AU611402B2 - Viral agent for nanb hepatitis, antigen and immunologic reagent - Google Patents
Viral agent for nanb hepatitis, antigen and immunologic reagent Download PDFInfo
- Publication number
- AU611402B2 AU611402B2 AU82734/87A AU8273487A AU611402B2 AU 611402 B2 AU611402 B2 AU 611402B2 AU 82734/87 A AU82734/87 A AU 82734/87A AU 8273487 A AU8273487 A AU 8273487A AU 611402 B2 AU611402 B2 AU 611402B2
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- AU
- Australia
- Prior art keywords
- nanb
- hepatitis
- viral
- document
- epidemic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/29—Hepatitis virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Communicable Diseases (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
I'PATENT AU-Al-82734/87 CT ORGANISAT M BurDE ZTE LECTUELLE P C T Burj u ini 2r i DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT) (51) Classification internationale des brevets 4 (11) Num6ro de publication internationale: WO 88/ 03410 A61K 39/29, G01N 33/576 Al (43) Date de publication internationale: 19 mai 1988 (19.05.88) (21) Numro de la demande internationale: PCT/FR87/00441 (74) Mandataires: ORES, Bernard etc.; Cabinet Ores, 6, avenue de Messine, F-75008 Paris (FR).
(22) Date de d6p6t international: 6 novembre 1987 (06.11.87) (81) Etats d6sign6s: AU, DK, JP.
(31) Numbros des demandes prioritaires: 86/15625 87/01738 Publike (32) Dates de priorit6: 7 novembre 1986 (07.1 1.86) Avec rapport de recherche internationale.
11 f6vrier 1987 (11.02.87) Avant 'expiration du dilai privu pour la modification des revendications, sera republide si de relies modifica- (33) Pays de priorit6: FR tions sont reques.
(71)(72) D6posant et inventeur: INSTITUT PASTEUR [FR/ FR]; 28, rue du Docteur-Roux, F-75724 Paris C6dex (FR).7 L 9 'A.Q.LP. -7 JUL3988 i (72) Inventeur: PILLOT, Jacques 14, chemin de Moulon, F-91190 Gif-sur-Yvette (FR).
AUSTRAUAN
1 JUN 1988 PATENT
OFFICE
(54) Title: VIRAL AGENT FOR NANB HEPATITIS, ANTIGEN AND IMMUNOLOGIC REAGENT (54) Titre: AGENT VIRAL DE L'HEPATITE NANB, ANTIGENE ET REACTIF IMMUNOLOGIQUE (57) Abstract New purified viral agent involved in viral non-A non-B, epidemic, sporadic or post-transfusional hepatitis, defined by the distribution profile of its antigenic activity, comprising at least one band at a density of 1.28-1.32 g/cm 3 and/or at least one band with a density of 1.17-1.19 g/cm 3 and the antigen associated with said viral agent. Method for isolating said viral agent and the antigen which is associated therewith. Utilization of said viral agent as diagnostic ieagent. Method for the detection of the non-A non-B antigen present in the faeces of patients suffering from non-A non-B viral hepatitis. Immunologic reagent for the detection of NANB viral, epidemic, sporadic or post-transfusional hepatitis and method for isolating anti-NANB antibodies capable of being used as immunologic reagents. Applications of said anti-NANB antibodies to the diagnosis of viral, epidemic, sporadic or post-transfusional NANB hepatitis and as therapeutic agents.
(57) Abrkg6 Nouvel agent viral purifi impliqu6 dans l'h6patite virale non-A non-B, 6pid6niique, sporadique ou post-transfusionnelle, defini par le profil de r6partition de son activit6 antig6nique, qui comporte au moins une bande A une densit6 de 1,28-1,32 g/cm 3 et/ou au moins une bande d une densit6 de 1,17-1,19 g/cm 3 et l'antig~ne associ6 i cet agent viral. Proc6d6 d'isolement dudit agent viral et de l'antig6ne qui lui est associ Utilisation dudit agent viral en tant que r6actif de diagnostic. Proc6d6 de d6tection d'antigene non-A non-B present dans les feces de patients atteints d'h6patite virale non-A non-B. R6actif immunologique pour la d6tection de l'h6patite virale NANB, pid6mique, sporadique on posttransfusionnelle et proc6d6 d'isolement d'anticorps anti-NANB aptes i tre utilis6s en tant que r6actifs immunologiques.
Applications desdits anticorps anti-NANB au diagnostic de l'h~patite virale NANB 6pid6mique, sporadique ou post-transfusionnelle et en tant qu'agents thgrapeutiques.
4 S WO 88/03410 PCT/FR87/00441 VIRAL AGENT OF NANB HEPATITIS, ANTIGEN AND IMMUNOLOGICAL REAGENT The present invention relates to the isolation and characterization of a viral agent involved in non-A non-B hepatitis both epidemic and sporadic or post-transfusional.
Viral hepatitis may be caused by the virus of hepatitis A (HAV), by the hepatitis B virus (HBV) and by an unknown number of non-A non-B (NANB) viruses as yet unidentified.
At the present time, in developed countries in which HBV prophylaxis is effective, the NANB are the main source of infectious hepatitis leading to hospitalization. This illness is considered as involving at least three viruses r. ;r -2two are transmitted by contaminated blood and contaminated blood products the third which is the origin of epidemic NANB hepatitis has been establishable by the inventor as being also responsible for 50 to 60% of hepatitis cases requiring hospitalization, reported in Western countries and its mode of transmission is unknown. In underdeveloped countries NANB seems also associated with large-scale epidemics linked to water-borne fecal-oral transmission. To the present time, five epidemics of this type have been reported since 1980 in India, in Siberia, in Nepal, in Algeria and recently in the Ivory Coast. In all these cases, a high mortality level was observed among pregnant women.
Our present knowledge relating to the viruses which are at the origin of NANB hepatitis are of discouraging imprecision and are limited to clinical and epidemiological data. Filtrable agents have clearly been isolated by inoculation with blood or blood products coming from subjects suffering from post-transfusional NANB hepatitis and infectious blood products are available. As regards epidemic NANB hepatitis, very few experimental infections have been realised the infection is established to be transmissible to marmosets (Saguinus mystax), to macaque monkeys (Macacus cynomolgus) and to a human volunteer and recently to African green monkeys (Cercopithecus aethops and Erythrocebus patas), but no definitive infectious preparation has been suggested.
As regards post-transfusional hepatitis, despite the plethora of reports on NANB antigen-antibody systems, ultrastructural modifications and particles similar to viruses, all potentially promising, no causal agent or any system or virus which fulfills the serological criteria for a specific causal association has been identified prior to 4 I -3the work which has resulted in the present invention. As regards epidemic hepatitis, particles similar to viruses of spherical shape and 27 nm in diameter have been shown in very rare faeces and the immunological characterization has been carried out in mediocre fashion by agglutination with the electron microscope. Viral replication has never been obtained in cell cultures for whatever NANB virus this may be French patent application no. 86 05347, in the name of the Institut Pasteur, and which mentions Monsieur Pillot as inventor, is directed to a reagent suitable for enabling the detection of epidemic NANB viral hepatitis and a diagnostic process employing this reagent the reagent according to this patent application is constitued by anti- NANB IgMs isolated from serums of monkeys infected artificially by faecal extracts from patients known to suffer from epidemic NANB hepatitis, which anti-NANB IgMs are advantageously fixed to solid supports, such as, particularly, PVC plates.
The continuation of his research from the invention according to the aforesaid patent application has enabled the inventor to achieve the following objectives 1) He has infected monkeys of which he has used the antibodies as well as Acs of human convalescents to develop an ELISA test for the specific detection of antigens (Ag) associated with the virus and the detection of anti-viral Acs 2) The inventor has succeeeded in replicating the virus in a continuous line of hepatocyte cells with a good production of antigenic material 3) He has arrived at making visible viral particles in purified preparations of samples of faeces and in infected hepatocyte lines I 7 7 a_ f -4- 4) He has been able to establish the implication of the same causal agent in NANB viral hepatitis both epidemic and sporadic.
By considering the role of the epidemic NANB agent in sporadic hepatitis the inventor has been in a position to verify that said agent can be not only carried by water, but also be transmitted by other routes than water and particularly the blood transfusion route.
The present invention has taken as an objective the identification and characterization of a novel viral agent, as well as an NANB antigen (Ag) associated with this novel viral agent and a method of diagnosis of NANB hepatitis both epidemic and sporadic and post-transfusional, by detection of the Ag associated with the virus of NANB, by using antibodies (Ac) which are the subject of the aforesaid Institut Pasteur patent application or through research of Acs of the patient, by using the virus or its fractions adsorbed on a suitable support.
It is also an object of the present invention to provide immunological diagnostic reagents useful in the diagnostic method of NANB hepatitis according to the present invention, as well as a kit ready for use for the performance of said diagnostic method.
An object of the present invention is a novel purified viral agent involved in epidemic, sporadic or posttransfusional viral NANB hepatitis, characterized in that it has a distribution profile of its antigen activity which comprises at least one band at a density of 1.28-1.32 g/cm3 and/or at least one band at a density of 1.12-1.19 g/cm3.
According to an embodiment of the purified viral agent involved in the epidemic, sporadic or post-transfusional NANB hepatitis, the latter is isolated from faeces of patients afflicted with NANB hepatitis by centrifugation SL j i and filtration through a filter whose porosity is of the order of 0.22 pm.
According another embodiment of the viral agent involved in epidemic, sporadic or post-transfusional NANB hepatitis, the latter is obtained from hepatocytes or continuous cell lines of hepatocytes inoculated with the faecal extract of a patient afflicted with NANB hepatitis or with a liver extract from an infected monkey.
