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  • 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
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/60—New or modified breeds of invertebrates
  • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
  • A01K67/65—Genetically modified arthropods
  • A01K67/68—Genetically modified insects
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • C12N2770/24251—Methods of production or purification of viral material

Definitions

• the present invention relates to the identification of novel systems for the amplification of Hepatitis C virus (HCV).
• HCV Hepatitis C virus
• HCV hepatitis C virus
• HCV Hepacivirus
• Flaviviridae which are enveloped single stranded RNA viruses, among which are viruses responsible for major epidemic diseases such as Yellow Fever (YF), Dengue (DEN) and Dengue haemorrhagic fever (DHF), Japanese encephalitis (JE), St. Louis encephalitis (SLE), West NiIe fever (WN) and Hepatitis C (HC) to name only the most important ones.
• YF Yellow Fever
• DEN Dengue
• DHF Dengue haemorrhagic fever
• JE Japanese encephalitis
• SLE St. Louis encephalitis
• WN West NiIe fever
• HC Hepatitis C
• Flaviviruses are transmitted by insect vectors, according to very different epidemiological modalities. Some diseases are typically human (or primate-related) and never affect animals such as DEN and DHF; other infections are more zoonotic and more or less accidentally affect humans, such as JE, SLE and WN. Finally, some Flaviviruses can circulate epidemically in both human and animal populations (YF). These different epidemiological modalities nevertheless have common bases such as viral amplification in insect cells.
• anti-HCV vaccine strategies are today based on two usual methods in the field: on the one hand recombinants Vaccinia or AAV (Adeno-Associated Virus); on the other hand, anti-HCV vaccine strategies are developed based on polypeptides derived from HCV proteins.
• HCV-JFH1 allows the production of complete infectious viral particles (after transfection in Huh-7 cells). However, at present, the system is not suitable for large biomass production.
• VLPs Virus Like Particles
• PPs retroviral pseudo-types
• the invention relates to a method of in vivo amplification of the hepatitis C virus (HCV) comprising the following steps: a / ingestion of the viral source by female mosquitoes of the genus Aedes; b / breeding of mosquitoes during the time necessary for the amplification of the virus.
• HCV hepatitis C virus
• the first step is to "pass" the virus into the mosquito in the most efficient way possible, in this case by ingestion.
• the virus enters the stomach where it will cross the stomach wall, then disperse into the haemocele and reach different organs, such as the ovaries and salivary glands, where it is likely to multiply.
• the virus can therefore be transmitted to subsequent generations of mosquitoes or to humans during a sting.
• Mosquitoes of the genus Aedes are Diptera, Nematocera, family Culicidae, subfamily Culicinae, Tribe Aedini. Females are characterized by short palps and nonplumose antennas. In the context of the present invention, Aedes vexans and Aedes caspius species have been used.
• the population used in the process comes from a rearing made from material (eggs, larvae or adults) taken directly from the field, in the privileged biotope of these two species.
• the individuals used advantageously belong to the FO, Fl or F2 generations.
• the contact between the viral source and the mosquitoes is by ingestion.
• the viral source is conditioned using red blood cells for example sheep, possibly supplemented with ATP, to increase the palatability of female mosquitoes.
• ingestion is done during a blood meal taken by female mosquitoes.
• the ingestion is carried out using the technique of the gorgement.
• This particularly effective technique is well known to entomologists and described for example in Fouque et al. (7).
• a blood meal is offered to female mosquitoes in a throat which keeps the meal at 37 ° C.
• the gorge is covered with a skin through which mosquitoes insert their proboscis and take the meal.
• the viral source comprises a "native" virus (virulent), or advantageously attenuated.
• a virulent virus replicates the infection and therefore offers a first-rate experimental model.
• an attenuated virus is of particular interest in the production of vaccines or antibodies.
• the viral load comes from a serum, advantageously of human origin, for example that of a chronic hepatitis carrier HCV genotype Ib.
• the conventional title of such a serum is usually around 2 E 6 copies / ml.
• the contacting should take place at least 1000 copies of the virus per mosquito, preferably 1500 copies of the virus per mosquito.
• the second step of the method according to the invention consists in raising the mosquitoes for a determined period.
• the breeding is carried out under controlled conditions, including temperature and humidity.
• the duration of the breeding is determined according to the following criteria:
• the amplification appears optimal between 15 and 30 days of breeding for the Aedes tested, with an average value advantageous a breeding time of about 20 days.
• the recovery of the virus, especially in the form of viral particles, may require their extraction of mosquito tissue.
• Preferred tissues are the ovaries and salivary glands.
• the invention thus provides a method of amplifying HCV, taking place in vivo in specific mosquito species, giving results quite unexpected and remarkable.
