RU2281327C2 - STRAIN OF HYBRID CULTURING Rattus norvegicus 122H9 CELLS WHICH IS PRODUCER OF CROSS-REACTIVE NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST PATHOGENIC FOR HUMAN ORTHOPOXVIRUSES - Google Patents

Abstract

FIELD: biotechnology and virology.

SUBSTANCE: strain of hybrid culturing Rattus norvegicus 122H9 cells is disclosed. Said strain is obtained by fusion of rat myeloma cells 210RC.Y3-Ag1.2.3 with rat spleen cells LOU, immunized with purified ectromelia virus of K-1 strain. Said strain is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses.

EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.

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Description

The invention relates to biotechnology and virology and relates to the production of a new strain of hybrid cells Rattus norvegicus, a producer of cross-reactive neutralizing monoclonal antibodies (MCAs) against orthopoxviruses. MCAs can be used in medicine, immunology and biotechnology with the aim of developing and improving vaccine prevention and therapy for diseases caused by orthopoxviruses pathogenic for humans, such as variola virus (VNO), smallpox monkeys (SBI), smallpox vaccines (WWII) and smallpox cows (Wok).

Foreign collections of murine monoclonal antibodies (MABs) to orthopoxviruses [1-5], produced by strains of hybrid cells of the animal Mus musculus, are known.

However, analogues of strains of hybrid cells of the animal Rattus norvegicus, producers of monoclonal antibodies to orthopoxviruses, are unknown.

The technical result of the claimed invention is to obtain a strain of hybrid cells Rattus norvegicus 122H9, secreting MCAs that have cross-reactivity with CE, BOB, wok and VNO and neutralizing activity against wok and variola virus. The specified technical result is achieved by fusion of rat myeloma cells 210RC.Y3-Ag1.2-3. with rat spleen cells LOU immunized with purified ectromelia virus (strain K-1), which was obtained by culturing and purifying the virus.

The inventive strain of hybrid cells Rattus norvegicus 122H9 obtained at the State scientific center of virology and biotechnology "Vector" (SSC WB "Vector") of the Ministry of Health of the Russian Federation and deposited in the Research Institute of cell cultures SSC WB "VECTOR" under No. NIIMB-281. The copyright name of the hybridoma cell line is 122H9.

The strain of Rattus norvegicus 122H9 is characterized by the following features:

The pedigree of the strain. The hybrid cultured cell strain was obtained by fusion of rat myeloma 210RC.Y3-Ag1.2.3 cells. (Y-3) with LOU rat spleen cells immunized with a purified ectromelia virus preparation. A 50% solution of Sigma polyethylene glycol with a molecular weight of 2000 was used as a merging agent. The strain was grown on GAT selective medium, then it was cloned three times using the limiting dilution method using rat peritoneal macrophages as LOU cells. The yield of positive clones in the last cloning was 100%.

The number of passages at the time of deposit is -12 passages.

Marker signs and methods for their assessment. The strain secretes rat immunoglobulins that specifically interact with EB. Analysis of rat immunoglobulins is carried out by enzyme-linked immunosorbent assay using 200 ng of purified EB as an antigen. The interaction of MCA with VE is detected using a conjugate of antibodies against rat IgG labeled with horseradish peroxidase.

Contamination by bacteria and fungi was not detected.

Morphological signs. The culture consists of large rounded cells, similar in morphology and size to the original parent Y-3 myeloma. The oval core is located eccentrically and occupies a significant part of the cytoplasm.

Cultural properties. The culture medium is Dulbecco's MEM Needle medium containing increased amounts of arginine up to 200 mg / l, folic acid up to 12 mg / l, asparagine up to 36 mg / l, as well as 0.05 mM 2-mercaptoethanol and 20 mM HEPES. The content of fetal serum in the growth medium is 10%. 80 μg / ml gentamicin sulfate is also added to the medium. Hybridoma 122H9 grows as a monolayer suspension culture. A sowing dose of 100-200 thousand cells per milliliter, the sowing rate is 1: 5-1: 6 after 3-4 days.

The cultivation of hybridomas in the body of the animal. Female rats Lou (SSC VB "Vector") are sensitized by intraperitoneal administration of 5 ml of paraffin oil or pristan. After 2-4 weeks, 10 million hybrid cells are injected into the animals. Ascitic tumor forms in 10-12 days. From one animal, 20-50 ml of ascitic fluid can be obtained. Hybridoma is vaccinated in 100% of cases.

