FR2794864A1 - METHOD OF EARLY DETECTION OF FLAVIVIRUSES AND ITS APPLICATIONS - Google Patents

Abstract

Early detection of infection by a flavivirus (FV) comprises detecting the non-structural glycoprotein, NS1, during the clinical phase of the infection by an immunological method that employs two of the same or different antibodies, one (Ab1) for capture and the other (Ab2) for detection. Early detection of infection by a flavivirus (FV) comprises detecting the non-structural glycoprotein, NS1, during the clinical phase of the infection by an immunological method that employs two of the same or different antibodies, one (Ab1) for capture and the other (Ab2) for detection. Ab1 is polyclonal preselected from immunocapture by NS1 in its hexameric form, or a mixture of monoclonal antibodies selected for high affinity for hexameric NS1 and then purified. Ab2 is polyclonal, directed against the hexameric form, or a mixture of monoclonals directed against this form. Independent claims are also included for the following: (1) kits for early diagnosis of FV infection; (2) purifying NS1 in hexameric form; (3) an immunogenic composition containing, as an active ingredient, NS1 in hexameric form, optionally also other proteins and at least one vehicle; (4) an immunogenic composition containing as an active ingredient, either nucleic acid that expresses all or part of NS1 of dengue virus, of any serotype, or an expression system containing this nucleic acid linked to a promoter functional in the host being treated, and at least one vehicle; and (5) producing the NS1 protein of dengue virus by expressing a 1.2 kb sequence (reproduced), linked to a promoter, in eukaryotic cells.

Description

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 The present invention relates to a method for the early detection of flaviviruses, in particular the dengue virus and to its applications.

 Dengue fever is an acute febrile tropical disease, the causative virus of which is an arbovirus, transmitted by mosquitoes. The vectors of the disease are mosquitoes of the genus Aedes, in particular Aedes aegypti whose breeding places are most often domestic and peridomestic. The responsible virus, isolated in 1951, has been classified into four different antigenic types (DENI, DEN2, DEN3 and DEN4). It belongs to the family of Flaviviridae, genus flavivirus.

 More than two billion people live in endemic areas and the number of people infected with the virus is estimated to be more than 60 million per year.

Dengue fever is responsible for 200,000 hospitalizations and several tens of thousands of annual deaths, most of them children.

 After an incubation of five to eight days, the onset of clinical signs is generally sudden and consists of the appearance of undifferentiated fever (DF dengue fever) accompanied by severe headache, low back pain, muscle and joint pain as well as chills. From the third to the fifth day of feverish phase, a congestive maculopapular rash may appear for three to four days (classic dengue).

 In its severe form, the infection can lead to the appearance of a hemorrhagic syndrome (DHF or dengue haemorrhagic fever), characterized by increased vascular permeability and dysregulation of hemostasis. While in the majority of cases, the evolution is generally favorable within a week, the outcome of the disease can be fatal in the event of hypovolemic shock (DSS or dengue shock syndrome). These complications are generally observed in the presence of pre-existing immunity, acquired in particular during a primary infection with a heterologous dengue virus (different serotype). In fact, two different types of serological response are identified in subjects infected with dengue: individuals who have never had a flavivirus infection and have not been vaccinated against another flavivirus (yellow fever virus, virus Japanese encephalitis for example) will present a primary response, characterized by a slow appearance of antibodies specific for the virus responsible for the infection; individuals who have ever had a

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 flavivirus infection (another dengue serotype, for example) or have been vaccinated against another flavivirus will show a secondary response, characterized by rapid onset of antibodies.

 The infectious agent is the dengue virus belonging to the family of Flaviviridae, to which also belong the yellow fever virus and that of Japanese encephalitis (TP Monath et al., (1996) Flaviviruses in BN Fields, DM Knipe, PM Howly et al. (Eds.) "Fields Virology" Philadelphia: Lippincott Raven Press Publishers). These viruses have a single-stranded RNA of positive polarity which comprises 11000 nucleotides and which codes for a polyprotein of approximately 3400 amino acids. It is individualized into three structural proteins and seven non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, during co- and post-translational cleavages by viral and cellular proteases. The non-structural protein NS1 was first identified in 1970 by P.K. Russel et al. (J. immunol., (1970), 105,838-845) and characterized in 1985 by G. W. Smith et al.

(J Gen Virol., (1985), 66,559-571). This highly conserved glycoprotein in the flavivirus family (T. P. Monath already cited), in particular in the four serotypes of the dengue virus, exists in an intracellular form, and in an extracellular form. The intracellular form is thought to intervene in the early phases of virus replication (J.M. Mackenzie et al., Virologv, (1996), 220,232-240). Before being transported to the plasma membrane, the NS1 protein undergoes dimerization. In mammalian cells, but not in insect cells, part of the NS1 protein is released into the extracellular medium either as a soluble protein or as a microparticulate. When it is in soluble form, the protein exists in the form of an oligomer, in particular a pentamer or a hexamer (Crooks AJ et al. J Chrom. (1990), 502,59-68 and J Gen. Virol.