According to yet another embodiment of the vital agent involved in epidemic, sporadic or post-transfusional NANB hepatitis, said viral agent with which is associated the specific antigen, is in the form of spherical viral particles of two principal types, namely particles of about nm diameter and particles of the order of 25 to 30 nm diameter, the first being to a major extent present in fractions of a CsC1 gradient of density varying between 1.1 and 1.6 g/ml, whose density is comprised between 1.28 and 1.32g/cm 3 and 40-41.5% fractions of a sucrose gradient of concentration varying from 10 to 60% weight/volume and the second being to a major extent present in the fractions of the CsCI gradient of density comprised between 1.12 and 1.19 g/cm 3 and the 26-28% fractions of said sucrose gradient.
It is also an object of the present invention to provide an antigen associated with the above-defined NANB virus, characterized in that it is obtained by treatment of a viral agent isolated from faeces of patients, cultivated on hepatocytes infected by the virus of NANB hepatitis, with physical or chemical dissociation agents.
According to an advantageous embodiment of the antigen associated with the above- mentioned NANB virus, said antigen is obtained by treatment with ultrasound of a viral agent isolated from continuous cell lines of hepatocytes AINT 0 4' I '^nj^ -6infected with the virus of NANB hepatitis.
According to another embodiment of the antigen associated with the NANB virus, its distribution profile in a CsCl gradient is in the form of a band with a density of 1.12-1.19 gcm3 and/or a band of density 1.28-1.32 and the antigen is present after ultra-centrifugation of the CsCl fractions in a sucrose gradient, in the 26-28% fraction of the sucrose gradient for the first band and in the 40-41.5% fraction of the sucrose gradient for the second band.
It is also an object of the present invention to provide a process for the isolation of a viral agent involved in epidemic, sporadic or post-transfusional viral hepatitis, which is characterized in that faeces of patients afflicted with NANB hepatitis are subjected to a centrifugation treatment, followed by a filtration treatment through a filtering element whose porosity is of the order of 0.22 pm, to extract said viral agent from the faeces.
According to one embodiment of the process of isolation of the viral agent above-mentioned, the centrifugation is advantageously constituted by a centrifugation operation at 5000 to 8000 g approximately for 10 minutes approximately.
According to another embodiment of the process of isolation of the above-said viral agent, prior to the above-mentioned centrifugation operations, the faecal extract is subjected to treatment with ultrasound or to a chemical dissociation treatment.
It is also an object of the present invention to provide another process for obtaining the above said viral agent, which is characterized in that hepatocytes or continuous hepatocyte cell lines are inoculated with a fecal extract from a patient afflicted with NANB hepatitis or with a liver extract from a monkey (preferably Saimiri) inoculated with a faecal extract of a patient suffering from NANB zI '1i -7hepatitis, and cultivated until confluence of the culture, from which a sub-culture is performed which is centrifuged after several passages to collect the desired viral agent.
According to an advantageous embodiment of this process, the centrifugation pellet is taken up again with a suitable buffer to be subjected to ultrasound treatment which releases into the supernatent, the NANB Ag.
It is in addition an object of the present invention to provide a process for the detection of NANB antigen present in the faeces of patients afflicted with epidemic, sporadic or post-transfusion viral NANB hepatitis, characterized in that it employs immunoglobulins isolated from serum of convalescent patients, or from monkeys previously infected with the virus of NANB hepatitis, which are incubated with faecal extracts of mammals presumed to be afflicted with viral NANB hepatitis to form antigen-antibody immunocomplex which is revealed by means of a suitable detection tests of the presence of the NANB antigen in said faeces, such as an ELISA test or a radioimnunological test, particularly.
According to an advantageous embodiment of the process of detection of NANB antigen in the faeces of patients afflicted with NANB hepatitis, the latter is characterized in that extracts of faeces of mammals presumed to be .nfected with NANB virus, obtained by centrifugation and filtration of the faeces of these mammals, are incubated with an anti-NANB IgM fraction isolated from serums of patients, or from monkeys infected artificially with faecal extracts of human patients known to be afflicted with NANB hepatitis which fraction is fixed to a solid support then with a purified NANB IgG immune anti-virus fraction purified according to the invention, from a mammal of the ~i-O Iw -8same species or of a neighbouring species, labelled with an enzymatic label such as B-galactosidase or peroxidase, the reaction being developed by means of a suitable developer substrate such as orthonitrophenyl-B-D-galactopyranoside or orthophenyl-diamine, in acid buffer. The labelling may also be done with a fluorescent or radioactive element by methods known to the technician skilled in the art.
According to the invention, strains of the viral agent according to the invention, isolated as described in the o1 foregoing and the following, and characterized as indicated below have been deposited at the Collection Nationale de Culture de Microorganismes maintained by L'Institut Pasteur, dated 30 October 1986, namely a strain of viral agent isolated from faeces from a patient afflicted with sporadic NANB hepatitis, called "Clamart strain", which was assigned deposit no: 1-617, a strain of a viral agent isolated from faeces of a patient suffering from epidemic NANB hepatitis called "Ivory Coast" strain, which was assigned deposit no: 1-616.
According to an embodiment of the process of detection of antibodies present in the blood of the patient or of the convalescent or in products used in blood transfusion or in fractions derived from blood, which have been in contact with the causal viral agent of non-A non-B hepatitis, as defined according to the present invention, including strains 1-617 and 1-616 and those having an immunological reaction crossed with the two reference strains, the serum of the patient or convalescent patient is placed in contact with the purified viral preparation (or with a fraction of the latter) as collected under the conditions mentioned above, after centrifugation and isolation of the band corresponding to a density of 1.30.
Said viral preparation is fixed, for example, to the NT 0< 1 rQ I
A
uu~usurmuur~"~~-: -9walls of a microplate according to known techniques.
The complex Ag/Ac is developed by a human antiimmunoglobulin preparation labelled with a suitable label, for example, an enzyme.
A similar system may be used to characterize an antibody activity in the igms of a patient for very early serological diagnosis of the disease, by using human anti- IgMs, or better by trapping the IgMs from a serum by letting them react with the virus or its fractions and by characterizing th Ag fixed to the IgM of the patient.
It is an object also of the present invention to provide immunological reagents for the detection of epidemic, sporadic or post-transfusional NANB hepatitis, which are characterized in that they are constituted by immunoglobulins isolated from the serum of human convalescent persons or from serum of monkeys previously infected with the virus of NANB hepatitis, which reagents are adapted to form an antigen-antibody immunicomplex when they are incubated with extracts of faeces containing NANB hepatitis antigens.
According to an advantageous embodiment of the immunological reagents according to the present invention, the latter are adapted to form an immunocomplex by incubation with extracts of faeces of individuals contaminated with NANB virus according to the invention and purified by centrifugation and filtration.
It is also an object of the present invention to provide an immunological reagent for the detection of epidemic, sporadic or post-transfusional viral NANB hepatitis, characterized in that it is constituted by NANB antihepatitis antibody prepared from serums of animals artificially infected by injection of extracts of faeces of patients afflicted with viral NANB hepatitis, or serums of i 1i I ii animals immunized by the NANB virus or its purified fractions.
According to an advantageous embodiment of this immunological reagent, the NANB anti-hepatitis antibody (IgM or IgG) is fixed to a solid support.
According to an advantageous modality of this embodiment, the solid support coated with NANB anti-hepatitis antibody is in addition coated with a protein such as BSA in particular, preferably in buffered solution.
The immunological reagent according to the present invention is employed in the process of detection of NANB antigen according to the invention by incubating it with extracts of faeces or blood products or serum of patients then with purified antibodies from serums of convalescent patients of epidemic NANB hepatitis or serums of animals artificially infected, immunized against this virus, said antibodies being labelled by a suitable enzyme such as pgalactosidase, for example. The reaction is developed by means of a suitable developer or substrate such as orthonitrophenyl-p-galactopyranoside.
The in vitro diagnostic method of the presence o.f the virus of NANB hepatitis according to the present invention consists therefore of placing in contact a serum or another biological medium of a patient of whom it is desired to establish the diagnosis, with at least one of 'he proteins or glycoproteins of the NANB virus or with a viral lysate or with a viral extract then to detect the immunological reaction by the ELISA method or by another immunoenzymatic method or a method by immunofluorescence, which can measure directly or indirectly the immunofluorescence or the immunoenzymatic reaction.
This is why the present invention encompasses also labelled viral extracts whether labelled by an enzyme, by the CsCI gradient fractions of a density between 1.12 and 1.19 g/jm 3 and in the 26-28% fractions of said G e /2 -11- /2 fluorescence or labelled by a radioisotope.
The immunodetection methods comprise as is known the deposition of quantities of specific extract or of viral proteins according to the invention, in or on a support such as the cups of a titration microplate the introduction of increasing dilutions of the serums to be diagnosed onto said support, such as said cups incubation of the support such as the above- said microplate careful washing of the support such as the microplate above-mentioned by means of a suitable buffer introduction of specific labelled human antiimmunoglobulin antibodies into or onto the support such as the cups of the above-said microplate, the labelling being performed by an enzyme selected from among those capable of hydrolysing a substrate so that the absorption of radiation of the latter is modified at least in a specific band of wavelengths, and -the detection preferably comparatively, with respect to a control, of the amount of hydrolized substrate both by measurement of the dangerous biological product and by the actual presence of the disease.
It is also an object of the present invention to provide a kit or outfit ready for use for the performance of the diagnostic method (search for antibodies or NANB virus) S according to the invention, defined above, which kit is characterized in that it comprises an extract or a purified fraction of the viral agent according to the invention, which extract or fraction is labelled, for example, by a radioisotope, an enzyme or by immunofluorescence human anti-immunoglobulins or a protein A possibly fixed to a water-insoluble support such as agarose beads or 1, isolated from the feces of a patient afflicted with sporadic NANB viral hepatitis, called "Clamart Strain" deposited on 30 October 1986 under no. 1-617 at the -12latex, magnetic or not buffers and, if necessary, substrates to display the labelling.
