VLDL, chylomicrons, and in certain instances, HDL and LDL as well; see the 2019 review from D. Marais in Pathology). To establish a calibration curve in the analytical format, purified NS1 was incubated in plasma from healthy donors obtained at ICAReB (Institut Pasteur), and concentration values ​​of the NS1-ApoA1, NS1-ApoB, or NS1-ApoE complexes were calculated based on the equivalent concentration of NS1 (Figure 5; Figure 11). The detection limits for the NS1, NS1-ApoA1, or NS1-ApoE assays were set as twice the mean signal obtained with normal human plasma, corresponding to equivalent NS1 concentrations of 0.5, 0.3, and 15 ng per milliliter, respectively (Figure 5; Figure 11).

[0086] Patients presenting with acute dengue-like illness—between June and October 2011 and 2012—were enrolled at Kampong Cham Referral Hospital. According to the WHO 1997 classification system, inclusion criteria were children aged 2 to 15 years who had fever or a history of fever at the time of presentation and at least two of the following symptoms within the preceding 72 hours: headache, retro-orbital pain, myalgia, arthralgia, rash, or any hemorrhagic signs. We conducted a prospective, single-center, cross-sectional study of hospitalized children with severe and nonsevere dengue. The first visit was performed upon hospital admission (visit 1, V1). The day of symptom onset was defined as day 0 of illness. Visit 2 (V2) was performed during the afebrile phase, characterized by the first day of a temperature ≤38°C. Finally, visit 3 (V3) was performed and considered as the discharge visit for patients who fully recovered or as a follow-up visit for patients in the critical phase. Clinical and biological follow-up, including abdominal / thoracic ultrasound recordings, was performed at each visit. DENV infection in hospitalized patients was confirmed by NS1 antigen detection using our NS1 capture ELISA (Alcon-LePoder et al. 2006) and / or qRT-PCR and / or virus isolation on Aedes albopictus C6 / 36 cells from plasma samples obtained at Visit 1 (Andries et al. 2015). Finally, disease severity in confirmed dengue patients was assessed according to the WHO 1997 and 2009 criteria using clinical and biological data recorded at admission (ADM), follow-up visit (F-VIS), or discharge (DIS). A total of 56 patients were retrospectively classified into three groups according to increasing grades of disease severity according to the 1997 classification: dengue fever (DF, n = 20), dengue hemorrhagic fever (DHF, n = 29), and dengue shock syndrome (DSS, n = 7). Of particular note, the 2009 classification separated patients into only two groups: dengue with dangerous symptoms (DWWS, n = 19) and severe dengue (SVD, n = 37), but no patients had classical dengue (DF).

[0087] We measured the concentrations of NS1-ApoA1 and NS1-ApoE complexes in a Cambodian cohort on the day after admission and discharge from the hospital. This corresponds to an average time difference of 4.3 days between the two blood sample collections (Figures 6 and 7). We separated patients whose final examination was a follow-up visit (F_VIS) from those tested at admission and discharge, as well as cases of DWWS identified as SVD. The vast majority (approximately 90%) of DWWS patients showed a sharp decrease in blood NS1-ApoA1 levels between their first and last sample collections, whereas half of the SVD patients showed a slight increase between the two time points (Figure 6). Conversely, NS1-ApoE concentrations significantly increased over time in the majority of samples (Figure 7), suggesting a dynamic process occurring during the acute phase of the disease.

[0088] We observed over 82% of samples positive for NS1-ApoA1 complexes at admission, with 36% remaining positive at the third visit (acute / discharge) (Table 1). The opposite trend was observed for NS1-ApoE complexes, whose concentrations clearly increased over time, rising from 25% to 50% between admission and final visit (Table 1). We found that approximately 70% of patients were NS1 antigen positive over the 2- to 6-day window after fever, confirming numerous reports, including ours, but only 42% of the same individuals were still NS1 positive at the time of their final visit or discharge (Table 1). Thus, NS1 concentrations were found to decrease over time (Table 1). Overall, NS1-ApoA1 complexes appear to be better diagnostic candidate markers than NS1 protein itself.