(1994), 75,3453-3460). At the moment the role of the NS1 protein is not known.

 Several studies suggest a role for the NS1 protein in the protective immune response against flavivirus infections. Experiments with yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis viruses have shown protection against a lethal dose of homologous virus in animals vaccinated with the NS1 protein subunit or

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 produced by vector viruses, vaccinia or adenovirus type (Schlesinger et al. (1986), 60,1153-1155;. J. Gen. Viral., (1987), 68,853-857; Falgout et al. J Virol., ( 1990), 64, 4356-4363; Jacobs et al., J Virol., (1992), 66,2086-2095). Passive immunization of mice with anti-NS monoclonal antibodies has also made it possible to obtain a certain degree of protection (Schlesinger et al. J Immunol. (1985), 135, 2805-2809; Gould et al. J Gen. Virol ., (1986), 67,591-595; Henchal et al. J Gen.

Virol., (1988), 69,2101-2107). The role of antiNSI antibodies in protection is not fully known, but it may be associated with lysis of infected cells (Schlesinger et al. Virology, (1993), 192,132-141). NS1 proteins on the surface of infected cells would be recognized by complement-binding antibodies.

 There is no specific treatment and the care given to the patient is only symptomatic. In the case of classic dengue fever, treatment is based on the administration of analgesics and antipyretics. In the case of DHF, the treatment consists of an infusion to compensate for the plasma leakage, associated with the correction of hydroelectric disorders and the revival of diuresis.

 There is no commercially available vaccine against the dengue virus.

On the other hand, protection trials with attenuated strains of the 4 serotypes of the dengue virus were carried out by N. Bhamarapravati et al. (Dengue and Dengue haemorrhagic fever (1997), 367-377) with unsatisfactory results. Prevention therefore rests solely on the fight against the vector. This struggle combines larval destruction and "adulticide" sprays.

 In the absence of a vaccine, it is necessary to monitor epidemics and prevent the above-mentioned complications; to do this, active surveillance programs have notably been set up by the World Health Organization and essentially include surveillance of cases of fever and vector insects and serological and virological screening of people with fever and suspected of being infected with the dengue virus.

 The etiology of dengue is sometimes tricky to assert when a patient has an undifferentiated febrile syndrome like "dengue-like" which can originate from another arbovirus, viruses causing eruptive fevers, the

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 flu, leptospirosis and even malaria. Only a laboratory examination can provide the diagnosis.

 There are currently several diagnostic tests for dengue.

However, to arrive at an interpretable result, it is necessary to combine several methods: - virus isolation, by classical virology techniques, in particular by infection of cell cultures or propagation in mice of mice or amplification by inoculation with mosquitoes and examination, for example by immunofluorescence. These methods have the drawback of being very difficult to implement and of depending on the earliness of the sample and good storage conditions; moreover, the first results cannot be obtained in less than a week; to overcome these drawbacks, one can have recourse to a test by RT / PCR (V. Deubel, L'eurobiologiste (1997), tome XXXI, 37-155); this method is not always reliable and cannot be used routinely in countries affected by dengue virus infection for reasons of cost and equipment; - serological tests; the earliest serological diagnosis consists in looking for IgM by the MAC-ELISA technique (immunoglobulin M Antibody Capture Enzyme-Linked ImmunoSorbent Assay). Their detection makes it possible to establish a diagnosis of probability of infection by a flavivirus from the defervescence of the fever; IgG testing requires two samples: one at the start of clinical signs, the other 10 to 28 days later, so as to demonstrate a serological conversion by hemagglutination inhibition reaction (IHA) or by ELISA .

 Simple and inexpensive immunological tests have also been proposed, usable in countries at risk, which use, as specific immunological reagent peptides derived from the non-structural protein NS1 characteristic of flaviviruses. Thus, US Pat. No. 5,824,506 describes a method using peptides derived from the non-structural protein NS1, which makes it possible to detect the antibodies induced by the presence of the dengue virus; however, the peptides selected essentially recognize the samples obtained from

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 convalescent subjects and also recognize better patients infected for the second time than those infected for the first time; these disappointing results could be explained either by the fact that the antibody titers are variable during the acute phase (clinical phase) of the disease, or by the fact that the peptides used are not representative of the antigenic characteristics of the protein and therefore lead to poor recognition of the antibodies sought.

 In all cases, only late confirmation of a flavivirus infection can be made.