• the process according to the invention offers very promising prospects, including for the massive in vitro production of HCV.
• the method according to the invention gives access to tissues, in particular of ovary or salivary gland, or Aedes cells, advantageously Ae cells. Caspius or vexans, carriers of HCV.
• the invention also relates to a method for obtaining in vitro producer cells of HCV. It is then a question of cocultivating Aedes cells carrying HCV, obtained using the process according to the invention, and AP61 or C6 / 36 type cells.
• the AP61 and C6 / 36 cells have been described by Germi et al. (9). These are mosquito cell lines, in particular Aedes pseudoscutellaris, continuous and well defined. Co-culture aims to transfer HCV into these cell lines.
• the invention therefore also relates to in vitro HCV-producing mosquito cells obtained from this process.
• FIG. 1 corresponds to a gel illustrating the detection of viral RNA in HCV infected Aedes vexans mosquitoes, after extraction of the total RNAs and standard RT-PCR with revelation of the DNA with ethidium bromide.
• FIG. 2 illustrates the detection of the viral RNA by qRT-PCR, after extraction of the total RNAs of Aedes mosquitoes (vexans + caspius) after 24 days of breeding.
• the standard curve was performed on a log dilution of a known title viral RNA extract (120,000 copies / run at 1,200 copies / run) (Fig. 2A).
• positive controls are identified by (OJ, T +), and individuals who may have replicated HCV after 24 days by (D20).
• For each sample, are indicated (i) the number of cycles at the end of which the viral RNA was detected (ii) and its initial concentration by compared to the standard curve.
• the curve of FIG. 2B shows the evolution of the signal detection for each sample.
• FIG. 3 illustrates the detection of the viral RNA by qRT-PCR, after extraction of the total RNA from Culex mosquitoes (pipiens) after 28 days of breeding.
• the standard curve was carried out on a logarithmic dilution of an RNA extract. virus of known title (800,000 copies / run at 8,000 copies / run) (Fig. 3A). For each sample, are indicated (i) the number of cycles at the end of which the viral RNA was detected (ii) as well as its initial concentration relative to the standard curve.
• the curve of FIG. 3B shows the evolution of the signal detection for each sample.
• the individuals tested come from a breeding made from mosquitoes (eggs, larvae and adults) collected on the ground in the urban area of Marseille for the species Culex pipiens and in the reserve of the Tour du Valat in the Camargue for species Aedes vexans and Aedes caspius. These farms were carried out at the ENSAM premises located at the Domaine du Merle, Salon de elle. These farms are made in a classic insectarium, with breeding standards. The larvae are fed with yeast and fish feed and the adults are fed a sucrose solution. The blood meals of Culex and Aedes are composed of red blood cells washed with sheep, the purpose of the washing being to eliminate the interference of the elements of the blood.
• the viral solution containing HCV virus (serum of patient with chronic hepatitis at 2 E 6 copies / ml) is transported by special transporter to the L2 + where are placed mosquitoes.
• the viral solution is provided by the Laboratory of Molecular and Structural Virology of the Faculty of Medicine of Grenoble.
• the viral solution is stored in the freezer at -80 ° C. of the L2 +. 4. Preparation of the infected blood solution
• the batches of females are kept at a constant temperature of about 28 ° C and with a controlled humidity of at least 70%. Each day, the females are fed with cotton soaked in a 10% glucose solution deposited on the metal cage. Spawning lodges are wetted through the cage with a squeeze bottle.
• mosquitoes are sacrificed by placing the metal cage in the cold. The mosquitoes are then placed in 1.5 ml tubes, identified and returned to the freezer until the RNA extraction step before PCR. Negative witnesses, drunk on non-viremic blood are also sacrificed. The lot of mosquitoes, sacrificed on the day of OJ and put in the freezer at -80 ° C., constitute the positive controls OJ of gorging, extraction and amplification of the viral RNA. // - Search for viral RNA
• the primers used as well as the probe (for qPCR) were designed for amplification of the conserved region of the 5 'non-coding end of the viral genome.
• the 2CH sense primer (5'-AAC TAC TGT CTC CTC GCA GAA-3 ') (SEQ ID 1), located between nucleotides -289 and -269, and the antisense primer ITS (5' GCG).
• ACC CAA CAC TCG TAC GCT-3 ') (SEQ ID 2), located between nucleotides - 70 and -90.
• the probe used for qPCR is TM416 (5'-6Fam-AAC CCG CTC AAT TGG GG A-Tamra-3 ') (SEQ ID 3) located between nucleotides -137 and -119.
• the RT-PCR was carried out under the following conditions: The retrotranscription is carried out for 30 minutes at 50 ° C., followed by a step of deactivation of the retrotranscriptase (15 minutes at 95 ° C.), then 45 cycles of PCR alternating one step elongation time (1 minute at 63 ° C.) and a denaturation step (30 seconds at 90 ° C.).
• a first series of experiments revealed the presence of viral RNA in Aedes vexans after 21 days of breeding. The results are shown in Figure 1, which shows detectable amounts of the virus at day 21.

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• 2006
  • 2006-12-22 FR FR0655935A patent/FR2910491B1/fr not_active Expired - Fee Related
• 2007
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  • 2007-12-20 JP JP2009542167A patent/JP2010513425A/ja not_active Withdrawn
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