[0089] We further demonstrated the presence of NS1-ApoB complexes in 5 of 7 patients, whereas 3 non-dengue control patients were found to be negative (Figure 11). Concentrations ranged from 162 ng equivalents of NS1 / mL to 1230 ng equivalents of NS1 / mL. Expanded studies are needed to determine the added value this parameter may offer in terms of dengue diagnosis and prognosis.

[0090] Table 1: Estimates of the percentage of positive plasma samples according to different virological parameters tested as diagnostic markers in hospitalized dengue virus-infected patients. Values ​​are reported for patients admitted to the hospital (admission) or at the third visit corresponding to the follow-up or discharge visit (acute / discharge). The percentage of positive samples is in bold text. The highest percentage corresponds to the assay condition with the best diagnostic readout. [Table 1]

[0091] Example 6 Quantification of viral or host factors in plasma of DENV-infected patients - Correlation level between clinical grade and virological variables Statistical analysis highlighted significant concentration changes of NS1-ApoA1 and NS1-ApoE complexes as a function of different degrees of severity according to the 2009 classification (Figure 8). An Anova test was used to assess differences in median concentrations across degrees of severity, and p-values ​​are reported in Figure 8. The p-values ​​relate to a test of equivalence between median concentrations of two degrees of severity (i.e., a small p-value corresponds to a denial of equivalence between the two degrees) (Figure 8). The amount of NS1-ApoA1 complexes in plasma samples correlates significantly with disease severity (p = 0.015), whereas the value of NS1-ApoE complexes has no predictive value for the date of hospital admission (p = 0.71). Furthermore, the concentration of NS1-ApoA1 complexes also correlates significantly with the intensity of plasma leakage (p = 0.005) (Figure 11). Plasma fluid loss is a crucial criterion used to assess the severity of dengue disease.

[0092] Example 7 Identification of NS1-HDL complexes of various flaviviruses To assess whether different recombinant proteins could interact with HDL, purified preparations of NS1 proteins from flaviviruses other than DENV, including Japanese encephalitis, West Nile, Zika, yellow fever, and tick-borne encephalitis viruses, were mixed with normal human plasma. At the end of the incubation period, the presence of putative NS1-HDL complexes was tested using the same capture ELISA format as for DENV NS1 protein bound to ApoA1. This approach was made possible by the broad cross-reactivity of the DENV NS1 MAb 17A12, which allows detection and immobilization of NS1 proteins from a variety of flaviviruses. Under our experimental conditions, we were able to demonstrate the formation of NS1-HDL complexes, albeit at various levels, for all mosquito-borne NS1 proteins tested, whereas the NS1 protein from tick-borne encephalitis virus showed no signal at all (Fig. 9). These differences may be due to variations in the affinity of MAb 17A12 for various NS1 proteins, which results in varying proportions of complexes immobilized on the ELISA plate, or to differences in the intrinsic ability of various flavivirus NS1 proteins to bind to HDL. These data indicate that NS1 binding to HDL is a common feature of flaviviruses.

[0093] Example 8 Dengue NS1-specific monoclonal antibodies (MAbs) can prevent NS1 binding to HDL We tested the ability of anti-NS1 MAbs to interfere with the binding of DENV NS1 to HDL. To this end, we incubated purified NS1 with each MAb at a molar ratio of 1 Ab:1 NS1 protomer and measured the residual binding of NS1 hexamers on immobilized HDL by BLI (Figure 12). The ability of various anti-NS1 MAbs to inhibit NS1-HDL interactions varied, from the lack of inhibition observed with MAb 8G6 to nearly complete inhibition by MAb 17A12, which showed signals comparable to the background noise generated by various MAbs tested in the absence of NS1. MAb 1A11 was also a potent inhibitor, whereas MAbs 6D2 and 4F7 only partially inhibited complex formation (Figure 12). The BLI signal generated by NS1 protein bound to MAb 8G6 was higher than that of NS1 protein alone due to the higher mass of the NS1-8G6 complex captured by the immobilized HDL particles and detected by the sensor. All other MAbs that bound to NS1 showed a decrease in signal intensity compared to NS1 alone, consistent with partial to complete inhibition.