To solve this problem, Andrew Falconar and his team (Report from the Sir Albert Sakzewski Virus Research Center Royal Children's Hospital,
1991) proposed to detect the presence of the non-structural glycoprotein NS1 itself. They have developed a double sandwich ELISA test, in which a rabbit serum containing anti-NSI polyclonal antibodies, used as capture antibody, is immobilized on a microtiter plate. The captured antigen is detected using mouse monoclonal antibodies directed either against the DEN2 type virus, or group specific; the formation of the antigen / antibody complex is revealed using anti-mouse goat IgG conjugated to peroxidase. By this method, the Authors have shown that by using purified or degraded dimeric NS 1 protein as standard, the detection sensitivity of the test is approximately 4 ng / ml with the DEN2 monoclonal antibodies as a revelation and about 60 ng / ml with group monoclonal antibodies.

 However, this test does not make it possible to detect the NS1ni protein in cases of primary infections in the acute or convalescent phase, or in secondary infections in the convalescent phase in which there is a high titer of anti-NS 1 antibodies; the Authors concluded from this that the NS1 protein should be present in significant quantities only in the case of secondary infections and this, transiently, during the infection.

 However, the inventors have developed a process for purifying the NS1 protein of a flavivirus in hexameric form, which has enabled them to select antibodies specific for this protein in hexameric, dimeric or monomeric form, or one of its fragments, and to show so

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 surprising that these antibodies are the tools of choice for demonstrating the different forms of the circulating NS1 protein in the context of infection with a flavivirus, in particular in the early phases where the response to specific antibodies is undetectable, especially during primary infections with the dengue virus.

 Consequently, the inventors have set themselves the goal of providing a method for the early detection of a flaviviral infection, which better meets the needs of the practice than the methods of the prior art, that is to say say a process which is reliable, rapid, inexpensive and which makes it possible to adapt medical care in time and to avoid large-scale epidemics by implementing rapid and massive anti-vector actions.

 The present invention therefore relates to a method for the early detection of a flaviviral infection, characterized in that it comprises the detection of the non-structural glycoprotein NS1 of a flavivirus in a biological sample, during the clinical phase of the infection, using an immunological method using at least two identical or different antibodies, - the first antibody or antibody for capturing the NS1 glycoprotein consisting of antibodies chosen from the group consisting of: - polyclonal antibodies previously selected by immunocapture on the NS 1 protein of said flavivirus, of hexameric, dimeric or monomeric form, said forms being non-degraded and - mixtures of anti-NSI monoclonal antibodies previously selected for their affinity for the protein NS1dudit flavivirus, of hexameric, dimeric or monomeric form, said being non-degraded, said anticorp s monoclonals then being purified, - the second antibody or revealing antibody being chosen from the group consisting of: - polyclonal antibodies directed against the protein NS 1 of the same type as that to be detected and - a mixture of monoclonal antibodies directed against a protein NS 1 of the same type as that to be detected.

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 For the purposes of the present invention, the term “non-degraded form of the NS1 protein of a flavivirus” means the native protein or one of its fragments, in hexameric, dimeric or monomeric form, comprising at least one epitope allowing immunocapture, in particular a epitopes collected in table 1 on page 319 of the AKI article Falconar et al., Arch; Virol., (1994), 137.

 Within the meaning of the present invention, the term “polyclonal and monoclonal antibodies” directed against the NS1 protein of a flavivirus, antibodies obtained by immunization of a non-human mammal, either by an NS1 protein of hexameric, dimeric or monomeric form, said forms being non-degraded, either by a living or inactivated flavivirus, said polyclonal antibodies being selected for their affinity for the NS protein in its various forms and purified in a single step and said monoclonal antibodies being selected for their affinity for the NS1 protein in its various forms and then purified by conventional techniques, in particular by affinity chromatography or ion exchange.

 Surprisingly, the use, for the detection of the NS1 protein in a biological sample, of polyclonal antibodies selected and purified by immunocapture on the NS1 protein of hexameric, dimeric or monomeric form, said forms being non-degraded or of monoclonal antibodies previously selected for their reactivity towards the NS1 protein of hexameric, dimeric or monomeric form, said forms being non-degraded, and purified, in place of a total hyperimmunized rabbit serum, makes it possible to significantly improve the sensitivity of the method and to highlight the NS1 protein circulating in the blood of patients, from the early stage of infection, both during a primary infection and a secondary infection.

 The method according to the present invention has a certain number of advantages: - it can be implemented early: the presence of the NS1 glycoprotein is revealed during the clinical phase, before the antibody response is detectable,

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 - it is sensitive: it is possible to detect up to less than 2 ng of protein / ml of serum, which makes it possible to detect the circulating NS1 protein in the early phase of primary infections, - it is rapid: one can obtain a response in the day, - it is relatively inexpensive and can therefore be used in countries at risk, - it makes it possible to distinguish vaccinated people from people recently infected with a flavivirus since the NS1 protein will be absent in vaccinated people in whom anticoprs could still be detectable ..

