Patent Publication Number: US-10309965-B2

Title: Method and kit for determining the probability that a patient will develop a severe case of dengue

Description:
This is a Division of application Ser. No. 15/117,765 filed Aug. 10, 2016, which is a National Stage Entry of PCT/FR2015/050352 filed Feb. 12, 2015, which claims priority to FR 1451274 filed Feb. 18, 2014. The disclosures of the prior applications are hereby incorporated by reference herein in their entireties. 
    
    
     The subject of the present invention is a method for the early prediction of severe dengue or hemorrhagic dengue using protein markers. 
     Over the past 30 years, dengue, a viral disease transmitted by urban hematophagous mosquitoes of the  Aedes  genus has worryingly spread throughout the world. It is currently a real public health problem for more than one hundred countries located in the subtropical zone, particularly in the Pacific West, South America and South-East Asia zones. The emergence of the disease is largely due to the population explosion and to chaotic urbanization. Climatic abnormalities also play a not insignificant role. 
     In this respect, dengue could emerge in the western regions of the world which until now have been spared the virus. Thus,  Aedes albopictus , one of the vectors of the disease, has recently been found in the North of Italy and in the South of France. Most recently, autochthonous cases of dengue have been recorded in the South of France. It is estimated that close to three billion people are exposed to the risks of dengue. Close to one million hospitalizations are registered yearly and there have been thousands of deaths. Children are the main victims of the disease. 
     The dengue virus is a single-stranded, positive-polarity enveloped RNA virus of the family Flaviviridae. The genome of the virus (11 000 nucleotides) encodes a polyprotein of approximately 3400 amino acids which undergoes co- and post-translational cleavage which results in structural proteins (C, prM, E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). There are 4 viral serotypes (DV1 to DV4), which can coexist in endemic zones. There is approximately 70% sequence homology between the various serotypes. Infection by a given serotype confers long-term immunity for this serotype. Cross-protection lasts only a few months: reinfection is therefore possible with a different serotype. 
     Infection begins with a bite from a mosquito infected with one of the dengue viruses. Incubation, the period during which the virus replicates in the blood without however giving rise to any symptoms, generally lasts from 4 to 10 days. The first signs occur after the incubation period. 
     In its conventional form (“conventional” dengue fever: DF), dengue is characterized by sudden-onset hyperthermia accompanied by one or more of the following symptoms: shivering, headaches, joint and/or muscle pain, nausea and vomiting. A rash may also appear, generally on around the 5th day of symptoms. This acute febrile stage, which corresponds to the viremic phase, generally lasts from 3 to 5 days (extremes: 2 to 7 days). More than 95% of cases will have no signs of severe illness and will recover with no complications in under 7 days. 
     In 2 to 4% of cases, the patient may develop a critical phase characterized by a more or less severe plasma leakage syndrome and an increased hematocrit level, leading to dengue hemorrhagic fever: DHF. This phase typically (but not necessarily) appears at the time of defervescence, around the 4th or 5th day. It is generally brief (24 to 48 h) but may develop into a severe form characterized by major hemorrhagic manifestations, a state of shock and/or the failure of one or more organs. Development into a severe form is most often signalled by one (or more) warning sign(s), such as:
         fever (temperature of greater than 39° C.) after the 5th day;   intense abdominal pain, persistent diarrhea, uncontrollable vomiting and complete refusal of food;   edemas and/or minor effusion;   bleeding of mucous membranes which does not stop automatically;   pronounced lethargy or restlessness;   thrombocytopenia;   signs of hemoconcentration.       

     In the most severe cases, the leaking of plasma can lead to deadly hypovolemic shock (Dengue Shock Syndrome: DSS) if the patient is not rapidly treated. Rare but deadly hepatic and neurological involvement is also associated with the severity of the disease. The mortality rate, which is variable according to epidemics, can reach 5% of established DHF cases. This rate can increase up to 20% without hospital care or appropriate treatments. 
     To simplify, these severe cases will be referred to as severe dengue, SevD in the remainder of the description, as opposed to conventional dengue, DF. 
     90% of cases of severe dengue occur during secondary infection with a heterologous serotype, and 10% during primary infection, generally in infants aged from 6 months to 1 year. There are several factors which influence the severity of the infection, such as the factors of the host, serotype and genotype of the virus, the order and time between successive infecting viruses, the quality and quantity of cross-reactive antibodies and the CD4/CD8 response. Studies have shown a correlation between viral load and severity of the disease. The exact causes of the occurrence of severe dengue are, however, still not known. Up until now, no specific determining factor for virulence has been demonstrated. Furthermore, since there is no vaccine against the dengue virus, the only treatments available are symptomatic treatments. Consequently, it is important to be able to monitor epidemics and to predict severe cases for appropriate hospital care. 
     The methods currently used to diagnose dengue do not make it possible to predict the development of severe dengue. At the very most, serological methods make it possible to distinguish between primary and secondary infections and molecular methods make it possible to detect the virus and to carry out serotyping [1, 2, 3, 4]. 
     The present invention provides a solution to the problems presented above by means of a method which allows both early and specific detection of proteins in a blood sample making it possible to predict patients developing severe dengue. Indeed, the inventors found, surprisingly, that proteins from the host were expressed more or less abundantly (overexpressed/underexpressed) in cases of patients developing severe dengue, compared to the amount or expression thereof in cases of patients remaining with conventional dengue (that is to say not developing severe dengue) in blood samples consisting, for example, of plasma. Most particularly, they have demonstrated for the first time and completely unexpectedly that platelet factor 4 (PF4) is underexpressed in the case of patients developing severe dengue and thus constitutes a marker for predicting severe dengue. 
     Thus, a subject of the present invention is a method for predicting, in vitro, the probability of a patient developing severe dengue, based on a blood sample, wherein:
         a) the quantity in said blood sample of at least one marker, which is platelet factor 4, is determined,   b) the quantity of platelet factor 4 determined in step a) is compared with a reference quantity of said marker obtained from a group of individuals who have been diagnosed with non-severe dengue, wherein, if the quantity of platelet factor 4 determined in step a) is less than the reference quantity established in step b), it is predicted that the patient will develop severe dengue.       

     According to the method of the invention, it is also possible to determine, in step a), the quantity in the blood sample of at least one other marker chosen from olfactomedin 4 (OLFM4) and α2-macroglobulin (A2M) or the respective quantities of the two markers and, in step b), the quantity of the marker or of the two markers of step a) is compared with a reference quantity obtained from a group of individuals who have been diagnosed with non-severe dengue and, if the quantity of olfactomedin 4 in step a) is greater than the reference quantity established in step b) and/or the quantity of α2-macroglobulin is less than the reference quantity established in step b), it is determined that the patient will develop severe dengue. 
     The invention also relates to a kit for the in vitro prediction of severe dengue, comprising:
         a binding partner for platelet factor 4,   a binding partner for the dengue virus NS1 protein.       