[0094] conclusion We demonstrate and report herein that NS1 hijacks endogenous ApoA1-positive HDL and ApoB-positive LDL lipoprotein particles during the acute phase of dengue disease. Dengue virus NS1 preferentially binds to HDL particles. Binding of NS1 to HDL alters its functional state and induces the activation of a proinflammatory response in human macrophages. During the later clinical phase, the concentration of NS1-ApoA1 complexes—representing NS1-HDL species—declines, and NS1 instead appears to colonize a broad range of ApoE-positive lipoprotein particles. This suggests that the interaction of the NS1 protein with host lipoproteins may be a highly dynamic process that changes over time, either as a means for the virus to control the fate of various lipoprotein particle species and their associated metabolic pathways or, to some extent, as a means for the host to eliminate toxic HDL particles.

[0095] Detection of the complex formed between DENV NS1 and host lipoproteins has greater diagnostic value than detection of NS1 itself, and quantification of its blood concentration should be included in a prognostic marker panel that helps clarify which patients are at risk of developing severe hemorrhagic fever and / or shock upon hospital admission. In the absence of specific therapy, creating a decision tree would have a major impact on the art for improving clinical management and reducing mortality.

[0096] NS1-HDL complexes can be formed in in vitro reconstitution assays for all mosquito-borne NS1 proteins of various flaviviruses, including DENV, Japanese encephalitis virus, West Nile virus, Zika virus, and yellow fever virus. This observation points to a common functional root for the NS1 proteins of the flavivirus genus. All NS1-HDL complexes may play an important role in viral pathogenesis and may have useful diagnostic and prognostic value for all flaviviruses. (References) TIFF0007783172000002.tif220170TIFF0007783172000003.tif220170TIFF0007783172000004.tif46170

Claims

1. An in vitro method for diagnosing flavivirus infection or an associated disease, comprising detecting the presence or level of a complex formed by flavivirus nonstructural protein 1 (NS1) and plasma lipoprotein particles positive for the plasma lipoproteins apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), or apolipoprotein E (ApoE) in a blood sample obtained from a subject.

2. An in vitro method for monitoring flavivirus infection or associated disease, comprising detecting the level of a complex formed by flavivirus nonstructural protein 1 (NS1) and plasma lipoprotein particles positive for the plasma lipoprotein apolipoprotein A1 (ApoA1) or apolipoprotein E (ApoE) in a blood sample obtained from a subject.

3. a. contacting the blood sample with an antibody specific for the flavivirus NS1 or an antibody specific for the plasma lipoprotein to form an immune reaction product; and b. detecting the presence of said immune reaction product; 3. The in vitro method of claim 1 or 2, comprising:

4. The in vitro method of claim 3, further comprising a step c after the detecting step in b, of quantifying the complex formed by NS1 and the plasma lipoprotein particles.

5. a. contacting the blood sample with an antibody specific for the flavivirus NS1 to form a first immune reaction product; b. contacting the first immune reaction product with an antibody specific for the plasma lipoprotein to form a second immune reaction product; and c. detecting the presence of said second immune reaction product 5. The in vitro method of claim 3 or 4, comprising:

6. The in vitro method of claim 5, further comprising a step d of quantifying the complex formed by NS1 and the plasma lipoprotein particles after the detecting step in c.

7. The in vitro method of claim 4 or 6, wherein the amount of the complex is determined by capture ELISA using the antibody specific for the flavivirus NS1 coated on a solid support and the antibody specific for the plasma lipoprotein as a detection antibody.

8. 6. The in vitro method of claim 5, wherein a third antibody raised against the antibody specific for the plasma lipoprotein and conjugated to a suitable label is used to detect the presence of the second immune reaction product.

9. 9. The in vitro method according to claim 3, wherein the antibody specific for the plasma lipoprotein is an antibody specific for ApoA1.