 According to an advantageous mode of implementation of said method, the flaviviral infection is an infection with the dengue virus.

 According to another advantageous embodiment of said method, the first antibody is preferably fixed on a suitable solid support and the second antibody is optionally conjugated to an appropriate marker.

 According to another advantageous embodiment of the said method, when the second antibody is not conjugated to a marker, its binding to the NS1 protein fixed on the solid support is then detected by a third antibody conjugated to a suitable marker , said third antibody being an antibody conventionally used, such as for example an IgG directed against the second antibody and produced in particular in goats, pigs or donkeys.

 Among the markers used, mention may be made, for example, of fluorescent markers, the biotin / streptavidin system, non-isotopic markers or enzymes, such as for example horseradish peroxidase or alkaline phosphatase.

 According to another advantageous embodiment of the said method, the said third antibody is conjugated to an enzyme.

 According to another advantageous embodiment of the said method, the first antibody or capture antibody consists of polyclonal antibodies of mice, selected by immunocapture on the NS1 protein of the dengue virus, said protein being in hexameric form , and

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 the second antibody or antibody for detecting the presence of NS1 in the biological sample to be analyzed, consists of polyclonal rabbit antibodies immunized with protein NS1 of the dengue virus, said protein being in hexameric form, the binding said second antibody being revealed by a third antibody consisting of antibodies, conjugated to peroxidase and directed against the second antibody.

 According to another even more advantageous embodiment of the said method, the polyclonal antibodies of mice are purified by immunocapture on the NS1 hexameric dengue protein of serotype 1.

 The present invention also relates to a kit or kit for the diagnosis of a flaviviral infection, characterized in that it comprises: - at least one capture antibody and at least one revealing antibody as defined above, - at least one positive control consisting of the NS1 protein of a flavivirus and / or of different serotypes according to the flavivirus, said protein being in hexameric, dimeric or monomeric form, said forms being non-degraded, and - at least one negative control consisting of a serum normal human.

 According to an advantageous embodiment of the diagnostic kit according to the invention, said NS1 protein in its various forms is obtained from a culture supernatant, either from infected mammalian cells, or from mammalian cells transfected with a recombinant plasmid, comprising the gene for the NS 1 protein or a fragment of the said gene or a fragment of the flaviviral genome, the said fragments being capable of expressing the NS 1 protein.

 According to another advantageous embodiment of the diagnostic kit according to the invention, the NS1 protein is that of the dengue virus.

 According to another even more advantageous embodiment of said diagnostic kit, the plasmid is the plasmid pCIneo-NS1.FGA which has been deposited with the National Collection of Cultures and Microorganisms held by the Institut Pasteur under the number 1-2220 , dated June 7, 1999.

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 The subject of the present invention is also a process for purifying the NSI protein of a flavivirus, in hexameric form, from a culture supernatant, either of infected mammalian cells or of mammalian cells transfected with a recombinant plasmid. , comprising the NSI protein gene of a flavivirus or a fragment of said gene or a fragment of the flaviviral genome, said fragments being capable of expressing the NS1 protein, characterized in that the soluble form of the NS1 protein is separated from the form microparticulate of said protein, - either by treatment with a precipitating agent and then centrifugation at a speed greater than or equal to 10,000 g, - or by centrifugation at a speed greater than or equal to 50,000 g, before the purification step carried out by classical techniques, in particular by affinity chromatography.

 For the purposes of the present invention, the term “precipitating agent” means an agent which specifically precipitates microparticulate proteins or cellular debris, such as for example polyethylene glycol, said agent being used under conventional conditions which make it possible to separate soluble proteins and microparticulate proteins. or cellular debris.

 In a preferred embodiment of said purification process, the hexameric protein NS1 is that of the dengue virus.

 The present invention also relates to an immunogenic composition, characterized in that it comprises an active principle selected from the group consisting of: - the NS1 protein of a flavivirus, of hexameric form, optionally associated with other proteins, - a DNA sequence coding for said NS 1 protein and - an expression system comprising at least one promoter capable of expressing in the host into which it is injected, the DNA coding for protein NS 1 of flavivirus, said gene expressing said protein, in association with at least one pharmaceutically acceptable vehicle.

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 Vaccination protocols using nucleic acids are in particular described in international application WO 90/11092.

 In a preferred embodiment of the immunogenic composition according to the present invention, the immunogenic composition comprises at least one mixture of the NS1 proteins of hexameric form corresponding to the various serotypes of the dengue virus.

 The present invention also relates to the use of an NS1 protein of hexameric form of a flavivirus, for the preparation of an immunogenic composition capable of inducing the production of antibodies in vivo.

 In a preferred mode of said use, the NS 1 protein is that of the dengue virus, in particular of serotype 1.