     The kit may also comprise a binding partner for OLFM4 and/or a binding partner for A2. 
     Definitions 
     The term “blood sample” is intended to mean whole blood, serum and plasma. 
     The term “group of individuals who have been diagnosed with non-severe dengue”, used to determine the reference quantity of the marker of interest, is intended of course to mean that the group of individuals has not developed severe dengue. Thus, in step b) the quantity of platelet factor 4 determined in step a) is compared with a reference quantity of said marker obtained from a group of individuals who have been diagnosed with dengue without having developed severe dengue. 
     The term “binding partner” is intended to mean, for example, receptors, antibodies, antibody fragments, antibody analogs and any other ligand capable of binding to a protein. 
     The binding-partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies. 
     The polyclonal antibodies may be obtained by immunization of an animal with the appropriate immunogen, followed by recovery of the desired antibodies in purified form, by taking the serum of said animal, and separation of said antibodies from the other serum constituents, especially by affinity chromatography on a column to which is bound an antigen specifically recognized by the antibodies. 
     The monoclonal antibodies may be obtained by the hybridoma technique, the general principle of which is summarized below. 
     Firstly, an animal, generally a mouse, is immunized with the appropriate immunogen, and the B lymphocytes of said animal are then capable of producing antibodies against this antigen. These antibody-producing lymphocytes are then fused with “immortal” myeloma cells (murine in the example) so as to give rise to hybridomas. Using the heterogeneous mixture of cells thus obtained, a selection of the cells capable of producing a particular antibody and of multiplying indefinitely is then carried out. Each hybridoma is multiplied in the form of a clone, each resulting in the production of a monoclonal antibody of which the recognition properties with respect to the protein may be tested, for example, by ELISA, by one-dimensional or two-dimensional Western blotting, by immunofluorescence, or by means of a biosensor. The monoclonal antibodies thus selected are subsequently purified, especially according to the affinity chromatography technique described above. 
     The monoclonal antibodies may also be recombinant antibodies obtained by genetic engineering, using techniques well known to those skilled in the art. 
     The term “antibody analogs” is intended to mean biological and/or chemical compounds which have the same binding capacities as the antibodies or antibody fragments or similar binding capacities. In particular, the antibody analogs include small proteins which, like antibodies, are capable of binding to a biological target thus making it possible to detect it, to capture it or quite simply to target it within an organism or within a biological sample. The fields of application of these antibody analogs are virtually as vast as those of antibodies. By way of example, mention may be made of the Nanofitins™, which are small proteins sold by the company Affilogic. 
     The binding partners specific for the desired protein can be used as a capture reagent, as a detection reagent or as capture and detection reagents. 
     The visualization of the immunological reactions, i.e. the protein/binding partner binding, can be carried out by any means of detection, via labeling, of the binding partner. 
     The term “labeling” is intended to mean the binding of a label reagent capable of generating a detectable signal, i.e. a compound, a substance or a particle which can be detected by visual, fluorescent or instrumental means. 
     A nonlimiting list of these label reagents consists of:
         metal or alloy particles, such as colloidal gold particles,   polymer particles, such as colored latex particles,   magnetic particles,   fluorescent molecules,   chemoluminescent molecules.
 
By way of example of immunological tests as defined above, mention may be made of “sandwich” and “competition” methods.
       

    
    
     
       FIGURES 
       The majority of the figures illustrate the validation of the results by a quantitative ELISA assay carried out on individual samples taken from patients during the acute febrile phase of the disease, before defervescence. The patients having remained with conventional dengue are denoted DF and the patients having then developed severe dengue are denoted SevD. In all cases, the reading is carried out at an optical density (OD) of 450 nm. The results were obtained on samples with different geographical origins: Columbia and Cambodia. On the graphs obtained (GraphPad Prism software, V4.03), the median calculated is represented by a horizontal line. The box illustrates the values encompassing 50% of the individuals. The maximum and minimum values are also illustrated. The values taken into account correspond to the mean of two independent tests carried out in duplicate. 
         FIG. 1  illustrates the presence of virus in the fractions purified from plasma from patients but not in the control (C), demonstrated with Western blotting using a monoclonal antibody directed against the viral protein E. 
         FIG. 2  illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the PF4 marker on plasma samples from Colombian patients. 
         FIG. 3  illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the PF4 marker on plasma samples from Cambodian patients. 
         FIG. 4  illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the OLFM4 marker on plasma samples from Colombian patients. 
         FIG. 5  illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the OLFM4 marker on plasma samples from Cambodian patients. 
         FIG. 6  illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the A2M marker on plasma samples from Cambodian patients. 
     
    
    
     Example 1: Characterization of the Samples 
     15 Colombian plasma samples positive for dengue were selected, from which 8 originate from patients remaining with conventional dengue without developing severe dengue (patients/samples referred to hereinafter as DF or conventional dengue) and 7 originate from patients having then developed severe dengue (patients/samples referred to hereinafter as SevD or severe dengue). The various plasmas were grouped together, composing respectively a pool of conventional dengue DF plasma and a pool of severe dengue SevD plasma for those having developed severe dengue. All the plasmas were taken during the acute febrile phase of the disease, before the critical phase, from patients having a secondary infection. The serotypes concerned were serotypes 1, 2 and 3. All the patients having developed severe dengue were hospitalized and had signs of hemorrhaging. No comorbidity was reported [5]. All the plasmas were verified as being NS1-positive (Platelia dengue kit, Bio-rad) and the viral load was also verified by Q-RT-PCR with a commercially available kit (PrimerDesign) following supplier instructions: the mean number of viral RNA copies was estimated at 4×10 6  and 4.1×10 7  for the conventional dengue (DF) pools and severe dengue (SevD) pools, respectively. The pools composed correspond to a volume of approximately 2 ml of plasma. Before purification, the plasma mixtures were centrifuged for 5 mins at 1000×g and at 4° C. so as to remove the impurities present in the sample and to obtain clarified samples. 
     The plasma selection criteria are described in table 1 below. The samples were taken after appearance of symptoms. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Colombian plasma 
                   
                 DF POOL 
                 SevD POOL 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 General 
                 Number 
                 8 
                 7 
               
               
                   
                 Age (mean) 
                 26.8 
                 33.7 
               
               
                   
                 M/F ratio 
                 6/2 
                 3/4 
               
               
                   
                 Day 
                 3.3 
                 2.7 
               
               
                   
                 Secondary infection 
                 Yes 
                 Yes 
               
               
                   
                 NS1 
                 Positive 
                 Positive 
               
               
                 Serotype 
                 DV1 
                 1 
                 0 
               
               
                   
                 DV2 
                 1 
                 3 
               
               
                   
                 DV3 
                 6 
                 4 
               
               
                   
               
            
           
         
       
     
     These plasma sample pools were then purified to obtain virus-enriched fractions as described below. 
     Example 2: Purification of the Samples 
     All the steps are carried out at 4° C. The clarified samples are supplemented with 8 ml of cold pH8 PBS (PBS8), then centrifuged for 2 h at 41 000×g in an Optima L90 ultracentrifuge (Beckman). The rotor used is the SW41 rotor (Beckman). After centrifugation, the supernatant is removed and the viral pellet obtained is resuspended in 200 microliters of PBS8 then loaded onto a discontinuous gradient composed of 5 ml of 60% (w/w) sucrose in PBS8 and 5 ml of 20% (w/w) sucrose in PBS8. After renewed centrifugation for 2 h at 41 000×g, a virion-enriched ring located at the interface between the two sucrose solutions is taken off with a pipette, diluted 10 times with PBS8 and finally centrifuged one last time for 2 h at 41 000×g. The pellet obtained is resuspended in 200 microliters of PBS8. 
     This resuspension is then purified using an insoluble polyelectrolyte, Viraffinity (BioSupportGroup, USA). For this purpose, 200 microliters of an MN buffer (60 mM MES pH 6.5, 150 mM NaCl) are added to the viral suspension along with 100 microliters of Viraffinity. The mixture is incubated for 5 min at room temperature then centrifuged for 10 min at 1000×g, following supplier instructions. The supernatant is removed and the polymer pellet is rinsed 3 times with 200 microliters of MN buffer. The viral proteins are recovered by heating the polymer for 5 min/70° C. in the presence of 50 microliters of a buffer containing SDS (Novex InVitrogen) then centrifugation for 5 min at 1000×g. The presence of the virus in the final samples was verified by immunoblotting with a monoclonal antibody directed against the envelope protein of the dengue virus (E protein). As illustrated in  FIG. 1 , strong signals at 60 KDa and 120 KDa, corresponding respectively to the monomeric and dimeric forms of the envelope protein, are specifically detected by the monoclonal antibody in the plasma pools. On the contrary, the envelope protein was not detected on a control corresponding to a pool of healthy (non-dengue) plasma purified in the same way as has been described above. 
     Example 3: Identification of the Specific Proteins for Each Plasma Pool, Conventional Dengue DF and Severe Dengue SevD, by Mass Spectrometry (MS) 
     Method: 
     The viral preparations and the control sample obtained according to example 2 are deposited on a non-denaturing polyacrylamide gel and migrated until the proteins penetrate into the gel, in order to desalify the sample. The band containing the proteins is excised manually then washed three times in a buffer containing 50% acetonitrile then finally dried in 100% acetonitrile. The gel is then rehydrated in a 7% H 2 O 2  solution before being washed again. A solution of trypsin diluted in 25 mM NH 4 HCO 3  is then added for hydrolysis at 37° C. overnight. The peptides thus obtained are extracted by 15 minute sequential extractions with 30 microliters of 50% acetonitrile, 30 microliters of 5% formic acid and 30 microliters of 100% acetonitrile. These sequential extractions are mixed, dried under vacuum and resuspended in a solution containing 5% acetonitrile and 0.1% trifluoroacetic acid. After quantification of the samples, a defined quantity of peptides is analyzed by nano liquid chromatography coupled together with mass spectrometry (Ultimate 3000, Dionex and LTQ-Orbitrap VelosPro, Thermo Fisher Scientific). The results are acquired by virtue of the Xcalibur software (Thermo Fisher) and automatically converted by the Mascot Daemon V2.2 software (Matrix Science). Searching is then carried out on the Swissprot and Trembl databases via Mascot 2.2. Each experiment was carried out twice, independently. The proteins were identified by the EDyP Service laboratory (CEA Grenoble, France). 
     Results: 
     The viral envelope protein E was repeatedly identified in the samples containing virus. The predominantly identified peptide sequence is GWGNGCGLLFKG. This result confirms the presence of the virus in the purified fraction. 
     For the proteins of cellular origin, identified by proteomics on purified plasma pools, the results obtained are summarized in tables 2a and 2b. In these tables, only those proteins having a variance of less than 25% for the number of peptides found from one experiment to the other have been considered. Similarly, for the severe dengue sample, a number of peptides of greater than 2 was required. According to these criteria, 189 proteins were finally selected. These proteins are described in tables 2a and 2b below. A ratio of “number of peptides in severe dengue (SevD) sample”/“number of peptides in conventional dengue (DF) sample” (SevD/DF) could be calculated for the majority of these proteins (cf. table 2a). Some proteins were only identified in the SevD sample (cf. table 2b); in this case, the SevD/DF ratio could not be calculated. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 2a 
               