The characterization of certain of the antigens which enter into the composition of the viral agent isolated according to the present invention has been carried out in a first phase on Clamart strain by proceeding as described below.
The Clamart strain was isolated from stools of a patient afflicted with sporadic NANB hepatitis.
The supernatent of said stools was centrifuged in a cesium chloride gradient of which the band containing the viral antigen purified as defined above was collected and its density determined as being 1.30.
The epitopes recognized in the above-defined diagnostic test are borne by these antigens which can in this respect be used in said test.
The ready-for-use reagent used for the detection of NANB hepatitis according to the present invention is prepared and used as described below Monkeys are artificially infected by ingestion of faeces of patients afflicted with NANB epidemic hepatitis.
The serums of monkeys used are those collected on the 27th day after infection, from monkeys artificially infected as indicated above. The serums are chromatographed on a dextran gel column in a Tris-NaCl 0.1 m buffer, pH 8 to isolate the desired antibodies which are anti-NANB IgMs.
The IgMs so isolated are fixed to solid supports preferably such as PVC plates, by placing the IgMs at concentrations of about 10 to 15 pg of proteins per ml of PBS, in contact with the solid support for 18 to 24 hours at +4C It is preferred for these PVC plates to be coated with NANB anti-hepatitis IgMs, to be then covered with a I I -13- PBS layer containing 1% of BSA (bovin serumalbumin) and 0.1% "Tween 20". The doubly coated PVC plates are then ready for use as a diagnostic reagent useful for the detection of the viral agent associated with NANB hepatitis.
The method of diagnosis according to the present invention commences preferably by incubation of said diagnostic plates with extracts of faeces to be diagnosed 0 for 1 hour at-37 C. The plates are then incubated with IgG fractions obtained by purification of serums of convalescent patients from epidemic NANB hepatitis by chromatography on a DEAE-Trisacryl column and labelled with an enzyme, preferably beta -galactosidase, hydrolysis of a developer substrate in the presence of which the reaction takes place and which is preferably orthonitrophenyl-beta D-galactopyranoside.
The method of detection according to the invention may also consist of seeking the presence of in NANB antibodies in serum or other biological medium of a patient or of a convalescent in such a case the serum is placed in contact with the viral preparation according to the invention- collected for example, after centrifugation and isolation of the band of corresponding density at 1.28-1.32 g/cm3 ,or with a fraction of this viral preparation this viral preparation, or one of its fractions, is placed in reaction with an IgM or IgG preparation which coats the walls of a microplate in a manner known to the technician skilled in the art The formation of an immunocomplex (virus or fraction of the latter anti-virus human antibodies) is then opposed to the development of the antigens that it contains bythe convalescent IgG labelled, for example, with an enzyme.
The search for antibodies by means of this method of -14detection may be carried out in the blood of patients or convalescents, in products used in blood transfusion and in fractions derived from persons who have been exposed to the causal agent of NANB hepatitis, among which the strains I- 617 and 1-616 and those showing an immunological reaction crossed with these two reference strains, particularly the strain isolated from patients afflicted with posttransfusional NANB hepatitis, whose immunological properties are identical with those of the strain 1-617, and which is identified as viral strain "H-SET" deposited February 1987 under no. 87.02.05.02 at the ECACC Collection (Great Britain).
Although the clinical and epidemiological data have been able to suggest that the epidemic form of NANB hepatitis shown by patients afflicted with epidemic NANB hepatitis is due to a virus different from that which is at the origin of post-transfusional NANB hepatitis transmitted by parenteral injection according to the present invention the inventor has been able to isolate from patients afflicted with post-transfusional NANB hepatitis a new strain of NANB virus of which the immunological properties are practically identical with those of the strains deposited 30 October, 1986 under nos. 1-616 and 1-617 at the CNCM of the Institut Pasteur. Very sensitive serological tests of the presence of HAV and HBV virus have permitted these two viruses to be excluded as etiological agents and to exclude, in the same way, other hepatotropic viruses such as cytomegalovirus Epstein-Barr virus and the virus of yellow fever.
It is also an object of the present invention to provide a novel purified viral agent involved in the sporadic, epidemic and post-transfusional forms of NANB hepatitis.
This virus is distinguished from HAV and HBV viruses as
I
I I f iI regards the antigen homology of its proteins as genetic material.
The compositions prepared according to the present invention (purified viral antigens, recombinant proteins obtained by expression of the NANB virus in procaryotic or eurcaryotic cells or synthetic peptides deduced from the sequence of the genome) may be used for the diagnosis of NANB hepatitis or for the vaccination proper to induce synthesis of a protective immune response to the host.
The present invention encompasses all the compositions of this type containing an antigen having equivalent immunological properties to those of the Clamart NANB virus (CNCM 1-617) or Ivory Coast virus (CNCM 1-616). In fact, two proteins or antigens are considered as equivalents within the scope of the present invention when they are capable of being recognised by the same antibodies. The products expressed by corresponding sequences of the genetic material coding corresponding genetic sequences are, consequently comprised within the scope of such equivalent antigens.
The present invention includes also serums which can be produced from animals by inoculation to the latter of NANB virus by means of the aforesaid compositions. In particular the present invention includes the polyclonal antibodies specifically directed against each of the antigens of the NANB virus It includes also monoclonal antibodies capable of being obtained by suitable methods and directed more specifically against the antigens of the NANB virus.
According to the present invention, the use of the virus or of its lysate or of a fraction of this lysate may be contemplated for the preparation of monoclonal antibodies and the viral lysate or fractions of the latter or purified proteins from this virus, of the lysate or its fractions _n a [1! -16may be used for the search for antibodies in the serum of patients by the employment of conventional tests of the, RIPA, ELISA or Western blot(immunoprint) type.
These polyclonal and monoclonal antibodies may be used in many applications among which may be mentioned neutralization of the corresponding antigen or of the total virus. It may be used to detect viral antigens in biological preparations or to purify corresponding antigens.
The present invention includes also any equivalent virus of NANB hepatitis having the same immunological characteri4tics. The studies carried out by the inventor have shown the immunological relationship between the antigenic materials isolated from extracts of faeces of patients afflicted respectively with epidemic, sporadic and post-transfusional NANB hepatitis.
Thus, the strain of Clamart type deposited under no.I- 617 at the CNCM and also deposited at the ECACC Collection undrer no.87.02.05.02) was isolated from stools of a patient who showed an acute NANB hepatitis duly characterized as transfusional NANB hepatitis. The stools contained the antigen according to the invention also characterized according to the invention, in epidemic hepatitis (Ivory Coast) and sporadic hepatitis (France).
An antigen associated with the virus of NANB hepatitis has been characterized over 15 days at the acute phase of the disease. Other similar cases were observed with characterization of the same antigen in the stools and isolation from a virus showing immunological characteristics identical with virusr 1-616 and 1-617.
Besides the foregoing features, the invention comprises also other features which will emerge from the description which follows.
'Kr p p -17- The invention will be better understood by means of the additional description which follows which refers to examples of the practice of the method according to the present invention.
It must be well understood however, that these examples of practice are given purely by way of illustration of the invention, of which they do not in any way constitute a limitation thereof.
In the course of the first step, two Cercopithecus aethops monkeys and an Erythrocebus patas monkey were inoculated orally with 2 ml of a mixture of a 10% aqueous extract of faeces of Ivory Coast patients presumed to be afflicted with epidemic NANB hepatitis, collected in the course of the first days following the appearance of symptons The mixture, having previously been centrifuged and filtered through Millipore filters of 0.22 um to remove bacteria and parasites. Specimens of serum and specimens of faeces of the monkeys were collected on the one hand before administration of the faecal extract and on the other hand the 27th day after inoculation. The hepatic functions of these monkeys were not studied these animals did not show obvious clinical manifestations. A Saimiri monkey was also inoculated under the same conditions with 0.5 ml of faecal extract coming from an Ivory Coast patient different from those used for the African green monkeys. A second Saimiri monkey which had been inoculated three months previously with a faecal preparation of hepatitis A virus and seroconverted by means of anti-HAV IgM antibodies on ,j the 29th day, was inoculated intraveneously with 0.5 ml of the same sample of filtered faeces. The two Saimiri monkeys were bled twice weekly to perform biological and serological examinations and their faeces were examined daily. The two Saimiri monkeys were dead at day 28 without I j.
-18having shown particular symptoms None of the monkeys showedconvincing biochemical proof ofa hepatic disfunction and histological examination carried out post mortem showed a congestion of the liver without lesion of the parenchyma.
The serums of these 5 monkeys were fractionated in Tris- NaC1 buffer 0.1M, pH 8, on "Sephacryl S 300" and the fractions containing IgM and IgG were collected. They were used as receptor antibodies in immunoenzymatic tests. The IgM was selected for its nigh detection capacity and to prevent falsely positive reactions due to a rheumatoid factor or similar to rheumatoid factor which was recognized as stimulating the NANB antigen.
In fact, it would also be possible to use IgG although its background noise is a little higher than that of IgM and its deficient specificity for certain preparations.
To reveal the fixation of the faecal antigen to the antibody on solid phase, IgGs coming from serums of convalescents were fractionated by chromatography on DEAEtrisacryl after precipitation with (NH 4 2
SO
4 The IgGs were conjugated with B-galactosidase or peroxidase.
Labelled IgGs were used for the detection of the antigens an IgG preparation coming from an Ivory Coast patient convalescent from epidemic NANB hepatitis an IgG preparation sampled from a cured Algerian patient in the course of the Medea epidemic in 1981-1982 finally the IgG of a Cercopithic monkey inoculated experimentally. The IgG preparations of the three origins gave equivalent results.