The characteristic of a useful product. Typing of hybridoma immunoglobulins was carried out by Ouchterloni immunodiffusion method. MCAs belong to the class of IgG. They specifically interact with the 12.8 kDa protein (214L gene) of ectromelia virus in an immunoblot reaction. The titer of MCA in ascites is at least 1: 2200000 in ELISA with VE. Stable MCA production is maintained for at least 10 passages in vitro. From one milliliter of ascitic fluid, 5-7 mg of purified monoclonal antibodies can be obtained. The strain stably produces MCA for one month of continuous inoculation in culture.

Cryopreservation. Freezing medium: DMEM (M) medium - 50%, fetal serum - 40%, dimethyl sulfoxide - 10%. A cell suspension in a volume of 0.5-1 ml is transferred into plastic cryovials and placed in a foam container with a wall thickness of 1-1.5 cm. The container is introduced into a pair of liquid nitrogen. After a day, the tubes are transferred to liquid nitrogen. Defrosting is carried out by lowering the tubes into water at a temperature of 37-41 ° C. Cells are diluted in 5 ml of DMEM (M) medium and centrifuged at 1000 rpm. The precipitate is resuspended in a growth medium and transferred to culture bottles at a concentration of 200-300 thousand cells per milliliter. The viability after thawing is 60-80% (staining with 0.25% trypan blue).

The method of obtaining the inventive strain

The strain of hybrid cells Rattus norvegicus 122H9 receive as follows.

Female rats LOU, weighing 180-200 g (vivarium GNTSVB "Vector") are immunized according to the scheme that is shown below in table 1.

150 million spleen cells and 50 million Y3 cells are used for fusion. The mixture of cells is centrifuged, the supernatant is thoroughly removed and 0.4 ml of a 50% solution of polyethylene glycol (PEG) with a molecular weight of 2000 is added to the cell pellet. The mixture is centrifuged for 15 minutes at 600g. After 3-5 min of pause, the PEG layer is slowly diluted with a versene solution, after which the precipitate is resuspended and the suspension is centrifuged. Cells are dispensed into five 96-well culture plates at 100 μl per well. Selection of hybrid cells is carried out in a NAT medium consisting of DMEM (M) nutrient medium, to which 15% fetal bovine serum, 0.1 mM hypoxanthine, 0.04 mM thymidine and 0.01 mM aminopterin are added.

Table 1
Immunization schedule Days Antigen Dilution Solution Dose of antigen (mcg / rat) Injection area 0 Freund's complete adjuvant 7.5 Intraperitoneally 7 Incomplete Freund's Adjuvant 7.5 Intraperitoneally 10 Saline 75 Intravenously eleven Saline 75 Intravenously fourteen Hybridization (Spleen Fence)

Specific hybrids are selected by enzyme-linked immunosorbent assay (ELISA), using 200 ng of purified ectromelia virus as antigen. Non-specific binding sites are saturated with a 0.5% casein solution (ICN). Then, 100 μl of the culture medium of the studied hybridomas are transferred to the wells and incubated for 45 min (at 37 ° C). After incubation, the wells are washed 3-5 times with saline containing 0.05% tween-20 (Sigma). 100 μl of antispecific conjugate (rabbit immunoglobulins against rat immunoglobulins labeled with horseradish peroxidase) were added to the tablets and incubated for 45 min at 37 ° C. Then the tablets are washed and an enzymatic staining reaction is carried out. The analysis results are determined on a MULTISCAN spectrophotometer at a wavelength of 492 nm.

The 122H9 hybrid strain was cloned twice by limiting dilution, transferred to mass culture and frozen in liquid nitrogen.

The following examples detail the essence of the invention.

Example 1. Cultivation of a strain of hybrid cells Rattus norvegicus 122H9, secreting MCA against orthopoxviruses in rats LOU.

The cultured 122H9 cells in the logarithmic growth phase are centrifuged sterile for 5-10 minutes at 1000 rpm in an OPN-3 centrifuge. The supernatant is removed and the precipitate is suspended in a sterile Earl or Hanks solution. Preliminarily, rats LOU (GNTSVB "Vector"), weighing 180-200 g, at least 10 days before vaccination of hybridoma cells, are injected intraperitoneally with 5 ml of paraffin oil or pristan. A suspension of cultured cells of producer strain 122H9, prepared as described above, is inoculated intraperitoneally with animals, 10 million cells in 1-3 ml of medium without serum or Earle's solution. 10-14 days after vaccination of hybridoma cells, 15-30 ml of ascitic fluid is formed, which is used to obtain monoclonal antibody preparations.