               
                   
               
               
                   
                   
                 Mean 
                 Mean 
                   
               
               
                   
                   
                 number 
                 number 
                   
               
               
                   
                   
                 of 
                 of 
                 SevD/DF 
               
               
                 Accession 
                   
                 peptides 
                 peptides 
                 peptides 
               
               
                 number 
                 Protein name 
                 DF 
                 SevD 
                 ratio 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 P09871 
                 Complement C1s subcomponent (C1 esterase) 
                 3.5 
                 14.5 
                 4.14 
               
               
                 P07225 
                 Vitamin K-dependent protein S 
                 1 
                 4 
                 4.00 
               
               
                 P01008 
                 Antithrombin-III (ATIII) (Serpin C1) 
                 3 
                 9.5 
                 3.17 
               
               
                 Q16610 
                 Extracellular matrix protein 1 
                 1 
                 3 
                 3.00 
               
               
                   
                 (Secretory component p85) 
                   
                   
                   
               
               
                 P27918 
                 Properdin (Complement factor P) 
                 1.5 
                 4 
                 2.67 
               
               
                 B4E1B2 
                 Serotransferrin 
                 11 
                 29 
                 2.64 
               
               
                 Q53H26 
                 Transferrin variant (Fragment) 
                 11 
                 29 
                 2.64 
               
               
                 B4DPQ0 
                 Complement C1r subcomponent 
                 5 
                 13 
                 2.60 
               
               
                 P01019 
                 Angiotensinogen (Serpin A8) 
                 1 
                 2.5 
                 2.50 
               
               
                 B4DDU2 
                 Tubulin alpha-ubiquitous chain 
                 2.5 
                 6 
                 2.40 
               
               
                 P01779 
                 Ig heavy chain V-III region TUR 
                 2.5 
                 6 
                 2.40 
               
               
                 P68366 
                 Tubulin alpha-4A chain (Alpha-tubulin 1) 
                 3 
                 6.5 
                 2.17 
               
               
                 P00734 
                 Prothrombin (Coagulation factor II) 
                 2 
                 4 
                 2.00 
               
               
                 P21333 
                 Filamin-A (Actin-binding protein 280) 
                 14.5 
                 28.5 
                 1.97 
               
               
                 P00450 
                 Ceruloplasmin (EC 1.16.3.1) (Ferroxidase) 
                 8.5 
                 16.5 
                 1.94 
               
               
                 E7EX29 
                 14-3-3 protein zeta/delta (Fragment) 
                 4.5 
                 8.5 
                 1.89 
               
               
                 P63104 
                 14-3-3 protein zeta/delta (KCIP-1) 
                 4.5 
                 8.5 
                 1.89 
               
               
                 B4E1D8 
                 cDNA FLJ51597, highly similar to 
                 10.5 
                 19 
                 1.81 
               
               
                   
                 C4b-binding protein 
                   
                   
                   
               
               
                 P18206 
                 Vinculin (Metavinculin) 
                 5 
                 9 
                 1.80 
               
               
                 P12814 
                 Alpha-actinin-1 (Alpha-actinin 
                 6.5 
                 11.5 
                 1.77 
               
               
                   
                 cytoskeletal isoform) 
                   
                   
                   
               
               
                 O43707 
                 Alpha-actinin-4 (F-actin cross-linking protein) 
                 6.5 
                 11.5 
                 1.77 
               
               
                 P06396 
                 Gelsolin (AGEL) (Actin- 
                 3.5 
                 6 
                 1.71 
               
               
                   
                 depolymerizing factor) 
                   
                   
                   
               
               
                 P05106 
                 Integrin beta-3 (Platelet membrane 
                 5.5 
                 9 
                 1.64 
               
               
                   
                 glycoprotein IIIa) (GPIIIa) 
                   
                   
                   
               
               
                 P08514 
                 Integrin alpha-IIb (GPIIb) 
                 12.5 
                 20 
                 1.60 
               
               
                   
                 (Platelet membrane glycoprotein IIb) 
                   
                   
                   
               
               
                 P01833 
                 Polymeric immunoglobulin receptor (PIgR) 
                 2.5 
                 4 
                 1.60 
               
               
                 Q9Y490 
                 Talin-1 
                 28.5 
                 43 
                 1.51 
               
               
                 A2J1N9 
                 Rheumatoid factor RF-ET12 (Fragment) 
                 3 
                 4.5 
                 1.50 
               
               
                 Q5NV90 
                 V2-17 protein (Fragment) 
                 3 
                 4.5 
                 1.50 
               
               
                 P01023 
                 α-2-macroglobulin (Alpha-2-M) 
                 56.5 
                 83 
                 1.47 
               
               
                 A2KBC6 
                 Anti-Factor VIII scFv (Fragment) 
                 6.5 
                 9.5 
                 1.46 
               
               
                 P01011 
                 Alpha-1-antichymotrypsin (ACT) (Serpin A3) 
                 3.5 
                 5 
                 1.43 
               
               
                 P00738 
                 Haptoglobin 
                 13.5 
                 19 
                 1.41 
               
               
                 P02743 
                 Serum amyloid P-component (SAP) 
                 2.5 
                 3.5 
                 1.40 
               
               
                 P01024 
                 C3 and PZP-like α-2-macroglobulin 
                 60.5 
                 84.5 
                 1.40 
               
               
                   
                 domain-containing protein 1 
                   
                   
                   
               
               
                 A8K008 
                 cDNA FLJ78387 
                 17.5 
                 24 
                 1.37 
               
               
                 A2IPI5 
                 HRV Fab 026-VL (Fragment) 
                 3 
                 4 
                 1.33 
               
               
                 Q6UX06 
                 Olfactomedin 4 (OLM4) (Antiapoptotic 
                 4.5 
                 6 
                 1.33 
               
               
                   
                 protein GW112) 
                   