For each monkey, IgM of pre-inoculation was used in solid phase as control of IgM post-inoculation specificity. Nonimmune labelled human IgG was used as an Ag labelling control. The faeces of inoculated monkeys were examined to establish the presence of an NANB presumed Ag in the ELISA test. An antigen activity was detected in the faeces of two "w^4 L t
I
-19- Saimiri monkeys (cf Table The application of treatment by ultrasound enabled the amount of antigen detected to be increased and the sensitivity of the method to be increased. The Ag appeared from the 4th day after inoculation and persisted up to the 13th day.
Table 1 shows the NANB Ag detected in monkey faeces infected experimentally with extracts of faeces coming from patients afflicted with epidemic NANBH Detection was carried out as follows, by employing the ELISA technique NUNC immunoplates were coated with 100 pl of 50 pg/ml of IgM fraction, sampled 27 days after inoculation, in a physiological phosphate buffer solution (PBS) for 1 hour at 370 C and overnight at 4° C. After several washings, the plates were covered with 200 p 1
PBS
containing 1% of bovin serum-albumin and 0.1% of "Tween for 3 hours at 37 C. After washing, 100 pl of faecal extracts (1/10/ V/V) filtered on an element of 0.22 pm porosity were incubated on the coating IgMs, at 37 C for 1 hour, then overnight at 4 C. After several washings, 100 Pl of immune monkey IgG labelled with peroxidase at 5 mcg/ml in 1% of PSA 0.1% of Tween 20 in Tris sodium buffer pH 7.6 (TNB) containing 0.1% of NaN were used for the detection of the Ag. After 1 hour incubation at 37 C and several washings, the reaction was developed by means of 100 pl of orthophenylene-diamine in 0.005 M of citric buffer, pH 5.6 with 1 pl/ml H 2 0 2 The results obtained are expressed by the ratio between th sample studied and the mean of 5 negative samples The background noise of the negative samples was of the order of 0.04-0.06 in optical density (OD) at 492 nm. It is higher if the NaN 3 has been omitted but, in this case, smaller quantities of reagents must be placed in reaction. The specimens of which the ratio P/N was 2.1, were consideed positive. The faeces
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(1/10 weight/weight) which were examined were of two types: those which had undergone an ultrasound treatment and those which had not undergone such treatment the treatment with ultrasound applied was 3 mins at 4 C, with an output intensity of 4 with the BRENSON apparatus for volumes of 2 to 3 ml before centrifugation and filtration through an element of porosity of 0.22 pm. The positive results are underlined in Table 1. The faeces of a Saimiri monkey which had received the faecal extract of a patient afflicted with a non-infectious disease of the liver served as a control and constantly gave negative results.
i I S0< Ji( -21- Number of days following inoculation P/N no ultrasound P/N with ultrasound 1st monkey (IV inocul.) 2nd monkey (oral inocul.) 1st 2nd monkey monkey 1.15 2.47 6.48 3.64 1.12 1.30 1.51 1.53 1.14 1.60 3.28 2.24 1.40 1.46 1.68 1.51 1.18 5.10 8.78 4.72 1.95 2.40 0.93 1.20 1.07 0.79 5.52 5.82 5.20 0.80 0.90 0.80 A second step consisted of studying the feces of patients afflicted with presumed acute NANB hepatitis, studied both for the sporadic form (French patients) and for the epidemic form (Ivory Coast patients) of NANB hepatitus. NANB hepatitis was diagnosed by the conventional immunological criteria for the three types of infection by hepatitis viruses B, delta): presence or absence of IgM and of anti-HAV IgG for virus A infection; HBsAg, anti-HBc and anti-HBc IgM, anti-HBs for HBV infection; delta and anti-delta.Ag for HDV infection where applicable. In addition, yellow fever markers were looked for in the Ivory Coast patients. All patients were assessed for infection by the cytomegalovirus by antibody IgM immunocapture and infection by the Epstein-
IC
-t 1
I
h, T -22- Barr virus by anti-VCA IgM detection in the immunofluorescent staining. The Ag associated with NANB was detected both in the epidemic and sporadic cases (Table No Ag was detected in control extracts of European patients presenting other gastro-intestinal difficulties. In most cases these results were corroborated several times with different solid phase IgG or IgM and with differently labeled IgG. No antigen was detected in the positive samples when tested against the control, pre-immune IgM.
Accordingly the detected Ag seems closely linked to the NANS virus.
Table 2 below illustrates the NANB Ag detection in the feces of patients suffering from both epidemic NANB hepatitis (Ivory Coast) and sporadic NANB hepatitis (France). Ninety feces from patients believed to have gastro-intestinal infections were used as controls. All the presumed NANB samples and seventeen of ne ninety controls were treated ultrasonically. Human antibody (Ab marked with peroxidase or with beta-galactosidase was used as the Ag tracer.
TABLE 2 Ivory Coast fecal French fecal French samples: epidemic samples: sporadic controls positive NANB ag 17 7 0 negative NANB ag 44 13 To confirm that the detected Ag is actually closely associated with the NANB virus, continuous cell lines of hepatocytic origin, PLC/PRF 5 and HE? G2, and normal human
V{
-1 t ,i -23fibroplast cells, MRC 5, were cultivated in DULBECCO's medium containing 10% fetal calf serum. The cell lines were inoculated with antigen by means of a fecal extract from an NANB hepatitis patient. Following culture confluence, a sub-culture was made after adding EDTA-trypsin. In some cases the toxic substances present in the feces caused premature cell death the first weekother feces, non-toxic to the cell lines, caused cell death after several passages. This specific lethal effect was observed solely with the cell line PLC/PRF 5, and not with either the cell line HEP G2 and the cells MRC 5. The cells were subjected to ultrasonic treatment and filtration with a porosity of 0.22 micron.
NANB Ag was detected in the supernatant of infected PLC/PRF but not in the supernatant of the HEP G2 cell line infected under the same conditions.
The PLC/PRF 5 cells inoculated with a 20% liver extract from infected Saimiri monkey evidenced positive antigen characteristics similar to that found in the fecal samples (Table 3); no cell toxicity following inoculation was observed.
Table 3 illustrates the detection of NAN3 Ag in the hepatic cell lines inoculated by either a fecal extract from an Ivory Coast patient afflicted with epidemic NANB hepatitis or by a liver extract from a Saimiri monkey (No. 1).
44 ii b I
I.
-24- The liver extract was prepared in PBS (10% weight/weight) and was homogenized using Ultraturax (Polylabo-France). Following centrifugation, the residue was suspended again in four times its weight of TNB and subjected to ultrasonic treatment in the presence of 0.005 mg/ml of phenylmethylsulfonide (PMSF) chloride.
A liver extract from a Saimiri monkey inoculated with a fecal extract from patients afflicted with a non-infectious liver disease was used as control.
Following three passages, :he inoculated cell cultures were centrifuged. The residue was placed in TNB and, following add:tion of PMSF, the cells were treated ultrasonically. The f:gures indicate the P/N ratio.
a2 t TABLE 3 Cells inoc. Cells inoc.
Cells inoc. with liver with liver Hepatoma with fecal extract from extract from noncell extract from infected from Saimiri con- inocstrain Iv. C, patient Saimiri monkey trol monkey ulated PLC/PRF 5 8.19* 4.39 2 1.01 HEP G2 1.62 1.14 2 1.07 *After 1/100 dilution, P/N 6.29.
As the object of the fourth step, the antigen associated with the virus was purified from fecal samples of different origins.
Three antigen distribution curves, shown in the attached Figure 1, were observed. In fresh samples, the antigen aotivity is found in the density band at 1.28-1.32 g/cm 3 Some of the fecal samples preserved at +4 0 C for several months or fresh samples aeposed to ultrasonication only showed antigen activity at a density of 1.12-1.19 g/cm 3 Other samples demonstrated activity at both densities.
Figure 1 shows the antigen distribution in the CsCl gradier following isopycnic ultracentrifugation which was obtained as follows: Three ml specimens of fecal samples (10% in RPMI) were filtered through a 0.22 micron porous element in a CsCl gradient between 1.1 and 1.6 g/ml. Isopycnic centrifugation (48 hours) a- 78,000 g was carried out in 12 ml tubes (1,2,3,2,0.5,0.5 ml of CsC cf densities respectively 1.i, 1.2, 1.3, 1.4, 1.5 and 1.6) .5 n L f fractions were collected and tested to determine the presence of NANB viral anticen after dilution at one-half.
In Figure 1: A denotes fresh feces, B and C Ii) -26are preserved or ultrasonically treated feces. Low-density antigen substances which occur under conditions of preservation or ultrasonication are assumed due to decomposition by enzymes contained in the feces or to physical treatment. The CsCl fractions testing positive for the antigen were ultracentrifuged in a sucrose gradient from 10 to 60% weight/volume and the reactive fractions were examined under the electron microscope. The NANB antigen was found at 40-41.5% sucrose and predominantly at 26-28% sucrose. Of the nine samples prepared, seven tested finally positive for the NANB antigen: one from an inoculated Saimiri monkey (the second monkey was not examined), two from two of the three patients afflicted with acute epidemic NANB hepatitis (Ivory Coast), four from four of the five patients afflicted with sporadic NANB hepatitis (France). Further, two types of spherical viral particles were observed: large part:cles, about 75 nm in diameter with a double-membrane structure, and smaller homogeneous particles about 25 to 30 nm in diameter.