 The present invention also relates to the use of at least one anti-NS1 monoclonal antibody previously selected for its reactivity with respect to the NS1 protein of hexameric, dimeric or monomeric form, said forms being non-degraded, said monoclonal antibodies then being purified, and modified for the manufacture of a medicament capable of inducing passive immunization.

 Advantageously, the modifications of the antibodies are in particular the selection of Fab fragments or the humanization of the antibodies.

 The present invention also relates to the use of the NS 1 protein of dimeric or monomeric hexameric form, said forms being non-degraded, for selecting in vitro antibodies specific for said NS1 protein.

 In an advantageous embodiment of said use, the antibodies are polyclonal antibodies.

 In another advantageous embodiment of said use, the antibodies are monoclonal antibodies.

 In another advantageous embodiment of said use, the protein is the NS 1 protein of the dengue virus, in particular of serotype 1.

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 The anti-NS1 monoclonal antibodies are advantageously obtained by fusion of spleen cells of mice immunized with the NS1 protein of hexameric form with appropriate myeloma cells.

 Other characteristics and advantages of the invention appear in the following description and examples illustrated by the figures in which: - Figure 1 represents the purified hexameric NS 1 protein extracellular obtained after exclusion chromatography (a) after chromatography exclusion, the protein is concentrated to 0.5 mg / ml by ultrafiltration and treated with dimethylsuberimidate (DMS) at 0.0.5, 5 and 50 mM. The products obtained are placed in a non-reducing Laemmli buffer, separated on a 4 to 20% gradient acrylamide gel and labeled with Coomassie blue. A sample treated with 50 mM DMS is heated for 3 min at 95 ° C. before electrophoresis to dissociate the non-covalent oligomers. (b) The NS1purified protein is treated overnight at 37 ° C. with 0.5% or 1% of n-octylglucoside (nOG) and optionally treated with 25 mM DMS for 1 hour. The proteins are separated without hot denaturation on a 4 to 20% gradient acrylamide gel and detected by immunoblotting with an anti-NS 1 monoclonal antibody from the literature or as defined above.

 FIG. 2 represents the sequence of the NS 1 protein of the dengue virus of serotype 1 obtained by the clone 4C of Example 2 below, as well as the corresponding coding sequence.

 FIG. 3 illustrates the results obtained by assaying the circulating NS1 protein by the method of detection by ELISA-capture in patients previously infected with a dengue virus whose sera were taken during the acute and convalescent phases, as well as comparison with the results obtained by the techniques of the prior art IHA (inhibition of hemagglutination of serotypes 1, 2,3 or 4 of the dengue virus) and MAC ELISA (immunoglobulin M Antibody Capture Enzyme-Linked ImmunoSorbent Assay); Dl corresponds to serotype 1 of dengue; D2 corresponds to serotype 2 of dengue; D3 corresponds to serotype 3 of dengue and D4 corresponds to serotype 4 of dengue; ID = identity of the patient; 1 corresponds to the first sample in the acute phase of the disease 2 corresponds to the

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 second convalescent phase sample (taken 2 to 4 weeks after the first); in the ELISA-capture test, the values are expressed in optical density obtained for the same serum diluted 10.30 or 90 times.

 - Figure 4 illustrates the detection of the NS1 protein by the ELISA-capture test on sera from patients infected with the dengue virus serotype 1 from Guyana. The figures indicated represent the number of patients divided by category (positivity or negativity in ELISA-capture and positivity or negativity in IgM).

EXAMPLE 1 Purification of the NSI Protein of the Dengue Virus, Serotype 1
1. Materials and methods
The protein is produced on Vero cells infected with the dengue virus, serotype 1, strain FGA / 89 (P. Després et al., Viral, (1993), 196,209-219), under the conditions suitable for the method described by AKI Falconar et al., (J. Viral.

Meth., (1990), 30,323-332).

 The culture medium is harvested 5 days after infection and centrifuged at 1500 g to remove the cell debris. The centrifugation supernatant is brought to 20 mM Tris-HCl, 1 mM sodium azide, concentrated 6 times by cold ultrafiltration and the viral particles are eliminated by a precipitation of 2 h at 4 ° C., at a concentration of 7.5%. in polyethylene glycol (PEG), followed by centrifugation for 30 min. at 10,000 g. The clarified supernatant, containing 7.5% PEG is treated with 0.05% Tween 20 and 1 mg / ml of aprotinin, then passed over an immunoaffinity column on which is fixed an anti-NS 1 monoclonal antibody. The protein is eluted according to the diethylamine technique, as described in Falconar et al., (cited above), concentrated by ultrafiltration and the elution solution exchanged with a 10 mM Tris-HCl pH 7.5 buffer containing 1 mM of azide sodium.

2. Results
The results are illustrated in Figure 1.

 The figure shows that the NS1 protein is indeed in hexameric form. The proportion of hexameric form increases with an increasing concentration of DMS (Figure la).