Example 2. The selection of purified monoclonal antibodies produced by a strain of hybrid cells Rattus norvegicus 122H9, from ascites fluid.

One volume of ascitic fluid containing MCA is diluted with 4 volumes of 0.6 M acetate buffer (0.04 M citric acid, 0.2 M sodium acetate), pH 4.0, and adjusted to pH 4.5 with 0.1 N sodium hydroxide solution. Caprylic acid is added dropwise to the diluted sample, with constant stirring, at the rate of 25 μl per 1 ml of solution and incubated for 30 min at + 4 ° C. Then it is centrifuged for 30 min at 8000 g and the precipitate is removed, and the supernatant is mixed with 10-fold phosphate-buffered saline (PBS) and the pH is adjusted to 7.4 with a solution of 1.0 N sodium hydroxide. An equal volume (V: V) of a saturated solution of ammonium sulfate is added to this solution, shaken and incubated overnight at 4 ° C or 30 min at 20-25 ° C. Centrifuge for 15 min at 5000 g.

The supernatant is drained and the precipitate is resuspended in PBS, pH 7.4. Residues of ammonium sulfate are removed by dialysis against 50-100 volumes of FSB, pH 7.4.

Example 3. Determination by ELISA of the cross-interaction of MAB produced by the Rattus norvegicus 122H9 strain with orthopoxviruses.

ELISA was carried out on polystyrene tablets; the antigen (purified virus) was sorbed in FSB, pH 7.4, in a volume of 100 μl / well. Non-specific binding sites were saturated for 45 minutes at 37 ° C with a 0.5% casein solution in TSB-Tween buffer (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva), pH 7.4). MABs were added to the plates (45 minutes at 37 ° C) and binding of MABs to the antigen was detected with peroxidase-labeled antibodies against rat immunoglobulins. Next, a chromogen, 0.1% O-phenylenediamine, was added in citrate-phosphate buffer (0.2 M citric acid, 0.5 M Na ₂ HPO ₃ , pH 5.0) with 0.03% hydrogen peroxide. The reaction was stopped by adding 100 μl per well of 1N HCl and the optical density of the samples was measured on a Multiscan spectrophotometer (Finland) using a light filter with a maximum transmission of 492 nm. As a negative and positive control, homologous non-immune (normal) and hyperimmune serums were used, respectively.

Example 4. Detection in the immunoblot of the interaction of MCAs produced by the producer strain Rattus norvegicus 122H9 with a 12.8 kDa protein (gene 214L) of ectromelia virus, strain K-1.

Viral proteins after 12% PAAG electrophoresis were transferred to the nitrocellulose membrane (Millipore, USA). Non-specific binding sites were saturated with a 0.5% casein solution in TSB-Tween buffer (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva), pH 7.4) 2 hours at 37 ° C. Then, individual membrane strips were incubated with monoclonal antibodies for 4 hours at 20-22 ° C. The specific binding of antibodies interacting with viral proteins was detected using a conjugate of anti-rat IgG antibodies labeled with horseradish peroxidase. As a negative control, Y3 ascitic fluid in 1/5000 dilution was used, as a positive control, serum to VE in 1/5000 dilution from rats used to obtain hybridomas was used. The results are presented in the drawing, concerning the identification by the method of immunoblot protein VE (K-1), interacting with antibodies of the immune serum against orthopoxviruses and MKA 122N9. Where the numbers represent the following notation:

1 - rat antiserum to VE (K-1) (1/5000), non-reducing conditions;

2 - rat antiserum to VE (K-1) (1/5000), reducing conditions;

3 - MKA 122N9 (1/5000), non-reducing conditions;

4 - MKA 122N9 (1/5000), reducing conditions;

5 - ascitic fluid of rat myeloma Y3 (1/5000);

6 - normal mouse serum (1/5000).

Dilutions of the preparations used are indicated in parentheses. The location of marker proteins 10, 20, 30, 40, 50, 60.70 kDa is indicated on the right, and the proteins of the ectromelia virus are indicated on the left.