A tubular structure 15 nm in diameter (or width) and with a length up to I micron and more or less attached to the par-icles was observed and is shown in -he attached Figure 3. Certai. pictures indicate that small tubes leave the envelope of the large spherical particles which were primarily observed in fractions with a density between 1.28 and 1.32 g/cm 3 and in the 40-41.5% sucrose fraction of the sucrose gradient. Smaller particles were observed only in CsC1 fractions with a density between 1.12 and 1.19 g/cm 3 and -n the fraction of the sucrose gradient at 26-28 1 -27sucrose. It has not been yet established whether the smaller and lower-density particles are some kind of release of the initial virus. In none of the six fecal samples, found negative for presence of the antigen and examined simultaneously to act as controls, was a structure found resembling the particles.
A last step enabled detection of the NANB Ag in feces to implicate the epidemic NANB virus in sporadic NANB hepatitis.
NANB Ag associated with the virus was detected in seven out of seventeen fecal samples of patients suffering from acute spc-adic NANB hepatitis (See Figure Serological tests corroborateL these results: out of eight sera available for study, seven included the anti-NANB antibody.
Figure 2 illustrates the characterization of viral particles using the electron microscope for the feces of monkeys infected with NANB and of NANB hepatitis patients.
Following isopycnic ultracentrifuging in the manner described above, the fractions positive for Ag were mixed, dialyzec against TNB, and zone-ultracentrifuged in a sucrose gradient (10-60% weight) at 78.000g for five hours. The positive fractions were mixed, dialyzed, concentrated, and examined under the electron microscope. A drop of viral preparation was spread on a carbon-covered 200 mesh grid and, following absorption on filter paper, a drop of 2% uranyl acetate in distilled water was added.
After two-minutes, the gr'd was wiped with filter paper and examined under the :EM 1CO C x 11 microscope.
The attached Figures 3 and 4 show electron microsections of PLC/PRF 1 -28inoculated cells, after dyeing with uranyl acetate the stool extract of a patient suffering from non-A non-B hepatitis after detection of the Ag by the above-described techniques.
Viral particles are present again in the nucleus and cytoplasm in the form of "crystal lattices"; the particle diameter is very constant: 74 to 75 nm. Figure 3 is an enlarged view x 16000 and Figure 4 a view enlarged by 54000.
In view of the prevalence of NANB hepatitis epidemics in developing countries, it was felt significant to check whether the same viruses are involved in the sporadic cases in those countries. Therefore the sera of twenty-eight Moroccan patients with sporadic acute NANB hepatitis were examined to ascertain whether they contained antibodies associated with the epidemic NANB virus. Sixty-eight percent of the patients had epidemic NANB anti-virus antibodies whereas the same antibodies were found only in 10% of the blood donors in the same country, i.e., in the control sample.
Table 4 below illustrates the detection of specific antibodies relative to the NANB an:igen in the sera of sporadic NANB hepatitis patients. inhibition of the reaction under the conditions listed in the above Table 1 demonstrated the prese-e.
of the antibodies. The reaction was carried out by incuba:ing 50 i ul of a reference extract from the feces of an infected money for 1 hour at 37 0 C and overnight at 4 0 C with 50 ul of the serum to be examined. All the other reaction steps were carried out in the same manner as for the detection of Ag. The P/N ratio of this S,
I
L z D If
SI
-29reference antigen was about six to eight. Twenty-four normal human and non-diluted sera were tested in an inhibition reaction against the reference Ag in relation to PBS; none of them inhibited the reaction. Ultimately 50 ul of a mixture of these sera were used to show the reaction to be 100% positive. The tested samples were considered positive when the inhibition exceeded The NANB Ag was detected in the feces of five acute sporadic NANB hepatitis patients; antibody was detected in the sera of four of the five patients. In two additional patients, the acute disease was respectively at nine and three months and again no Ag was detected in the feces. The last of the five patients had NANB hepatitis in the'past.with a detectable antibody which disappeared after 3 years.
Table 4 Serum Blood Lab Hemodialysis Sporadic NANB source donors personnel patients hepatitis Country MOROCCO 8/78 0/12 1/12 19/28 FRANCE 0/26 7/8 As Table 4 also illustrates, neither hemodialysis or lab work handling human blood appear to increase the danger of infection by the NANB virus. The fact that Acs prepared frominfected amk! by an epidemic NANB virus are used for the detection of Ag connected with a sporadic NANB virus, shows that it relates to the same virus.
-oI I Y The diverse research studies described above enabled the inventor to characterize a new viral agent involved in NANB hepatitis. This viral agent also appears to be a causative factor in the epidemic, sporadic and post-transfusion forms of NANB hepatitis.
This new viral agent differs from the HAV virus: 1. an infection by the epidemic NANB virus was observed in one monkey and in patients showing anti-HAV immunity; 2. the conventional markers for the acute infection (anri- HAV TgM) were not detected; 3. this virus induced a specific immune response and the antibodies did not react with a viral HAV preparation; sera from hepatitis A convalescents gave negative results in the detection test for epidemic NANB antivirus antibodies.
The NANB v:rus :s also distinct from the HBV and consequently from the HDV: 1. the ailment was diagnosed in some epidemic NANB patients carrying anti-HBs Ag antibodies and further appeared in a vaccinated European patient with a strong anti-HBV immunity; 2. as a rule no HBs Ag and HBe Ag were detected; some Ivory Coast patients tested positive for some HBV markers, probably due to a prior infection, but there as no anti-HBc igM; 3. IgM isolated from monkeys infected by the epidemic NANB virus and used in detecting viral antigens react neither with HBV' or with purified HBs Ag preparations, nor with the sera from patients actively replicating HBV; anti-HBs antibodies are noninterfering in the epidemic NANB anti-virus antigen detection test.
I J
I
-31- The new purified virus characterized as described above appears to be isolatable from widely distributed sources. In addition to the cases described above, the antigen associated with the virus also was found in a Senegalese patient and antibodies were found in the serum of a convalescent of an Algerian epidemic. The epidemic NANB agent is considered to be a water-borne virus. Nevertheless, its role in sporadic hepatitis particularly in a developed country with sound sanitary cond:tions is as yet unclear. This does not rule out other trans::ssion modes, in particular through the blood.
Morgover, the involvement of the epidemic NANB virus :n cases of sporadic and post-transfusion NANB hepatitis may requaye a re-examination of -he use of the qualifier "epidemic".
All the same, the implication of the epidemic NANB virus in cases of sporadic or post-transfusional NANB hepatitis, should result in reconsideration of the adjective "epidemic".
It is as a result in fact of the researches of the Inventor that the virus identified according according to the present invention, called epidemic sporadic and transfusional NANB hepatitis virus (in short: "H-SET") is also responsible for the appearance of hepatitis of type not A not B in transfused patients.
A comparative study comprising polytransfused patients without infectious eepatitis and patients afflicted with A or B hepatitis has shown the absence of antigen associated with the virus in t-h4 their stools and the absence of virus cultivatable under the conditions in which the virus "H-SET" grows.
The strain "H-SET" 1-617 isolated from the stools of the patients -32suffering from transfusional NANB hepatitis, was cultivatable on PLC/PRP5 cells under the conditions described above. Lesions corresponding particularly to numerous syncitial formations appear at D6-D7 preceding the detachment of the infected cells.
An identical method of culture is usable for isolation of the virus from a blood sample. The infected cultures may be used as a source of unpurified antigen for characterization at their surface of the antibody molecules coming from a subject who has contracted the infection (the virus-antibody complex being revealed by means of labelled human anti-immunoglobulins).
The Clamart strain is established to be infectious for two rhesus monkeys (inoculation by ingestion (by mouth) with ml of a 10% stool extract) with the appearance of the antigen in the stools between.the 20th and 24th days after inoculation and with increase in the transaminases between the 28th and 32nd days (characteristic sign of hepatic attack).
1
Claims (27)
1. A novel purified viral agent involved in NANB viral hepatitis, wherein said viral agent: a) may be associated with a specific antigen; b) is in the form of spherical viral particles of two main types: particles aboit 75 nm in diameter and particles about 25 to 30 nm in diameter; the first being predominant in the fractions of a cesium chloride (CsCI) gradient with a density varying from 1.1 to 1.6 g/ml of which the density is between 1.28 and 1.32 g/cm 3 and the fractions of 40-41.5% of a sucrose gradient of a concentration varying between 10 and 60% in weight-volume, and the second being predominant in the CsCl gradient fractions of a density between 1.12 and 1.19 g/cm 3 and in the 26-28% fractions of said sucrose gradient, said density bands corresponding to the S* distribution curve of its antigen activity; c) is involved in epidemic, sporadic or post-transfusion viral hepatitis; and d) is either isolated from the feces of patients afflicted with NANB hepatitis by centrifugation and filtration through a filter of which the porosity is of the Sorder of 0.22 pm, or is prepared from hepatocytes, or from continuous hepatocyte cell lines, inoculated with the fecal extract from a NANB hepatitis sufferer or with a liver extract from an infected monkey.
2. An antigen associated with the viral agent according to te claim 1, said antibody being obtained from a viral agent, isolated from patient feces cultivated on infected hepatocytes by physical or chemical treatment.
3. An antigen according to claim 2, obtained from a viral agent isolated from infected continuous cell lines of hepatocytes by ultrasonication treatment. oLoe- o C\aS t-P
4. An antigen according to s~aZ_ 1 or laim 3, whose distribution curve in a CsCl gradient is in the form of a band with a density of 1.12 1.19 g/cm 3 and/or a band of density 1.28 1.32, and which is present after centrifugation of the CsCl fractions in a sucrose gradient, 4 in the 26-28 fraction of the sucrose gradient for the F i 1 i 34 first band and in the 40 41.5 fraction of the sucrose gradient for the second band. A method of isolating a viral agent according to claim 1, comprising the steps of subjecting the feces of patients afflicted with NANB hepatitis to a centrifugation treatment, followed by a filtration treatment though a filtering element whose porosity is of the order of 0.22 pm, to extract said viral agent frm the feces.