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 The NS1 extracellular hexameric protein can be transformed into dimeric subunits in the presence of the nonionic detergent, n-octylglucoside (nOG) (Figure 1b). After a night at 37 ° C. in the absence or presence of n-octylglucoside (nOG) and treatment with 25 mM DMS, the presence of bands corresponding to the dimer, the tetramer and the hexamer and in the presence of nOG, partial or complete dissociation of the hexamer as a function of the nOG concentration (FIG. 1b).

EXAMPLE 2 Protein NS1 of the Dengue Virus Serotype 1 by Vero Cells
1. Material and method
The plasmid pCIneo-NSl.FGA deposited with the National Collection of Cultures and Microorganisms (CNCM) of the Pasteur Institute under the n 1-2220, dated June 7, 1999) containing the gene of the protein NS1 including the coding gene for its signal peptide, preceded by a translation initiation codon and followed by a translation termination codon, is introduced into the competent bacterium Escherichia coli (Epicurian SURE from Stratagene). This plasmid is amplified in bacterial culture and purified according to the conventional technique of preparation of plasmid DNA. The purified DNA is used for the sequencing of different clones (the sequence of clone 4C is illustrated in FIG. 2) and to transfect Vero cells using either an adequate mixture with cationic liposomes, such as DOTAP (Boehringer-Mannheim), or with a non-liposomal agent, such as FuGENE (Boehringer-Mannheim). FuGENE and DNA are preincubated in serum-free medium for 15 min, then the mixture is brought into contact with a cell mat of Vero cells for 24 h. The cells are then rinsed using PBS (phosphate saline buffer), fixed 20 min at room temperature with a PBS solution containing 3% of paraformaldehyde, permeabilized 5 min with PBS containing 0.5% Triton X-100. The presence of NSI antigen is then revealed using specific antibodies, recognized by a conjugated antibody labeled with fluorescein.

 2. Results

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It is thus possible to demonstrate by a high fluorescent signal, specific for the viral antigen NS1, in approximately 20% of the transfected cells.

 The expression of NS1 is thus demonstrated and stable lines can be established in the presence of neomycin, a marker for selection of the transfected cells.

 FIG. 2 illustrates the sequence of the NS 1 protein of the dengue virus of serotype 1 thus obtained as well as the corresponding coding sequence.

Example 3: Implementation of the ELISA-capture technique according to the invention in the context of an infection with the dengue virus of serotype 1 and comparison with the methods of the prior art - 1. Principle of the ELISA-capture technique
The NS1 viral antigen is captured by polyclonal antibodies from monospecific mice previously purified by immunocapture on the purified hexameric NS protein of dengue, serotype 1.

 The presence of NS 1 is revealed by rabbit antibodies immunized with the purified hexameric NS11 protein, themselves recognized by antibodies conjugated to horseradish peroxidase.

2. Materials and methods - Purification of polyclonal antibodies of mice directed against the protein NS 1 a - Fixation of the protein NSI on membrane
The purified NS1 protein is fixed by adsorption on an amphoteric nylon membrane (Nytran, Schleicher & Schuell). The surface of the membrane is then saturated with bovine albumin present at a concentration of 3% in a saline solution buffered by phosphate (PBS: phosphate buffer saline; 10 mM phosphate; pH7.2; 150 mM NaCl). After 2 rinses in PBS, the membrane is treated with PBS containing 0.25% glutaraldehyde for 15 min. at room temperature. After 3 rinses in PBS, the membrane is neutralized with a buffer of 100 mM glycine, 3% bovine albumin, rinsed 2 times in PBS then stored at 4 C in PBS with 1 mM sodium azide.

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 b - Purification of monospecific polyclonal antibodies from mice, (capture antibody) [alpha]) production of polyclonal ascites: the brains of Swiss mice infected with the dengue virus and dying are ground in 9 ml of PBS buffer. The product is centrifuged for 10 min at 10,000 g at 4 C.

 The viral suspension is injected into Swiss mice, according to the following schedule: - OJ: 0.5 ml of antigen subcutaneously in the thigh, - D3: 0.4 ml of antigen and 0.1 ml of complete Freund's adjuvant intraperitoneally, - D25: 0.5 ml of antigen intraperitoneally, - D26: 0.5 ml of mouse ascites TG180, and - D28: 0.5 ml of antigen intraperitoneally .

The ascites are harvested on D42.

After collecting the ascites, the coagulum is left to form for 1 hour at room temperature and then centrifugation is carried out for at least 30 minutes at 1500 g. The supernatant is left overnight at 4 C. The pH of the supernatant is adjusted to 4.8 with 2 M acetic acid and then the supernatant is centrifuged again under the same conditions. The pH of the supernatant is then brought to 7.0 - 7.2 by addition of a 2N sodium hydroxide solution. The supernatant can be stored at - 20 C.