                   
                   
               
               
                 P03952 
                 Plasma kallikrein (EC 3.4.21.34) 
                 1.5 
                 2 
                 1.33 
               
               
                   
                 (Fletcher factor) 
                   
                   
                   
               
               
                 P08567 
                 Pleckstrin (Platelet 47 kDa protein) 
                 1.5 
                 2 
                 1.33 
               
               
                   
                 (p47) 
                   
                   
                   
               
               
                 P07996 
                 Thrombospondin-1 
                 19.5 
                 26 
                 1.33 
               
               
                 P07437 
                 Tubulin beta chain (Tubulin beta-5 chain) 
                 4.5 
                 6 
                 1.33 
               
               
                 A2MYD4 
                 V2-7 protein (Fragment) 
                 3 
                 4 
                 1.33 
               
               
                 P00488 
                 Coagulation factor XIII A chain (EC 2.3.2.13) 
                 3.5 
                 4.5 
                 1.29 
               
               
                 Q71U36 
                 Tubulin alpha-1A chain (Alpha-tubulin 3) 
                 3.5 
                 4.5 
                 1.29 
               
               
                 P68363 
                 Tubulin alpha-1B chain (Alpha-tubulin ubiquitous) 
                 3.5 
                 4.5 
                 1.29 
               
               
                 Q9BQE3 
                 Tubulin alpha-1C chain (Alpha-tubulin 6) 
                 3.5 
                 4.5 
                 1.29 
               
               
                 Q9H4B7 
                 Tubulin beta-1 chain 
                 3.5 
                 4.5 
                 1.29 
               
               
                 Q96K68 
                 cDNA FLJ14473 fis, clone 
                 12 
                 15 
                 1.25 
               
               
                   
                 MAMMA1001080 
                   
                   
                   
               
               
                 P08107 
                 Heat shock 70 kDa protein 1A/1B 
                 2 
                 2.5 
                 1.25 
               
               
                 P34931 
                 Heat shock 70 kDa protein 1-like 
                 2 
                 2.5 
                 1.25 
               
               
                 P11142 
                 Heat shock cognate 71 kDa protein 
                 2 
                 2.5 
                 1.25 
               
               
                 Q6N089 
                 Putative uncharacterized protein 
                 15 
                 18.5 
                 1.23 
               
               
                   
                 DKFZp686P15220 
                   
                   
                   
               
               
                 P02675 
                 Fibrinogen beta chain 
                 11 
                 13.5 
                 1.23 
               
               
                 P00739 
                 Haptoglobin-related protein 
                 12 
                 14.5 
                 1.21 
               
               
                 Q86UX7 
                 Fermitin family homolog 3 (Kindlin-3) 
                 5 
                 6 
                 1.20 
               
               
                 P02751 
                 Fibronectin (FN) 
                 61.5 
                 72.5 
                 1.18 
               
               
                 B7ZLE5 
                 FN1 protein 
                 61.5 
                 72.5 
                 1.18 
               
               
                 Q6MZM7 
                 Putative uncharacterized protein 
                 61.5 
                 72.5 
                 1.18 
               
               
                   
                 DKFZp686O12165 
                   
                   
                   
               
               
                 Q68CX6 
                 Putative uncharacterized protein 
                 61.5 
                 72.5 
                 1.18 
               
               
                   
                 DKFZp686O13149 
                   
                   
                   
               
               
                 P68032 
                 Actin, alpha cardiac muscle 1 
                 8.5 
                 10 
                 1.18 
               
               
                   
                 (Alpha-cardiac actin) 
                   
                   
                   
               
               
                 Q562R1 
                 Beta-actin-like protein 2 (Kappa-actin) 
                 8.5 
                 10 
                 1.18 
               
               
                 P0C0L4 
                 Complement C4-A (Acidic complement C4) 
                 52 
                 61 
                 1.17 
               
               
                 P0C0L5 
                 Complement C4-B (Basic complement C4) 
                 52 
                 61 
                 1.17 
               
               
                 BOUZ85 
                 Complement component 4B (Childo blood group) 
                 53 
                 62 
                 1.17 
               
               
                 Q6MZQ6 
                 Putative uncharacterized protein 
                 15 
                 17.5 
                 1.17 
               
               
                   
                 DKFZp686G11190 
                   
                   
                   
               
               
                 P07477 
                 Trypsin-1 (EC 3.4.21.4) (Serine protease 1) 
                 3 
                 3.5 
                 1.17 
               
               
                 Q4TZM4 
                 Hemoglobin beta chain (Fragment) 
                 6.5 
                 7.5 
                 1.15 
               
               
                 P04004 
                 Vitronectin (VN) (S-protein) (V75) 
                 6.5 
                 7.5 
                 1.15 
               
               
                 B2RUT6 
                 Complement component 4A (Rodgers blood group) 
                 53.5 
                 61.5 
                 1.15 
               
               
                 P01031 
                 Complement C5 
                 3.5 
                 4 
                 1.14 
               
               
                 Q9UL78 
                 Myosin-reactive immunoglobulin light 
                 3.5 
                 4 
                 1.14 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 Q5NV62 
                 V3-4 protein (Fragment) 
                 6 
                 7 
                 1.17 
               
               
                 A6H8M8 
                 C4A protein (Complement C4 gamma chain) 
                 53.5 
                 61 
                 1.14 
               
               
                 P02768 
                 Serum albumin 
                 51.5 
                 58.5 
                 1.14 
               
               
                 Q7Z351 
                 Putative uncharacterized protein 
                 15 
                 17 
                 1.13 
               
               
                   
                 DKFZp686N02209 
                   
                   
                   
               
               
                 P02649 
                 Apolipoprotein E (Apo-E) 
                 4 
                 4.5 
                 1.13 
               
               
                 P01009 
                 Alpha-1-antitrypsin (Alpha-1 
                 8.5 
                 9.5 
                 1.12 
               
               
                   
                 protease inhibitor) (Serpin A1) 
                   
                   
                   
               
               
                 Q08380 
                 Galectin-3-binding protein (Basement 
                 12 
                 13 
                 1.08 
               
               
                   
                 membrane autoantigen p105) 
                   
                   
                   
               
               
                 Q6N092 
                 Putative uncharacterized protein 
                 12 
                 13 
                 1.08 
               
               
                   
                 DKFZp686K18196 (Fragment) 
                   
                   
                   
               
               
                 P63261 
                 Actin, cytoplasmic 2 (Gamma-actin) 
                 20 
                 21.5 
                 1.08 
               
               
                 P02671 
                 Fibrinogen alpha chain 
                 9.5 
                 10 
                 1.05 
               
               
                 P60709 
                 Actin, cytoplasmic 1 (Beta-actin) 
                 19.5 
                 20.5 
                 1.05 
               
               
                 A2MYE1 
                 A30 (Fragment) 
                 4 
                 4 
                 1.00 
               
               
                 Q96SA9 
                 Anti-streptococcal/anti-myosin 
                 3.5 
                 3.5 
                 1.00 
               
               
                   
                 immunoglobulin kappa light chain 
                   
                   
                   
               
               
                 P06576 
                 ATP synthase subunit beta, 
                 2 
                 2 
                 1.00 
               
               
                   
                 mitochondrial (EC 3.6.3.14) 
                   
                   
                   
               
               
                 O43866 
                 CD5 antigen-like (CT-2) (IgM-associated peptide) 
                 15 
                 15 
                 1.00 
               
               
                 P12259 
                 Coagulation factor V (Activated protein C cofactor) 
                 1.5 
                 1.5 
                 1.00 
               
               
                 P02747 
                 Complement C1q subcomponent subunit C 
                 5.5 
                 5.5 
                 1.00 
               
               
                 P04196 
                 Histidine-rich glycoprotein (HPRG) 
                 1.5 
                 1.5 
                 1.00 
               
               
                 P01892 
                 HLA class I, A-2 alpha chain (MHC 
                 3 
                 3 
                 1.00 
               
               
                   
                 class I antigen A*2) 
                   
                   
                   
               
               