6. A method according to claim 5, wherein the centrifugation treatment is a centrifugation operation at 5000 8000 g.
7. A method according to claim 5 or claim 6, wherein prior to the centrifugation and filtration operations, the fecal extract is subjected to a physical dissociation treatment, or to a chemical dissociation treatment.
8. A method according to claim 7, in which the physical dissociation treatment is ultra-sound treatment.
9. A process for the preparation of a viral agent according to claim 1, comprising the steps of inoculating hepatocytes or continuous cell lines of hepatocytes with a fecal extract from NANB hepatitis patients or with a liver extract from a monkey previously inoculated with a fecal extract from NANB hepatitis patients; cultivating the cells to confluence, centrifuging the cells after several passages to collect the desired viral agent, and optionally I subculturing the viral agent. A process according to claim 9, further comprising the step of isolating the viral agent involved in epidemic, sporadic or post-transfusion NANB viral hepatitis.
11. A process according to claim 9 or claim 10, wherein the centrifugation pellet is resuspended in a suitable buffer and then subjected to ultrasonication to release NANB antigen into the supernatant. S A !I '0 i f /U3<
12. A process for the detection of NANB antigen as defined in claims 1 to 4,said antigen being present in feces of NANB viral hepatitis patients, comprising the step of incubating fecal extracts from mammals presumed afflic- ted with NANB viral hepatitis with an anti-NANB antibody comprising immunoglobulins isolated from the serum of either convalescents or monkeys previously infected with the viral agent according to claim 1 to form an antibody-anti- gen immune complex whose presence is revealed by means of a suitable detection method, to demonstrate the presence of the NANB antigen.
13. The detection process of claim 12 wherein fecal extracts from mammals presumed infected by the NANB virus, obtained by centrifugation and filtration, are incubated with an anti-NANB antibody made up of IgM fractions isolated from the sera of monkeys artificially infected with fecal extracts from human patients known to suffer from NANB hepatitis, wherein those fractions have been previous- ly fixed on a solid support, and then with an immune fraction of IgG of a mammal of the same species or a nearby species, which has been labeled with a fluorescent, radio- active, or enzyme label.
14. A method of detection of anti NANB antibodies in the blood of patients or in blood products, wherein the serum of a patient or of a convalescent person is contacted with the purified viral preparation according to cl=im 1 S. or with a fraction of the latter after centrifugation and isolation of the band corresponding to a density of 1.29 1.32, and development of the antigen-antibody complex with a human antiimmunoglobulin preparation labelled with an appropriate label. A method according to claim 14, wherein the detec- tion of said antibodies takes place by characterization of an antibody activity in the IgMs of a patient permitting very early diagnosis of the disease, by detecting the I.]Ms of a pat Lent's serum, by reacting them with a viral jjb_ MMWM ;i 1I 1 e 3b preparation purified according to claim 1 or with a fraction of the latter, and by characterizing the antigen fixed to the IgM of the patient by means of human anti-immunoglobulin immunoglobulins or fragments of the latter, labelled with a suitable label.
16. Strain of a purified viral agent according to claim 1, isolated from the feces of a patient afflicted with sporadic NANB viral hepatitis, called "Clamart Strain", deposited on 30 October 1986 under no. 1-617 at the COLLECTION NATIONALE DE CULTURE DE MICROORGANISMES, as hereinbefore defined.
17. Strain of a purified viral agent according to claim 1, isolated from the feces of a patient afflicted with epidemic NANB viral hepatitis, so-called "Ivory Cost S Strain", deposited on 30 October 1986 under no. 1-616 at the COLLECTION NATIONALE DE CULTURE DE MICROORGANISMES, as hereinbefore defined.
18. Strain of a purified viral agent according to claim 17, constituted by a culture of the viral of NANB hepatitis, derived from the feces of a patient afflicted with epidemic NANB viral hepatitis, on hepatocyte cells.
19. Strain of viral agent according to claim 18, constituted by a culture of the virus of non A non B hepatitis, derived from the feces of a patient afflicted with epidemic NANB viral hepatitis on a continuous hepatocyte cell line. Strain of viral agent according to claim 19, wherein Sthe continuous hepatocyte cell line is PLC/PRF 5 hepatocytes known under the name "Alexander line".
21. Immunological reagent for the detection of posttrans- fusional, sporadic or epidemic NANB viral hepatitis, by the method of detection according to claim 12, which comprises immunoglobulins isolated from human convalescent serum or from animal serum previously artificially infected with a viral agent according to claim 1. 4, i V/ 1 A- a /sT 'I I *t *b7 j
22. Immunological reagent according to claim 21, comprising an antibody formed of immunoglobulins prepared from serums of animals artificially infected by ingestion of extracts of feces of patients afflicted with epidemic NANB hepatitis.
23. Immunological reagent according to claim 22, wherein the serum of animals artificially infected is serum of monkeys, collected from the 27th day after infection by fecal extracts.
24. Method of detection of antibodies in the blood of patients or in blood products comprising the steps of contacting a sample of blood or of blood pr ,duct with a reagent constituted by viral antigen according to any one of claims 1 to 4 and identifying the presence of antibodies of viral NANB hepatitis in said blood or blood product specimen.
25. Diagnostic method for sporadic, epidemic or post- transfusion NANB viral hepatitis in fecal extracts, comprising the steps of incubating plates coated with an immunological reagent according to claim 21, consis- ting of IgM antibodies with fecal extracts for about 1 hour at about 37 0 C,then with fractions of IgMs or V IgGs purified from serums of patients convalescent from epidemic NANB hepatitis and labelled with an enzyme, in the presence of a suitable developer substrate.
26. Diagnostic kit for the diagnosis of epidemic sporadic or post-transfusion NANB viral hepatitis comprising at least one viral antigen according to any one of claims 1 to 4.
27. Diagnostic kit for epidemic, sporadic or post-transfu- sion NANB viral hepatitis, comprising at least an antibody against the viral agent according to any one of claims 1 to 4.
28. Therapeutic agent for the treatment of epidemic, sporadic or post-transfusion NANB viral hepatitis, compri- sing a purified antibody directed against the viral agent according to any one of claims 1 to 4.
29. Therapeutic agent according to claim 28, wherein said antibody is IgM. Therapeutic agent according to claim 28, comprising a monoclonal antibody produced by a hybridoma resulting from the fusion of myeloma cells and of spleen cells of mice inoculated with viral agents of NANB hepatitis according to any one of claims 1 to 4.
31. Strain of a purified viral agent according to claim 1 isolated from the feces of a patient afflicted with post-transfusion NANB viral hepatitis, so called "H-Set strain" deposited on 5 February 1987 under no. 87.02.05.02 at the ECACC Collection (Great Britain), as hereinbefore defined. 0:00 4** *S* S DATED THIS 6TH DAY OF MARCH 1991 S INSTITUT PASTEUR By its Patent Attorneys GRIFFITH HACK CO. S Fellows Institute of Patent Attorneys of Australia. i 4 r-f I *I i 14 INTERNATIONAL SEARCH REPORT International Application No PCT/FR 87/00441 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC 4 CIB4: A 61 K 39/29; G 01 N 33/576 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols CIB 4 A 61 K; G 01 N Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, 11 with indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 1 Y,P EP, A, 0242300 (INSTITUT PASTEUR) 21 1-45 October 1987, see page 3, lines 8-44 cited in the application Y Biological Abstracts, volume 82, No. 6, 1-45 1986, Biological Abstracts Inc. (Philadelphia, US) J.L. SARTHOU et al.: "Characterization of an antigen- antibody system associated with epidemic non-A, non-B hepatitis in West Africa and experimental transmission of an infectous agent to primates", see page AB-442, abstract 53850, ANN.INST PASTEUR VIROL 137 225-232, 1986 Y,P Biological Abstracts, volume 83, No. 12, 1-45 1987, Biological Abstracts Inc. (Philadelphia, US), J. Pillot et al.: "Immunological characterization of a viral agent involved in epidemic Special categories of cited documents: 'o later document published after the international filing date ment ing t ge ate the art which s not or priority date and not In conflict with the application but document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance Invention earlier document but published on or after the international document of particular relevance; the claimed invention filing date cannot he considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance' the claimed Invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the dorument referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of tills International Search Report 23 February 1988 (.23.02.88) 12 April 1988 (17 International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE Form PCT/ISA/210 (second sheet} (January 1985) I International Appf'.tin Jo. CT/FR 87/00441 1 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM ThE SICOND SHEET) Category Cttatlon of Document, with Indication, whee approprite, of tte ievarnt pasage Relevant to Claim No r and sporadic non-A, non-B hepatitis", see page AB-576, abstract 119271, ANN INST PASTEUR VIROL 138(1):