P) Ryn.flciiP.n # d.?. S ...? FitiÇ.9.rPs..de [..mouse; specifics of serum 1 dengue yijrus:
The membrane is incubated for one hour at room temperature in a mixture of polyclonal ascites directed against the 4 serotypes of the dengue virus prepared as described above.

After 3 rinses of the membrane in PBS, the antibodies fixed on the NS protein are eluted with a solution of diethylamine pH 11.4 (Dubelco medium modified by Iscove (Gibco) containing 100 mM diethylamine). The antibodies are concentrated by ultrafiltration and replaced in a PBS buffer containing 1 mM sodium azide.

 - Preparation of polyclonal rabbit antibodies directed against the NS 1 protein (revealing antibody):

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The rabbits were immunized by 3 or 4 successive injections of 30 g of hexameric NS1 protein purified according to the method of Example 1, performed on D0, D7, D21 and optionally on D49 and followed by a white bleeding at D83. The serum is depleted of non-specific anti-mouse antibodies by incubation with Sepharose beads carrying a monoclonal antibody described in the literature or prepared as described above.

- ELISA-capture process a - Standard curve
For each ELISA-capture plate intended for testing human sera, a standard range is produced from a solution of NS1 protein purified according to the method described in point 1, the initial concentration of which is 0.5 μg / ml and diluted from 3 to 3. b - Detection of the circulating NS1 protein during the acute phase:
The purified mouse polyclonal antibodies obtained according to the method described above (capture antibody) are fixed on a plate, diluted in a PBS solution and incubated overnight at 4 C. After 3 rinses of 5 minutes with a solution of PBS / Tween 0.05%, the plate is saturated with a mixture of PBS, Tween 0.05% and 3% milk, for 30 minutes at room temperature. After 3 rinses with a 0.05% PBS / Tween solution, several dilutions of the test sera are deposited and left to react for one hour, still at room temperature. The 1/10, 1/30 and 1/90 dilutions are made in a 0.05% PBS / Tween solution. After 3 rinses, the second antibody specific for NS1 is added (revealing antibody obtained in point 3 above), after having diluted it in a mixture of 0.05% PBS / Tween and 3% milk, and allowed to incubate for 45 minutes at 37 C. After 3 rinses, the anti-IgG antibody directed against the second antibody and labeled with peroxidase, said antibody being prepared under conventional conditions known to those skilled in the art, is added and the incubation carried out for 45 minutes at 37 C. After 3 rinses, it is revealed for 10 minutes with a TMB solution (3.3 ′, 5,5 ′-tetramethylbenzidine, Kierkegaard & Perry Lab).

The colorimetric reaction is stopped with sulfuric acid. c - Determination of the detection window

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The samples are taken from patients infected with the dengue virus serotype 1, between OJ, marking the appearance of clinical signs (initially an undifferentiated fever) and D66 corresponding to the end of the convalescent phase.

 In the sera of these patients, the presence of circulating NS1 is sought according to the ELISA-capture method described in point 4 and the result obtained is compared with the specific IgM positivity measured in MAC-ELISA, when the data are available.

3. Results
They are illustrated in FIGS. 3 and 4. a -Detection of the circulating NSI protein during the acute phase:
The ELISA-capture technique according to the invention makes it possible to detect the presence of the NS1 protein in the acute phase of the disease, regardless of whether the patients have a primary or secondary infection.

 The results confirm that the presence of the NS1 protein is transient, since in the samples taken during the convalescence phase, this protein is not detected (FIG. 3).

 93% of samples in the acute phase of the disease were found to be negative by the MAC ELISA test, while 100% of samples taken in the convalescent phase were found to be positive in this same test (Figure 3).

Similarly, the haemagglutination inhibition test (IHA) does not detect infection with dengue virus serotype 1 in 80% of cases in the acute phase of the disease, but this test is positive. in 100% of samples taken during the convalescent phase (Figure 3). b - Determination of the detection window
The results are collated in FIG. 4.

 Between D1 and D6, the possibility of detecting the circulating NS1 protein ranges from 83% (on D2) to 100% (on D5) of infected patients. Beyond D10, the circulating NS1 protein is no longer detected, while the antibody response becomes predominant.

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 The detection of the circulating NS1 protein does not seem to be dependent on the presence of specific IgMs which appear in certain cases on D3 and culminate from D5.

 Thus, the window for detection of the NS1 serum antigen by the ELISA-capture technique according to the present invention is preferably situated between D1 and D6 after the appearance of the clinical signs.