                 P30447 
                 HLA class I histocompatibility 
                 3 
                 3 
                 1.00 
               
               
                   
                 antigen, A-23 alpha chain 
                   
                   
                   
               
               
                 F6IQP2 
                 MHC class I antigen (Fragment) 
                 3 
                 3 
                 1.00 
               
               
                 F6IR35 
                 MHC class I antigen (Fragment) 
                 3 
                 3 
                 1.00 
               
               
                 A2NKM7 
                 NANUC-2 heavy chain (Fragment) 
                 3 
                 3 
                 1.00 
               
               
                 P26022 
                 Pentraxin-related protein PTX3 
                 2 
                 2 
                 1.00 
               
               
                   
                 (Pentaxin-related protein PTX3) 
                   
                   
                   
               
               
                 P02760 
                 Protein AMBP 
                 2.5 
                 2.5 
                 1.00 
               
               
                 A2J1M8 
                 Rheumatoid factor RF-IP12 
                 3 
                 3 
                 1.00 
               
               
                   
                 (Rheumatoid factor RF-IP13) 
                   
                   
                   
               
               
                 A2J1N0 
                 Rheumatoid factor RF-IP14 (Fragment) 
                 6 
                 6 
                 1.00 
               
               
                 Q9HCC1 
                 Single chain Fv (Fragment) 
                 7.5 
                 7.5 
                 1.00 
               
               
                 H0YLA9 
                 Uncharacterized protein 
                 2 
                 2 
                 1.00 
               
               
                 P02774 
                 Vitamin D-binding protein (DBP) (VDB) 
                 2 
                 2 
                 1.00 
               
               
                 P02647 
                 Apolipoprotein A-I (ApoA-I) 
                 11 
                 10.5 
                 0.95 
               
               
                   
                 (Apolipoprotein A1) 
                   
                   
                   
               
               
                 P68871 
                 Hemoglobin subunit beta (Beta-globin) 
                 8.5 
                 8 
                 0.94 
               
               
                 A2NYQ9 
                 Anti-folate binding protein (Fragment) 
                 7 
                 6.5 
                 0.93 
               
               
                 A2JA14 
                 Anti-mucin1 heavy chain variable 
                 6.5 
                 6 
                 0.92 
               
               
                   
                 region (Fragment) 
                   
                   
                   
               
               
                 B6EDE2 
                 Epididymis luminal protein 180 (Fragment) 
                 6.5 
                 6 
                 0.92 
               
               
                 Q14477 
                 Hbbm fused globin protein (Fragment) 
                 6.5 
                 6 
                 0.92 
               
               
                 Q670S4 
                 Hemoglobin Lepore-Baltimore (Fragment) 
                 6.5 
                 6 
                 0.92 
               
               
                 P02042 
                 Hemoglobin subunit delta (Delta-globin) 
                 6.5 
                 6 
                 0.92 
               
               
                 Q9UL90 
                 Myosin-reactive immunoglobulin heavy 
                 6.5 
                 6 
                 0.92 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 A2NZ55 
                 Variable immnoglobulin anti- 
                 6.5 
                 6 
                 0.92 
               
               
                   
                 estradiol heavy chain 
                   
                   
                   
               
               
                 Q15485 
                 Ficolin-2 (37 kDa elastin-binding protein) 
                 5.5 
                 5 
                 0.91 
               
               
                 Q9UL88 
                 Myosin-reactive immunoglobulin heavy 
                 5 
                 4.5 
                 0.90 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 Q6ZVX0 
                 cDNA FLJ41981 fis, clone 
                 9.5 
                 8.5 
                 0.89 
               
               
                   
                 SMINT2011888 
                   
                   
                   
               
               
                 Q7Z374 
                 Putative uncharacterized protein 
                 9.5 
                 8.5 
                 0.89 
               
               
                   
                 DKFZp686C02218 
                   
                   
                   
               
               
                 Q6MZX9 
                 Putative uncharacterized protein 
                 9.5 
                 8.5 
                 0.89 
               
               
                   
                 DKFZp686M08189 
                   
                   
                   
               
               
                 P02679 
                 Fibrinogen gamma chain 
                 12.5 
                 11 
                 0.88 
               
               
                 Q6P5S8 
                 antioxidant activity; very-low-density lipoprotein particle 
                 16 
                 14 
                 0.88 
               
               
                   
                 remodeling 
                   
                   
                   
               
               
                 P27105 
                 Erythrocyte band 7 integral membrane 
                 4 
                 3.5 
                 0.88 
               
               
                   
                 protein (Stomatin) 
                   
                   
                   
               
               
                 P69905 
                 Hemoglobin subunit alpha (Alpha-globin) 
                 4 
                 3.5 
                 0.88 
               
               
                 Q6GMX0 
                 Putative uncharacterized protein 
                 16 
                 14 
                 0.88 
               
               
                 P02792 
                 Ferritin light chain (Ferritin L subunit) 
                 3.5 
                 3 
                 0.86 
               
               
                 Q9UL71 
                 Myosin-reactive immunoglobulin heavy 
                 7 
                 6 
                 0.86 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 Q07954 
                 Prolow-density lipoprotein receptor- 
                 7 
                 6 
                 0.86 
               
               
                   
                 related protein 1 (LRP-1) 
                   
                   
                   
               
               
                 A1A508 
                 PRSS3 protein 
                 3.5 
                 3 
                 0.86 
               
               
                 Q6P5S3 
                 Putative uncharacterized protein 
                 7 
                 6 
                 0.86 
               
               
                 Q65ZC9 
                 Single-chain Fv (Fragment) 
                 7 
                 6 
                 0.86 
               
               
                 Q5CZ94 
                 Putative uncharacterized protein 
                 6.5 
                 5.5 
                 0.85 
               
               
                   
                 DKFZp781M0386 
                   
                   
                   
               
               
                 P19823 
                 Inter-alpha-trypsin inhibitor heavy 
                 9.5 
                 8 
                 0.84 
               
               
                   
                 chain H2 (ITI-HC2) 
                   
                   
                   
               
               
                 Q16195 
                 Keratin (Fragment) 
                 9.5 
                 8 
                 0.84 
               
               
                 P30464 
                 HLA class I, B-15 alpha chain (MHC 
                 3 
                 2.5 
                 0.83 
               
               
                   
                 class I antigen B*15) 
                   
                   
                   
               
               
                 P03989 
                 HLA class I, B-27 alpha chain (MHC 
                 3 
                 2.5 
                 0.83 
               
               
                   
                 class I antigen B*27) 
                   
                   
                   
               
               
                 Q9UL83 
                 Myosin-reactive immunoglobulin light 
                 3 
                 2.5 
                 0.83 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 P35579 
                 Myosin-9 (Myosin heavy chain 9) 
                 8.5 
                 7 
                 0.82 
               
               
                 A0A5E4 
                 Putative uncharacterized protein 
                 8.5 
                 7 
                 0.82 
               
               
                 P10909 
                 Clusterin (Apolipoprotein J) (Apo-J) 
                 5.5 
                 4.5 
                 0.82 
               
               
                 Q5EFE6 
                 Anti-RhD monoclonal T125 kappa light chain 
                 16 
                 13 
                 0.81 
               
               
                 Q86TT1 
                 Full-length cDNA clone CS0DD006YL02 
                 28 
                 22.5 
                 0.80 
               
               
                   
                 of Neuroblastoma 
                   
                   
                   
               
               
                 O14791 
                 Apolipoprotein L1 (ApoL-I) 
                 2.5 
                 2 
                 0.80 
               
               
                 A2NB45 
                 Cold agglutinin FS-1 L-chain (Fragment) 
                 2.5 
                 2 
                 0.80 
               
               
                 A2NB44 
                 Cold agglutinin FS-2 H-chain (Fragment) 
                 2.5 
                 2 
                 0.80 
               
               
                 B1N7B8 
                 Cryocrystalglobulin CC1 kappa light 
                 2.5 
                 2 
                 0.80 
               
               
                   
                 chain variable region 
                   
                   
                   