145-158, 1987 WO, A, 8202774 (BAXTER TRAVENOL LABORATORIES INC.) 19 August 1982, see the whole document WO, A, 8002598 (THE UNITED STATES OF AMERICA) 27 November 1980, see the whole document EP, A, 0061974 (TREPO CHRISTIAN) 6 October see the whole document EP, A, 0093438 (THE BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE) 9 November 1983 EP, A, 0068465 (EISAI CO. LTD) 5 January 1983 EP, A, 0066296 (EISAI CO. LTD) 8 December 1982 WO, A, 8203330 (TREPO CHRISTIAN) 1.-October 1982 1-45 1-45 1-45 Form PCT/ISA/210 (extra sheet) (January 1985) 0'Iw 4 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. FR 8700441 SA 19455 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 18/03/88 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date EP-A- 0242300 21-10-87 FR-A- 2597606 23-10-87 JP-A- 62249999 30-10-87 WO-A- 8202774 19-08-82 EP-A,B 0071640 16-02-83 GB-A,B 2108527 18-05-83 CA-A- 1168579 05-06-84 WO-A- 8002598 27-11-80 EP-A- 0029063 27-05-81 US-A- 4356164 26-10-82 AU-A- 6055380 15-01-81 CA-A- 1147647 07-06-83 US-A- 4395395 26-07-83 CA-A- 1155764 25-10-83 EP-A- 0128995 27-12-84 AU-B- 540153 01-11-84 JP-T- 56501173 20-08-81 EP-A- 0061974 06-10-82 US-A- 4542016 17-09-85 EP-A- 0093438 09-11-83 JP-A- 59042455 09-03-84 CA-A- 1208554 29-07-86 US-A- 4702909 27-10-87 EP-A- 0068465 05-01-83 JP-A- 58000753 05-01-83 CA-A- 1177749 13-11-84 EP-A- 0066296 08-12-82 JP-A- 57198867 06-12-82 CA-A- 1184846 02-04-85 WO-A- 8203330 14-10-82 FR-A,B 2502154 24-09-82 EP-A,3 0074986 30-03-83 3 For more details about this annex see Official Journal of the European Patent Office, No. 12/82 4? A .4 qI. RAPPORt *E RECHERCHE INTERNATIONALE Demands Internationale N' PCT/FR 87/00441 1. CLASSEMENT DE L'tNVENTION (sI plusieurs symboles de classiication sont applicables, les indliquer tous) Selon Is classification Internationale des brevets (CIS) ou A Ia fois selon as classification nationale et la CIB CIB 4 A 61 K 39/29; G 01 N 33/576 11. DOMAINES SUR LESQUELS LA RECHERCHE A PORTE Documentation minimale consulte I Documentation consult~e autre que la documentation minlmsle dans [a mesure oOi de tels documents font partle des domnalnes sur Iesquels la recherche a porlilA Ill, DOCUMENTS CONSIDtRtS COMME PERTINENTS CtgreIdentification des documents cittis, 11 avec Indication, si ndcessaire, N* des revendications Calgoiedes Passages pertinents 12 visdss 13 Y,P EP, A, 0242300 (INSTITUT PASTEUR) 21 -1-45 octobre 1987, voir page 3, lignes 8-44 cit.6 dans la demande Y Biological Abstracts, volume 82, no. 6, 1-45 1986, Biological Abstracts Inc. (Philadelphia, US) J.L. SARTHOU et'al.: "Characterization of an antigen- antibody system associated with epidemic non-A, non-B hepatitis in West Africa and experimental transmission of an infectous agent to primates", voir page AB-4.42, abr6g6 53850, ANN INST PASTEUR VIROL 137(2)- 225-232, 1986 Y,P Biological Abstracts, volume 83, no. 12, 1-45 1987, Biological Abstracts Inc. (Philadelphia, US), J. Pillot et al.: "Immunological characterization of a viral agent involved in epidemic.1 Cat~gories sp~ciales de documents citds: "a Tan document ultkrieur oublitiPost~rieurementh ladatede ddo6t a A a) document d~finissant l'6tat g~n~ral de Ia technique, non international ou A Ia date de PrioritL% at nappartenant pas 61l,6at dej]a technique pertinent, mats citA pour comprenare considdrd comme particulibrement pertinent le principe ou as thdorie constituant In base do l'invention E Y) document ant~rieur, mais publi6 A Ia date de ddpt interna- a Xa) docu ment perticuli~rement pertinent: Invention revandi- tional ou apr~s cetta date qude ne peut Mtrs considrde comme nouvelle ou comme a La) document pouvant later un doute cur une revendication da Impliquant une ectivitd inven, priort ou cit6 pour d~terminer Ia date oes publication dune ac Yei document particulihrement pertinent: l'inventfon raven- a utre citation ou pour une raison apdcisle (telle qu'Indlqu~e) cliques ne peut Wte considLr~e comma implicuant une 0 at document se r~f~rant A une divulgation orale, un usage, A activit6 Inventive lorsaue Is delumnent eat ass=ci A ui, ou une exposition ou tous autres moyane plusieure autras documents .u mtme nature, caitte combi- at P document oubli~i avant Ia date de d~o6t international, mats nasson diant 6vidente pour une personne du mter, post~riecrement A Ia date de priorit revendiqude ac document qui fait Partle de Is m~ma famille de brevets IV. CERTIFICATION Date A laquelle Is recherch~e Internationale a 6t0 effectlvement Date d'eoadition du present rapport de recherch~e internationale achevde 23 f6vrier 1988 1 P AR 13 Administration chargae de Ia recherche internationale S7,9iaW. ncti ntta a autorise OFFICE EUROPEEN DES BREVETS y EPUTTfEN Formulaira PCTIISA/2IO (deuxi~me feuille (janvie 1985) 3 C, 'Kr C '13 i i i i Demands Internationale N* PCT/FR 87/00441 (SUITE DES RENSEIGNEMENTS INDIQUtS SUR LA III. DOCUMENTS CONSID9RtS COMME PERTINENTS DEUX19ME FEUILLE) Ca~ol ntfts desdocuments Wk'. a k~dkanron. si niceessiro, W des revondcawns Catdpsorisage$ p I8 yrrt~nsrsta redi and sporadic non-A, non-B hepatitis", voir page AB-576, abr~g6 119271, ANN INST PASTEUR VIROL 138(1): 145-158, 1987 WO, A, 8202774 (BAXTER TRAVENOL LABORATORIES INC.) 19 aoat 1982, voir le document en entier WO, A, 8002598 (THE UNITED STATES OF AMERICA) 27 novembre 1980, voir le document en entier EP, A, 0061974 (TREPO CHRISTIAN) 6 octobre voir le document en entier EP, A, 0093438 (THE BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE) 9 novembre 1983 -EP, A, 0068465 (EISAI CO. LTD) 5 janvier 1983 1-45 1-45 1-45 EP, A, 0066296 (EISAI CO. LTD) 8 1982 WO, A, 8203330 (TREPO CHRISTIAN) 14 octobre 1982 d6cembre Formulaire PCTiISA.210 (leuille addltionnelle) (Janvwr1965) Ww ANNEXE AU RAPPORT DE RECHERCHE INTERNATIONALE RELATIF A LA DEMANDE INTERNATIONALE NO. FR 8700441 SA 19455' La pr~sente annexe indiquc Ces mcmbrcs dc la famille, de brevets relatifs oux documents brevets cit6s dans le rapport de recherche international vjs6 ci-dessus. Lesdits members sont contenus au ichier informotique de l'Office europ 6 en des brevets i la date du 18/03/88 Les rensci.gnemcnts fournis sont donnis i titre indicotif et n'cngnogent pas [a rcsponsabilit6 dc ['Office curopt~en des brcv-t-;. Document brevet cit6 Dote de Nferhre(s) de lo Dote de ou rapport de recherche pulcto oil obevet(s) ulcto EP-A- 0242300 21-10-87 FR-A- 2597605 23-10-87 UP-A- 62249999 30-10-87 WO-A- 8202774 19-08-82 EP-A,B 0071640 16-02-83 rB-A,B 2108527 18-05-83 CA-A- 1168579 05-06-84 WO-A- 3002598 27-11-80 EP-A- 0029063 27-05-81 US-A- 4356164 26-10-82 AU-A- 6055380 15-01-81 CA-A- 1147647 07-06-83 US-A- 4395395 26-07-83 CA-A- 1155764 25-10-83 EP-A- 0128995 27-12-84 AU-B- 540153 01-11-84 JP-T- 56501173 20-08-81 EP-A- 0061974 06-10-82 US-A- 4542016 17-09-85 EP-A- 0093438 09-11-83 UP-A- 59042455 09-03-84 CA-A- 1208554 29-07-86 US-A- 4702909 27-10-87 EP-A- 0068465 05-01-83 UP-A- 58000753 05-01-83 CA-A- 1177749 13-11-84 EP-A- 0066296 08-12-82 UP-A- 57198867 06-12-82 CA-A- 1184846 02-04-85 WO-A- 8203330 14-10-82 FR-A,B 2502154 24-09-82 EP-A,B 0074986 30-03-83 ['our tout renseignement concernunt cette annexe voir Journal Officiel dc ['Office curop~en des brevets, No.12,182
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
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| FR8615625 | 1986-11-07 | ||
| FR8615625A FR2606515B1 (en) | 1986-11-07 | 1986-11-07 | PROCESS FOR ISOLATING A NEW VIRAL AGENT AND THE ANTIGEN ASSOCIATED WITH IT, VIRAL AGENT AND ANTIGEN OBTAINED THEREBY AND METHOD FOR DETECTING THE SAME ANTIGEN USING AN APPROPRIATE IMMUNOLOGICAL REAGENT |