Claims (16)

1) Method for the early detection of a flaviviral infection, characterized in that it comprises the detection of the non-structural glycoprotein NS1 of a flavivirus in a biological sample, during the clinical phase of the infection, by an immunological method using at least two identical or different antibodies, the first antibody or antibody for capturing the NS1 glycoprotein being constituted by antibodies chosen from the group consisting of: polyclonal antibodies previously selected by immunocapture on the NS 1 protein of said flavivirus, of hexameric, dimeric or monomeric form, said forms being non-degraded and - mixtures of anti-NSI monoclonal antibodies previously selected for their affinity for the protein NS1dudit flavivirus, of hexameric, dimeric or monomeric form, said forms being non-degraded, said antibodies monoclonals then being purified, - the second the antibody or revealing antibody being chosen from the group consisting of: - polyclonal antibodies directed against the NS 1 protein of the same type as that to be detected and - a mixture of monoclonal antibodies directed against an NS 1 protein of the same type as that to detect.  2) Detection method according to claim 1, characterized in that the flaviviral infection is an infection with the dengue virus.  3) Detection method according to any one of claims 1 or 2, characterized in that the first antibody is fixed on a suitable solid support and the second antibody is optionally conjugated to an appropriate marker.  4) Detection method according to claim 3, characterized in that when the second antibody is not conjugated to a marker, its binding to the NS 1 protein fixed on the solid support, is then detected by a third antibody conjugated to a suitable marker. <Desc / Clms Page number 21>  5) Method of detection according to claim 4, characterized in that the third antibody is conjugated to an enzyme.  6) Detection method according to claim 5, characterized in that: - the first antibody or capture antibody consists of polyclonal antibodies of mice, selected by immunocapture on the NS1 protein of the dengue virus, said protein being in the form hexameric, and - the second antibody or antibody for detecting the presence of NS1 in the biological sample to be analyzed, consists of rabbit polyclonal antibodies immunized with protein NS1 of the dengue virus serotype 1, said protein being under hexameric form, the binding of said second antibody being revealed by a third antibody consisting of antibodies, conjugated to peroxidase and directed against the second antibody.  7) Box for early diagnosis of a flaviviral infection, characterized in that it comprises: - at least one capture antibody and at least one revealing antibody as defined in any one of claims 1 to 6, - at at least one positive control consisting of the NS1 protein of a flavivirus and / or of different serotypes according to the flavivirus, said protein being in hexameric, dimeric or monomeric form, said forms being non-degraded, and - at least one negative control consisting of a normal human serum.  8) A diagnostic kit according to claim 7, characterized in that said NS1 protein, in its various forms is obtained from a culture supernatant, either from infected mammalian cells, or from mammalian cells transfected with a recombinant plasmid , comprising the gene for the NS1 protein of a flavivirus or a fragment of said gene or a fragment of the flaviviral genome, said fragments being capable of expressing the protein NS 1. <Desc / Clms Page number 22>  9) Kit for early diagnosis of a flaviviral infection according to any one of claims 7 or 8, characterized in that the protein NS1 is that of the dengue virus.  10) Box for early diagnosis of a flaviviral infection according to any one of claims 8 or 9, characterized in that said plasmid has been deposited with the National Collection of Cultures and Microorganisms held by the Institut Pasteur under the number 1-2220, dated June 7, 1999.  11) Method for purifying the NS1 protein of a flavivirus, in hexameric form, from a culture supernatant, either from infected mammalian cells or from mammalian cells transfected with a recombinant plasmid, comprising the gene for NS1 protein or a fragment of said gene or a fragment of the flaviviral genome, said fragments being capable of expressing the NS1 protein, characterized in that the soluble form of the NS1 protein is separated from the microparticulate form of said protein, - either by treatment with a precipitating agent and then centrifugation at a speed greater than or equal to 10,000 g, - either by centrifugation at a speed greater than or equal to 50,000 g, before the purification step.  12) Immunogenic composition, characterized in that it comprises an active principle selected from the group consisting of: - the NS1 protein of a flavivirus, of hexameric form, optionally associated with other proteins, - a DNA sequence coding for said NS1 protein - an expression system comprising at least one promoter capable of expressing, in the host where it is injected, the DNA coding for the NS 1 protein of the flavivirus, said gene expressing said protein, in association with at at least one pharmaceutically acceptable vehicle.  13) Immunogenic composition according to claim 12, characterized in that it comprises at least a mixture of proteins NS1 of hexameric form corresponding to the different serotypes of the dengue virus. <Desc / Clms Page number 23>  14) Use of the NS1 protein of hexameric form or one of its expression systems, for the preparation of an immunogenic composition, capable of inducing the production of antibodies in vivo.  15) Use of at least one anti-NSI monoclonal antibody previously selected for its reactivity with respect to the NS1 protein of hexameric, dimeric or monomeric form, said forms being non-degraded, said monoclonal antibodies then being purified, and modified for the manufacture of a medicament capable of inducing passive immunization.  16) Use of the NS1 protein of dimeric or monomeric hexameric form, said forms being non-degraded, to select specific anti-NS 1 antibodies in vitro.