10. 9. The in vitro method according to claim 3, wherein the antibody specific for the plasma lipoprotein is an antibody specific for ApoB.

11. 9. The in vitro method according to claim 3, wherein the antibody specific for the plasma lipoprotein is an antibody specific for ApoE.

12. 12. An in vitro method for monitoring a flavivirus infection or an associated disease according to any one of claims 2 to 9 and 11, performed on blood samples obtained from said subject at different time points during the disease.

13. An in vitro method for diagnosing flavivirus infection or an associated disease according to any one of claims 1 and 3 to 11, wherein the presence of a complex formed by the flavivirus NS1 and the plasma lipoprotein particles in the sample indicates flavivirus infection.

14. 14. The in vitro method for diagnosing flavivirus infection or an associated disease according to claim 13, wherein the complex formed by the flavivirus NS1 and ApoA1-positive lipoprotein particles is quantified.

15. An in vitro method for diagnosing flavivirus infection or an associated disease as described in claim 13 or 14, which is repeatedly performed on a blood sample obtained from the subject to quantify flavivirus NS1-Apo1-positive lipoprotein particle complexes, flavivirus NS1-ApoE-positive lipoprotein particle complexes, and / or flavivirus NS1-ApoB-positive lipoprotein particle complexes.

16. 16. The in vitro method according to any one of claims 1 to 15, wherein the flavivirus NS1 is NS1 from a dengue virus and / or the flavivirus infection is a dengue virus infection.

17. 17. The in vitro method of claim 1, wherein the blood sample is whole blood, plasma or serum.

18. 18. The in vitro method of any one of claims 1 to 17, wherein the subject is a human.

19. A biomarker for the diagnosis of flavivirus infection or associated disease, comprising a complex formed by flavivirus NS1 and ApoA1-positive, ApoB-positive, or ApoE-positive plasma lipoprotein particles.

20. A biomarker for monitoring flavivirus infection or associated disease, comprising a complex formed by flavivirus NS1 and ApoA1-positive or ApoE-positive plasma lipoprotein particles.

21. 21. The biomarker of claim 19 or 20, wherein the flavivirus NS1 is NS1 from a dengue virus and / or the flavivirus infection is a dengue virus infection.

22. 20. Use of the presence or level of the biomarker of claim 19 in a blood sample obtained from a subject for the diagnosis of a flavivirus infection or an associated disease.

23. Use of the presence or level of a biomarker described in claim 20 in a blood sample obtained from a subject for monitoring flavivirus infection or an associated disease.

24. 24. The use according to claim 22 or 23, wherein the blood sample is whole blood, plasma or serum.

25. 25. The use according to any one of claims 22 to 24, wherein the subject is a human being.

26. 1. A kit for diagnosing a flavivirus infection or an associated disease in a blood sample obtained from a subject, comprising: a. Antibodies specific for flavivirus NS1; b. one or more antibodies specific for plasma lipoproteins selected from antibodies specific for ApoA1, antibodies specific for ApoB, and antibodies specific for ApoE; c. A means for detecting the production of an immune reaction product between said antibody and said complex formed by said flavivirus NS1 and plasma lipoprotein particles positive for ApoA1, ApoB, or ApoE. Includes a kit.

27. A kit for monitoring flavivirus infection or an associated disease in a blood sample obtained from a subject, comprising: a. Antibodies specific for flavivirus NS1; b. one or more antibodies specific for plasma lipoproteins selected from antibodies specific for ApoA1 and antibodies specific for ApoE; c. A means for detecting the production of an immune reaction product between said antibody and said complex formed by said flavivirus NS1 and plasma lipoprotein particles positive for ApoA1 or ApoE. Includes a kit.

28. 28. The kit of claim 26 or 27, wherein the flavivirus NS1 is NS1 derived from a dengue virus.

29. 29. The kit of any one of claims 26 to 28, wherein the blood sample is whole blood, plasma or serum.

30. 30. The kit of any one of claims 26 to 29, wherein the subject is a human.

Images