| FR8701738A FR2609807B2 (en) | 1987-02-11 | 1987-02-11 | IMMUNOLOGICAL REAGENTS FOR THE DETECTION OF NANB VIRAL HEPATITIS, THEIR PREPARATION METHODS AND THEIR APPLICATIONS IN METHODS OF DETECTION OF NANB VIRAL HEPATITIS AND AS THERAPEUTIC AGENTS |
| FR8701738 | 1987-02-11 |
Publications (2)
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| AU8273487A AU8273487A (en) | 1988-06-01 |
| AU611402B2 true AU611402B2 (en) | 1991-06-13 |
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| AU82734/87A Ceased AU611402B2 (en) | 1986-11-07 | 1987-11-06 | Viral agent for nanb hepatitis, antigen and immunologic reagent |
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| JP (1) | JP2602517B2 (en) |
| AU (1) | AU611402B2 (en) |
| DE (1) | DE3784484T2 (en) |
| ES (1) | ES2054696T3 (en) |
| NZ (1) | NZ222465A (en) |
| WO (1) | WO1988003410A1 (en) |
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| US5714596A (en) * | 1987-11-18 | 1998-02-03 | Chiron Corporation | NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus |
| US6171782B1 (en) | 1987-11-18 | 2001-01-09 | Chiron Corporation | Antibody compositions to HCV and uses thereof |
| YU48038B (en) * | 1987-11-18 | 1996-10-18 | Chiron Corp. | PROCEDURE FOR DIAGNOSIS OF NO-A, NO-V HEPATITIS |
| US5698390A (en) | 1987-11-18 | 1997-12-16 | Chiron Corporation | Hepatitis C immunoassays |
| US6861212B1 (en) | 1987-11-18 | 2005-03-01 | Chiron Corporation | NANBV diagnostics and vaccines |
| CN1074422C (en) * | 1987-11-18 | 2001-11-07 | 希龙股份有限公司 | Method for preparing isolated polypeptides containing HCV epitopes |
| US5712088A (en) * | 1987-11-18 | 1998-01-27 | Chiron Corporation | Methods for detecting Hepatitis C virus using polynucleotides specific for same |
| US5350671A (en) * | 1987-11-18 | 1994-09-27 | Chiron Corporation | HCV immunoassays employing C domain antigens |
| US5824649A (en) * | 1988-06-17 | 1998-10-20 | Genelabs Technologies, Inc. | DNA sequences of enterically transmitted non-A/non-B hepatitis viral agent and characteristic epitopes thereof |
| US5789559A (en) | 1988-06-17 | 1998-08-04 | Genelabs Technologies, Inc. | DNA sequences of enterically transmitted non-A/non-B hepatitis viral agent |
| DE3837616A1 (en) * | 1988-11-05 | 1990-05-10 | Behringwerke Ag | ONE-STEP IMMUNITY TEST FOR DETERMINING ANTIGEN-SPECIFIC ANTIBODIES OF ALL IMMUNOGLOBULIN CLASSES AND MEANS THAT ARE SUITABLE FOR THIS |
| US6027729A (en) * | 1989-04-20 | 2000-02-22 | Chiron Corporation | NANBV Diagnostics and vaccines |
| US7166287B1 (en) | 1989-12-18 | 2007-01-23 | Glaxo Wellcome Inc. | Viral agent |
| FI906208A7 (en) | 1989-12-18 | 1991-06-19 | The Wellcome Foundation Ltd | Viral agents |
| EP0447984A1 (en) * | 1990-03-20 | 1991-09-25 | Abbott Laboratories | Hyperimmune globulin against hepatitis C virus and method for making same |
| KR100241221B1 (en) * | 1991-08-26 | 2000-03-02 | 에드워드 콜린스 제임스 | Swine infertility and respiratory system heavy vaccine and diagnosis |
| US6080570A (en) * | 1991-08-26 | 2000-06-27 | Boehringer Ingelheim Vetmedica, Inc. | Method of producing a vaccine for Swine Infertility and Respiratory Syndrome |
| US6982160B2 (en) | 1991-08-26 | 2006-01-03 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions that include SIRS virus |
| US5846805A (en) * | 1991-08-26 | 1998-12-08 | Boehringer Ingelheim Animal Health, Inc. | Culture of swine infertility and respiratory syndrome virus in simian cells |
| WO1993006211A1 (en) * | 1991-09-16 | 1993-04-01 | Collins James E | Vaccine for mystery swine disease and method for diagnosis thereof |
| EP0839912A1 (en) | 1996-10-30 | 1998-05-06 | Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) | Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof |
| JP3961222B2 (en) | 1999-03-08 | 2007-08-22 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハー | PRRSV vaccine |
| PT2251419E (en) | 1999-04-22 | 2012-09-03 | Us Agriculture | Porcine reproductive and respiratory syndrome vaccine, based on isolate ja-142 |
| MXPA06015105A (en) | 2004-06-18 | 2007-03-26 | Univ Minnesota | Identifying virally infected and vaccinated organisms. |
| US7632636B2 (en) | 2004-09-21 | 2009-12-15 | Boehringer Ingelheim Vetmedica, Inc. | Porcine reproductive and respiratory syndrome isolates and methods of use |
| KR101321153B1 (en) | 2005-06-24 | 2013-10-22 | 리전츠 오브 더 유니버스티 오브 미네소타 | Prrs viruses, infectious clones, mutants thereof, and methods of use |
| AR078253A1 (en) | 2009-09-02 | 2011-10-26 | Boehringer Ingelheim Vetmed | METHODS TO REDUCE ANTIVIRICAL ACTIVITY IN PCV-2 COMPOSITIONS AND PCV-2 COMPOSITIONS WITH BETTER IMMUNOGENICITY |
| UA112768C2 (en) | 2011-02-17 | 2016-10-25 | Бьорінгер Інгельхайм Ветмедіка Гмбх | EUROPEAN PRRSV STRAIN |
| ES2553879T3 (en) | 2011-02-17 | 2015-12-14 | Boehringer Ingelheim Vetmedica Gmbh | Procedure for the production on a commercial scale of PRRSV |
| US9187731B2 (en) | 2011-07-29 | 2015-11-17 | Boehringer Ingelheim Vetmedica Gmbh | PRRS virus inducing type I interferon in susceptible cells |
| WO2013017568A1 (en) | 2011-07-29 | 2013-02-07 | Boehringer Ingelheim Vetmedica Gmbh | INFECTIOUS cDNA CLONE OF EUROPEAN PRRS VIRUS AND USES THEREOF |
| EP2968513A2 (en) | 2013-03-15 | 2016-01-20 | Boehringer Ingelheim Vetmedica, Inc. | Porcine reproductive and respiratory syndrome virus, compositions, vaccine and methods of use |
| DK3083947T3 (en) | 2013-12-20 | 2019-07-08 | Boehringer Ingelheim Vetmedica Gmbh | PRRS VIRUS VARIATION, CDNA CLONE OF EUROPEAN PRRS VIRUS, AND USES THEREOF |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6055380A (en) * | 1979-05-21 | 1981-01-15 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Detection of non-a, non-b hepatitis associated antigen |
| EP0061974A2 (en) * | 1981-03-27 | 1982-10-06 | Institut Merieux | New vaccine and therapy against non-A non-B viral hepatitis, and their preparation process |
| EP0092249A2 (en) * | 1982-04-21 | 1983-10-26 | Eisai Co., Ltd. | Non-A, non-B hepatitis-associated monoclonal antibody and diagnostic reagent |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4356164A (en) * | 1979-05-21 | 1982-10-26 | Govt. of the U.S., as represented by the Secretary, Dept. of Health & Human Services | Detection of non-A, non-B hepatitis associated antigen |
| CA1168579A (en) * | 1981-02-11 | 1984-06-05 | Philippe Maupas (Deceased) | Non-a, non-b hepatitis virus |
| FR2502154A1 (en) * | 1981-03-30 | 1982-09-24 | Trepo Christian | PROCESS FOR PREPARING ANTIGENS FOR VIRAL HEPATITIS NANB, AND APPLICATION TO THE REAGENT FOR THE DIAGNOSIS AND PROGNOSIS OF INFECTIONS CAUSED BY VIRAL HEPATITIS VIRUSES |
| JPS57198867A (en) * | 1981-06-02 | 1982-12-06 | Eisai Co Ltd | Non-a, non-b hepatitis related antigen and diagnostic therefor |
| JPS58753A (en) * | 1981-06-25 | 1983-01-05 | Eisai Co Ltd | Non-a and non-b type hepatitis related antibody and detection reagent |
| EP0093438B1 (en) * | 1982-05-05 | 1988-07-20 | Louisiana State University and Agricultural and Mechanical College | Non-a, non-b hepatitis antigen |
| FR2597606B1 (en) * | 1986-04-16 | 1989-10-06 | Pasteur Institut | NOVEL REAGENT FOR THE DETECTION OF NON TO NON B VIRAL HEPATITIS AND METHOD OF DIAGNOSING SUCH HEPATITIS BY IMMUNOENZYMATICS |
-
1987
- 1987-11-05 NZ NZ222465A patent/NZ222465A/en unknown
- 1987-11-06 EP EP87402519A patent/EP0277437B1/en not_active Expired - Lifetime
- 1987-11-06 JP JP62506994A patent/JP2602517B2/en not_active Expired - Lifetime
- 1987-11-06 WO PCT/FR1987/000441 patent/WO1988003410A1/en not_active Ceased
- 1987-11-06 ES ES87402519T patent/ES2054696T3/en not_active Expired - Lifetime
- 1987-11-06 DE DE8787402519T patent/DE3784484T2/en not_active Expired - Fee Related
- 1987-11-06 AU AU82734/87A patent/AU611402B2/en not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6055380A (en) * | 1979-05-21 | 1981-01-15 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Detection of non-a, non-b hepatitis associated antigen |
| EP0061974A2 (en) * | 1981-03-27 | 1982-10-06 | Institut Merieux | New vaccine and therapy against non-A non-B viral hepatitis, and their preparation process |
| EP0092249A2 (en) * | 1982-04-21 | 1983-10-26 | Eisai Co., Ltd. | Non-A, non-B hepatitis-associated monoclonal antibody and diagnostic reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01501205A (en) | 1989-04-27 |
| NZ222465A (en) | 1992-11-25 |
| DE3784484D1 (en) | 1993-04-08 |
| ES2054696T3 (en) | 1994-08-16 |
| EP0277437A1 (en) | 1988-08-10 |
| DE3784484T2 (en) | 1993-07-01 |
| JP2602517B2 (en) | 1997-04-23 |
| EP0277437B1 (en) | 1993-03-03 |
| AU8273487A (en) | 1988-06-01 |
| WO1988003410A1 (en) | 1988-05-19 |
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