               
               
                 Q865X2 
                 Full-length cDNA clone CS0DL004YM19 
                 2.5 
                 2 
                 0.80 
               
               
                   
                 of B cells 
                   
                   
                   
               
               
                 Q7Z3Y6 
                 Rearranged VH4-34 V gene segment (Fragment) 
                 2.5 
                 2 
                 0.80 
               
               
                 A0N5G5 
                 Rheumatoid factor D5 light chain (Fragment) 
                 2.5 
                 2 
                 0.80 
               
               
                 H0YK52 
                 Uncharacterized protein 
                 2.5 
                 2 
                 0.80 
               
               
                 Q14624 
                 Inter-alpha-trypsin inhibitor heavy 
                 16 
                 12.5 
                 0.78 
               
               
                   
                 chain H4 (ITI-HC4) 
                   
                   
                   
               
               
                 P31946 
                 14-3-3 protein beta/alpha (KCIP-1) 
                 4.5 
                 3.5 
                 0.78 
               
               
                 P62258 
                 14-3-3 protein epsilon (14-3-3E) 
                 4.5 
                 3.5 
                 0.78 
               
               
                 Q04917 
                 14-3-3 protein eta (Protein AS1) 
                 4.5 
                 3.5 
                 0.78 
               
               
                 P61981 
                 14-3-3 protein gamma (KCIP-1) 
                 4.5 
                 3.5 
                 0.78 
               
               
                 P27348 
                 14-3-3 protein theta (Protein HS1) 
                 4.5 
                 3.5 
                 0.78 
               
               
                 A2NUT2 
                 Lambda-chain (AA −20 to 215) 
                 9 
                 7 
                 0.78 
               
               
                 P02776 
                 Platelet factor 4 (PF-4) 
                 4 
                 3 
                 0.75 
               
               
                 P10720 
                 Platelet factor 4 variant (CXCL4L1) 
                 4 
                 3 
                 0.75 
               
               
                 P02746 
                 Complement C1q subcomponent subunit B 
                 9.5 
                 7 
                 0.74 
               
               
                 P48740 
                 Mannan-binding lectin serine 
                 7.5 
                 5.5 
                 0.73 
               
               
                   
                 protease 1 (EC 3.4.21.—) 
                   
                   
                   
               
               
                 Q96JD0 
                 Amyloid lambda 6 light chain 
                 9 
                 6.5 
                 0.72 
               
               
                   
                 variable region SAR (Fragment) 
                   
                   
                   
               
               
                 Q6GMV8 
                 Putative uncharacterized protein 
                 9 
                 6.5 
                 0.72 
               
               
                 Q8NEJ1 
                 Putative uncharacterized protein 
                 9 
                 6.5 
                 0.72 
               
               
                 A0N5G3 
                 Rheumatoid factor G9 light chain (Fragment) 
                 9 
                 6.5 
                 0.72 
               
               
                 P02745 
                 Complement C1q subcomponent subunit A 
                 3.5 
                 2.5 
                 0.71 
               
               
                 P60660 
                 Myosin light polypeptide 6 (MLC-3) 
                 3.5 
                 2.5 
                 0.71 
               
               
                 Q6MZU6 
                 Putative uncharacterized protein 
                 17.5 
                 12.5 
                 0.71 
               
               
                   
                 DKFZp686C15213 
                   
                   
                   
               
               
                 Q6N093 
                 Putative uncharacterized protein 
                 17.5 
                 12.5 
                 0.71 
               
               
                   
                 DKFZp686I04196 (Fragment) 
                   
                   
                   
               
               
                 P61224 
                 Ras-related protein Rap-1b (GTP-binding protein smg p21B) 
                 3 
                 2 
                 0.67 
               
               
                 P19105 
                 Myosin regulatory light chain 12A (MLC-2B) 
                 4 
                 2.5 
                 0.63 
               
               
                 P05387 
                 60S acidic ribosomal protein P2 
                 2.5 
                 1.5 
                 0.60 
               
               
                   
                 (Renal carcinoma antigen NY-REN-44) 
                   
                   
                   
               
               
                 P19338 
                 Nucleolin (Protein C23) 
                 10 
                 6 
                 0.60 
               
               
                 O75636 
                 Ficolin-3 (Collagen/fibrinogen 
                 3.5 
                 2 
                 0.57 
               
               
                   
                 domain-containing lectin 3 p35) 
                   
                   
                   
               
               
                 F8VWA4 
                 Uncharacterized protein 
                 3.5 
                 2 
                 0.57 
               
               
                 B7Z539 
                 cDNA FLJ56954 
                 8 
                 4.5 
                 0.56 
               
               
                 Q96CX2 
                 BTB/POZ domain-containing protein 
                 4.5 
                 2.5 
                 0.56 
               
               
                   
                 KCTD12 (Pfetin) 
                   
                   
                   
               
               
                 P02730 
                 Band 3 anion transport protein(AE 1) 
                 17.5 
                 9.5 
                 0.54 
               
               
                 P23528 
                 Cofilin-1 (p18) 
                 3 
                 1.5 
                 0.50 
               
               
                 P06748 
                 Nucleophosmin (NPM) 
                 3 
                 1.5 
                 0.50 
               
               
                 Q6N091 
                 Putative uncharacterized protein 
                 7 
                 3.5 
                 0.50 
               
               
                   
                 DKFZp686C02220 
                   
                   
                   
               
               
                 Q6N041 
                 Putative uncharacterized protein 
                 7 
                 3.5 
                 0.50 
               
               
                   
                 DKFZp686O16217 (Fragment) 
                   
                   
                   
               
               
                 P11166 
                 Solute carrier family 2, facilitated 
                 2 
                 1 
                 0.50 
               
               
                   
                 glucose transporter member 1 
                   
                   
                   
               
               
                 P04275 
                 von Willebrand antigen 2 
                 61.5 
                 28.5 
                 0.46 
               
               
                 P62805 
                 Histone H4 
                 2.5 
                 1 
                 0.40 
               
               
                 Q13201 
                 Multimerin-1 (EMILIN-4) 
                 11 
                 4 
                 0.36 
               
               
                 B7Z1F8 
                 cDNA FLJ53025, highly similar to 
                 52 
                 16 
                 0.31 
               
               
                   
                 Complement C4-B 
                   
                   
                   
               
               
                 P04114 
                 Apolipoprotein B-100 (Apo B-100) 
                 25 
                 7.5 
                 0.30 
               
               
                 Q8TCG3 
                 TPMsk3 (Fragment) 
                 5 
                 1.5 
                 0.30 
               
               
                 P16452 
                 Erythrocyte membrane protein band 4.2 (P4.2) 
                 5.5 
                 1 
                 0.18 
               
               
                 P11277 
                 Spectrin beta chain, erythrocyte (Beta-I spectrin) 
                 23 
                 3 
                 0.13 
               
               
                 P02549 
                 Spectrin alpha chain, erythrocyte 
                 33 
                 1.5 
                 0.05 
               
               
                   
                 (Erythroid alpha-spectrin) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 2b 
               
               
                   
                   
               
               
                   
                   
                   
                 Mean 
               
               
                   
                   
                   
                 number 
               
               
                   
                   
                   
                 of 
               
               
                   
                 Accession 
                   
                 peptides 
               
               
                   
                 Number 
                 Protein name 
                 SevD 
               
               
                   
                   
               
             
            
               
                   
                 P00751 
                 Complement factor B (C3/C5 
                 4.5 
               
               
                   
                   
                 convertase) 
                   
               
               
                   
                 P00752 
                 Complement component C8 alpha chain 
                 2 
               
               
                   
                 P00753 
                 Peroxiredoxin-1 (Natural killer 
                 2 
               
               
                   
                   
                 cell-enhancing factor A) 
                   
               
               
                   
                 P00754 
                 Moesin (Membrane-organizing 
                 2 
               
               
                   
                   
                 extension spike protein) 
                   
               
               
                   
                 P00755 
                 Chloride intracellular channel 
                 2 
               
               
                   
                   
                 protein 1 
                   
               
               
                   
                 P00756 
                 Ferritin heavy chain (Ferritin H 
                 2 
               
               
                   
                   
                 subunit) 
               
               
                   
                   
               
            
           
         
       
     
     Example 4: Confirmation Elisa 
     Method: 
     So as to confirm the mass spectrometry results, specific quantitative ELISAs were carried out in duplicate on individual plasmas. The proteins selected and tested, from those identified in tables 2a/2b, are those with a severe dengue (SevD)/conventional dengue (DF) ratio of greater than or equal to 1.33 and less than or equal to 0.75 with a mean number of peptides of greater than 1 for each sample and a potential link to dengue pathogenesis. This first screening made it possible to only assay those proteins most of interest. According to these criteria, the following proteins were selected:
         ceruloplasmin,   protein S,   properdin complement factor,   antithrombin-III,   secretory component p85,   complement C1r protein,   complement C1s protein,   angiotensin,   factor II,   CFB,   anti-factor VIII,   serum amyloid P-component,   olfactomedin 4 (OLFM4),   thrombospondin,   platelet factor 4 (PF4),   complement Clq protein,   moesine, and   complement C8 protein.       