Example 5. The study of the neutralizing activity of MCA 122H9 in a Vero cell culture. The neutralization reaction (PH) was used for vaccinia viruses (strain LIVP) and smallpox (strain India Za). Working with smallpox virus was carried out under conditions of maximum protection (BSL-4) using protective suits with positive pressure in a WHO collaborating center, Koltsovo. Novosibirsk region. The nature of the work with variola virus was previously approved by WHO. Virus infectivity was determined by plaque on a Vero cell culture. Virus neutralizing activity of MAB was determined in a monolayer of Vero cell culture, in 96-well plates, using ascitic fluid or purified MAB. Serial two-fold dilutions of the MCA in the support medium (50 μl) were added to the well of the plate containing 100 μl of the supporting medium, after which 1000 PFU of the infectious virus were added in a volume of 50 μl. The initial dilution of the MCA in the hole was 1: 40-1: 50 for WWII and 1: 400-1: 500 for VNO. The plates were incubated for 3 days in a CO ₂ incubator at 37 ° C with BOB and at 35 ° C with smallpox virus. After this, the cells were stained with 0.1% crystalline violet solution with 10% formalin and the number of plaques was taken into account visually. The presence of neutralization was judged by the absence or decrease in the number of plaques. J2D5 monoclonal antibody, kindly provided by Ichihashi Y [4], was used as a positive control, and ascites NSO myeloma was used as a negative control. The results are shown in Table 2. As can be seen from the data presented, MAB 122H9 secreted by the producer strain Rattus norvegicus NIIMB-281 inhibited the reproduction of BOB and VNO in Vero cell culture.

table 2
Determination of the neutralizing activity of MKA 122H9 ICA Target Protein (kDa) Immunogen virus The titer of the MCA in the PH (arr. Values) *. WWII UPO one hundred% ≥50% ≥50% 122H9 12.8 Renewable energy fifty 12000 500 * - in%, suppression of plaque formation is indicated.

The above properties of the Rattus norvegicus 122H9 hybrid cell strain allow us to conclude that for the first time on the basis of rat myeloma, the Rattus norvegicus 122H9 hybridoma, a producer of cross-reactive neutralizing MABs to orthopoxvirus proteins, was obtained. The strain of hybrid cells provides the production of rat IgG class immunoglobulins in the amount of 7.5 mg of purified antibodies from a milliliter of ascitic fluid. The use of rat-derived hybridomas to obtain monoclonal antibodies has its advantages over murine hybridomas in the amount of ascitic fluid produced. It is known that the yield of ascitic fluid from one mouse is from 3 to 5 ml. Using a strain of hybrid cells Rattus norvegicus 122H9 allows you to receive 15-30 ml of ascitic fluid from one animal. Purified MAB 122H9 specifically reacted in ELISA with ectromelia virus (MKA titer was 1: 2200000) and 12.8 kDa VE protein, strain K-1, (gene 214L) was detected in the immunoblot. the obtained MCAs also showed cross-reactivity in ELISA with vaccinia, cowpox and smallpox viruses. In addition, as shown by the results of the neutralization reaction, MKA 122H9 had a virus-neutralizing activity against BOB and VNO (strain India Za), the most pathogenic representative of orthopoxviruses. The above properties of the strain Rattus norvegicus 122H9 distinguish it from all previously described hybridomas producing MAB to proteins of orthopoxviruses.

Information sources

1. Baker O.R., Bray M., Huggins J.W. // Antiviral. Research - 2003 - Vol. 53 - P.13-23.

2. Czemy C.P., Johann S., Holzle L., Meyer H. // Virology - 1994 - Vol.200, N2 - P.764-77.

3. Dallo S, Rodriguez JF, Esteban M.A. // Virology - 1987. - Vol.159, No. 2 - P.423-32.

4. Ichihashi Y., Oie M. // Virology - 1988. - Vol.163, No. 1 - P.133-44.

5. Hooper J.W., Schmaljohn A.I., Schmaljohn C.S. // Prophylactic and therapeutic monoclonal antibodies. - Patent Number: 6, 451, 309 B2 (US)., Sep. 17, 2002.

6. Meyer H, Osterrieder N, Czemy CP. // Virology - 1994 - Vol.200, No. 2 - P.778-83.

Claims (1)

The strain of hybrid cells Rattus norvegicus 122H9, deposited in the Research Institute of cell cultures of the SSC VB "Vector" under No. NIIMB-281, used to obtain cross-reactive neutralizing monoclonal antibodies against orthopoxviruses, pathogenic for humans.