     Multimerin-1, apolipoprotein B-100 and von Willebrand factor were also assayed. 
     It should be noted that these proteins are predominantly elements of the coagulation pathway or the complement cascade. 
     These ELISAs were carried out by virtue of commercially available kits (USCN, China), following supplier instructions. Statistical analyses (Mann-Whitney test and ROC/AUC curve) were carried out by means of GraphPad Prism V4.03 software. 
     Each candidate marker was assayed on individual plasma samples. These samples are plasma samples taken during the acute febrile phase of the disease (viremic phase), these samples either originating from patients having remained with conventional dengue DF, without developing severe dengue, or from patients having developed severe dengue SevD. All the patients had secondary dengue. Only serotypes 1, 2 and 3 were represented (no serotype 4). These samples originated from l&#39;Universidad Industrial de Santander (Bucaramanga, Colombia) [5] or from the Institut Pasteur in Cambodia (Phnom-Penh). The latter were part of a prospective study carried out in agreement with the local ethics committee. The characteristics of the two sampling sources are given in tables 3 and 4 below. The samples were collected after appearance of symptoms. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                 DF (n = 15) 
                 SevD (n = 15) 
                 P value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Mean age +−  
                 29.3 +−  
                 0.52 
                 28.3 +−  
                 0.5 
                 ns 
               
               
                 SD (years) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
            
               
                 % male 
                 60% 
                 53% 
                 ns 
               
               
                 Sampling (day) 
                 3.06 
                 2.37 
                 ns 
               
            
           
           
               
               
               
               
               
               
            
               
                 Mean weight +−  
                 60.2 +−  
                 8.6 
                 50.3 +−  
                 15.6 
                 ns 
               
               
                 SD (years) 
                   
                   
                   
                   
                   
               
               
                 Total cholesterol (g/l) 
                 1.44 +−  
                 5.4 
                 1.29 +−  
                 10.1 
                 0.03  
               
               
                 AST (U/L) 
                 90.2 +−  
                 9.75 
                 142.7 +−  
                 29.8 
                 0.001 
               
               
                 ALT (U/L) 
                 69.7 +−  
                 11.4 
                 90.8 +−  
                 24.5 
                 ns 
               
            
           
           
               
               
               
               
            
               
                 Positive tourniquet test 
                 8/15 (60%) 
                 10/15 (66%) 
                 ns 
               
            
           
           
               
               
               
               
               
               
            
               
                 Viral load (copies/ml) 
                 4.05 10 6  +−  
                 3.5 
                 4.1 10 7  +−  
                 5.22 
                 0.006 
               
            
           
           
               
               
               
               
            
               
                 Serotype 
                 DV2-DV3 
                 DV2-DV3 
                 — 
               
               
                 Secondary dengue 
                 Yes 
                 Yes 
                 — 
               
               
                 Comorbidity 
                 No 
                 No 
                 — 
               
               
                   
               
            
           
         
       
     
                                 TABLE 4                   DF (n = 23)   SevD (n = 26)   P value                                                        Mean age +−    8.6 +−    0.38   7.3 +−    0.45   ns       SD (years)                                                 % male   56%   35.6%   ns       Sampling (day)   2.6   3.2   ns                                     Mean weight +−    20.48 +−    0.93   18.86 +−    0.98   ns       SD (years)                           Total cholesterol   3.32 +−    0.14   2.37 +−    0.12   &lt;0.0001                             (mmol/l)                                                 HDL (mmol/l)   0.82 +−    0.07   0.31 +−    0.02   &lt;0.0001       Triglycerides (g/l)   1.37 +−    0.17   2.8 +−    0.2   &lt;0.0001       AST (IU/L)   133.3 +−    23.18   302.5 +−    50.7   0.0043       ALT (IU/L)   66.9 +−    13.4   106.3 +−    21.2   ns       Viral load    7.1 10 8  +−    6   2.06 10 8  +−    2   ns       (copies/ml)                                                 Positive    19/23 (82%)   18/26 (82.6%)   ns                                 tourniquet test                                                     HGB   11.63 +−    0.15   13.3 +−    0.29   &lt;0.0001       Hematocrit   38.23 +−    0.57   41.62 +−    0.8   0.0013                             Hepatomegaly    8/12 (66%)   16/26 (61.5%)   ns       (ultrasound)                   Serotype   DV1   DV1   —       Secondary dengue   Yes   Yes   —       Comorbidity   No   No   —               SD: standard deviation       ns: non-significant p value            
Results:
 
     For the majority of the ELISA-assayed markers, no difference in plasma concentration was observed between the DF and SevD plasmas, whether Colombian or Cambodian (p&gt;0.1). 
     On the other hand, for two markers, the results make it possible to clearly distinguish those patients who then developed severe dengue SevD from those who remained solely with conventional dengue without developing severe dengue. The first marker is PF4 (platelet factor 4). 
     For the Colombian samples, a difference in plasma concentration in favor of the DF samples is observed (p&lt;0.001) ( FIG. 2 ). The AUC is 0.88 (95% CI: 0.7305-1). 
     This is confirmed for the Cambodian samples for which there is a significant difference in plasma concentration in favor of the DF samples (p&lt;0.0001) ( FIG. 3 ). The AUC is 0.94 (95% CI=0.87-1). The ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 5 below: for this marker and for a sensitivity of 95%, a specificity of close to 78% is reached. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                   
                 For a sensitivity of: 
                 The best specificity is: 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 PF4 
                 100 
                 61 
               
               
                   
                   
                 95 
                 77.9 
               
               
                   
                   
               
            
           
         
       
         
         
           
             The second marker is OLFM4 (olfactomedin 4) 
           
         
       
    
     For the Colombian samples, the plasma concentration of the marker is higher in the SevD samples compared to the DF samples (p=0.07; cf.  FIG. 4 ). 
     This is confirmed on the Cambodian samples with an extremely significant difference in concentration (p&lt;0.0003) and a median that is more than twice as high for the SevD samples compared to the DF samples ( FIG. 5 ). The AUC is 0.858 (95% CI=0.7307-0.985). The ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 6: for this marker and for a sensitivity close to 95%, a specificity of greater than 72% is reached. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   
                 For a sensitivity of: 
                 The best specificity is: 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 OLFM4 
                 100 
                 61 
               
               
                   
                   
                 94.4 
                 72.2 
               
               
                   
                   
               
            
           
         
       
     
     In parallel, another marker, α-2 macroglobulin (A2M) was identified from unpurified Cambodian plasma samples by a SILAC-type differential proteomic method (Stable Isotope Labelling by Aminoacids in cell Culture) [6]. The identification of this third marker is described in the following examples. 
     Example 5: Characterization of the Samples 
     The composition of each plasma pool or group used in this experiment is summarized in table 7. All the Cambodian plasmas selected to compose the pools were taken during the acute febrile phase of the disease, before the critical phase, from patients having a secondary infection. The serotype concerned was serotype 1. All the SevD patients were hospitalized and had signs of hemorrhaging. No comorbidity was reported. All the plasmas were verified as being NS1-positive (Platelia dengue kit, Bio-rad) and the viral load was also verified by Q-RT-PCR with a commercially available kit following supplier instructions. The pools composed correspond to a final volume of approximately 2 ml of plasma. The plasma groups are inactivated beforehand with heat (56° C./20 minutes) then preclarified by centrifugation for 5 mins at 1000×g and at 4° C., so as to remove impurities present in the sample. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                 DF (n = 6) 
                 SevD (n = 6) 
               
               
                   
                   
               
             
            
               
                   
                 Serotype 
                 DV1 
                 DV1 
               
               
                   
                 Secondary dengue 
                 YES 
                 YES 
               
               
                   
                 Age in years (mean) 
                 6-12 (8.6) 
                 6-8 (7) 
               
               
                   
                 Male/female 
                 2/4 
                 2/4 
               
               
                   
                 NS1 positive/virus positive 
                 YES/YES 
                 YES/YES 
               
               
                   
                 Mean day of sampling 
                 4 
                 3.85 
               
               
                   
                 Severity grade 
                 0-1 
                 3-4 
               
               
                   
                 Comorbidity 
                 NO 
                 NO 
               
               
                   
                   
               
            
           
         
       
     
     Example 6: Differential Proteome Analysis 
     The method used is a semi-quantitative proteomic method of SILAC type (Stable Isotope Labelling by Aminoacids in cell Culture) [6] developed by Pronota (Ghent, Belgium) using the MASStermind™ platform and carried out on conventional dengue DF or severe dengue SevD plasma groups. Each group is composed of a mixture of 6 samples, as detailed in example 5. 
     These plasma mixtures have been depleted beforehand in the 14 most abundant plasma proteins by affinity chromatography. The quantity of proteins recovered in the end was obtained by a colorimetric assay based on bicinchoninic acid (BCA assay Thermo Fisher Scientific Inc., USA). 
     The MASStermind™ study compared each sample to a reference sample which groups together all the samples. This method provides information on the relative levels, and presence or absence, of peptides/proteins in the severe dengue SevD samples compared to the conventional dengue DF samples. The differential analysis is carried out by mixing the samples labelled with different isotopes and by analyzing, by mass spectrometry, each matched peak. The isotope label is introduced by tryptic hydrolysis which incorporates 2  18 O atoms (“heavy” labeling) onto the C-terminal arginine of a peptide, which leads to a mass difference of 4 daltons to the same peptide labeled with  16 O (“light” labeling). The reference sample is labeled with  16 O, whereas the individual samples are labeled with  18 O. The MS/MS data are then submitted to the MASCOT software for identification of the peptides and proteins in each sample. 
     Following MS/MS analysis, more than 250 quantifiable proteins were identifiable, 10 proteins of which had at least 1 peptide found to be differential. For each protein identified, the SevD/DF ratio is calculated as the weighted mean of the coefficients of all the peptides identified for the given protein. Overall, the results showed a high degree of similarity between the two proteomes and only a few proteins were found to be expressed differentially. For three of these ten proteins, the peptides identified are systematically expressed differentially and have a mean SevD/DF ratio which deviates from 1 (cf. table 8). The three proteins identified are: α-2 macroglobulin (A2M), complement C3f and heparin cofactor 2 These proteins are predominantly elements of the coagulation pathway or the complement cascade. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                   
                   
                 95% CI on 
               
               
                   
                 Accession 
                   
                 mean of 
               
               
                 Proteins 
                 number 
                 SevD/DF ratio 
                 ratios 
               
               
                   
               
             
            
               
                 α-2 macroglobulin 
                 A2MG_HUMAN 
                 0.33 
                 0.25-0.44 
               
               
                 Complement C3f 
                 CO3_HUMAN 
                 0.89 
                 0.68-1.17 
               
               
                 Heparin cofactor 2 
                 HEP2_HUMAN 
                 2.25 
                 1.71-2.95 
               
               
                   
               
            
           
         
       
     
     Example 7: Confirmation Elisa 
     Method: 
     So as to confirm the mass spectrometry results, specific quantitative ELISAs were carried out in duplicate on individual plasmas. The proteins assayed are those identified in example 6: α-2 macroglobulin (A2M), complement C3 protein, and heparin cofactor 2. 
     These ELISAs were carried out by virtue of commercially available kits (USCN, China), following supplier instructions. Statistical analyses (Mann-Whitney test and ROC/AUC curve) were carried out by means of GraphPad Prism V4.03 software. 
     Each candidate marker was assayed on individual samples. These samples were plasma samples taken from patients during the acute febrile phase of the disease (viremic phase). Clinical follow-up of the patients showed that some finally remained with conventional dengue DF without developing severe dengue, whereas others had developed severe dengue SevD. All the patients had secondary dengue. Only serotype 1 was represented. These samples originated from the Institut Pasteur in Cambodia (Phnom-Penh) and were part of a prospective study carried out in agreement with the local ethics committee. The characteristics of the sampling source are given in table 9. 
                                 TABLE 9                   DF (n = 23)   SevD (n = 26)   P value                                                        Mean age +−    8.6 +−    0.38   7.3 +−    0.45   ns       SD (years)                                                 % male   56%   35.6%   ns                                 Sampling (day)   2.6       3.2   ns                                     Mean weight +−    20.48 +−    0.93   18.86 +−    0.98   ns       SD (kg)                           Total cholesterol    3.32 +−    0.14   2.37 +−    0.12   &lt;0.0001       (mmol/l)                           HDL (mmol/l)   0.82 +−    0.07   0.31 +−    0.02   &lt;0.0001       Triglycerides (g/l)   1.37 +−    0.17   2.8 +−    0.2   &lt;0.0001       AST (IU/L)   133.3 +−    23.18   302.5 +−    50.7   0.0043       ALT (IU/L)   66.9 +−    13.4   106.3 +−    21.2   ns       Viral load    7.1 10 8  +−    6   2.06 10 8  +−    2   ns       (copies/ml)                                                 Positive    19/23 (82%)   18/26 (82.6%)   ns       tourniquet test                                                 HGB   11.63 +−    0.15   13.3 +−    0.29   &lt;0.0001       Hematocrit   38.23 +−    0.57   41.62 +−    0.8   0.0013                             Hepatomegaly     8/12 (66%)   16/26 (61.5%)   ns       (ultrasound)                   Serotype   DV1   DV1   —       Secondary dengue   Yes   Yes   —       Comorbidity   No   No   —               HBG: plasma hemoglobin       SD: standard deviation       ns: non-significant p value            
Results:
 
     For the majority of the ELISA-assayed markers, (complement C3f and heparin cofactor 2) no difference in plasma concentration was observed between the DF and SevD plasmas (p&gt;0.1). 
     However, for the Cambodian samples ELISA-assayed for A2M, there was a significant difference in concentration (p&lt;0.0004) and a median approximately twice as high for the DF samples compared to the SevD samples ( FIG. 6 ). The AUC was 0.89 (95% CI=0.75-1). 
     For A2M, the ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 10: for a sensitivity close to 94%, a specificity of greater than 83% is reached. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                   
                 For a sensitivity of: 
                 The best specificity is: 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 A2M 
                 100 
                 72 
               
               
                   
                   
                 93.8 
                 83.3 
               
               
                   
                   
               
            
           
         
       
     
     LITERATURE REFERENCES 
     
         
         1. S B Halstead. The lancet 2007; 370: 1644-52 
         2. A S Leong et al. Semin. Diagn. Pathol. 2007; 24(4):227-236 
         3. K. Clyde et al. J. Virol. 2006; 23: 11418-11431 
         4. Fields Virology, Fifth edition, Knipe D M ed., LWW 
         5. Villar-Centeno L A et al. Am. J. Trp. Med. Hyg. 2008; 78: 370-374 6. R. Drissi et al. FEBS J. 2013