RU2565543C2 - Method of obtaining immunoenzymatic test system for determination of epitopes of envelope protein of puumala virus with protective orientation in vaccine preparations against haemorrhagic fever with renal syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed invention relates to field of biotechnology and deals with method of obtaining immunoenzymatic test-system for determination of epitopes of envelope protein of Puumala virus with protective orientation in vaccine preparations against haemorrhagic fever with renal syndrome (HFRS). Characterised method includes obtaining murine monoclonal antibodies, possessing neutralising activity with respect to Puumala virus; sorption of said antibodies on immune panel; separation of IgG from blood serum of HFRS reconvalescents with high titres of specific antibodies to Puumala virus; preparation of conjugate of specific IgG with horse radish peroxidise.

EFFECT: claimed invention makes it possible to detect envelope protein of both live and inactivated Puumala virus with similar accuracy, which serves for control of content of said virus, which is a causative agent of haemorrhagic fever with renal syndrome, in vaccine preparation.

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Description

The invention relates to the field of biotechnology, in particular to obtaining a vaccine for the prevention of hemorrhagic fever with renal syndrome (HFRS), and in particular, to a method for producing test systems designed to control the content of specific viral proteins in the vaccine preparation - inducers of the immune response against hantaviruses - pathogens HFRS.

Hemorrhagic fever with renal syndrome is an acute, viral disease of zoonotic nature, characterized by systemic lesions of small blood vessels, hemorrhagic diathesis and a peculiar kidney damage, high morbidity, severe course (often fatal) and persistent disability. The absence of specific treatment and prophylaxis agents determines the high social and medical significance of HFRS in Russia and in many countries of the world. Cases of HFRS disease were detected in 57 administrative regions of the Russian Federation and make up 8-10 thousand patients annually, with 98% of the incidence recorded in the European part of Russia, of which 98% are associated with the Puumala virus.

The most effective method of fighting HFRS is vaccination, which has been demonstrated in China and South Korea. Currently, commercial HFRS vaccines are produced only in China, however, none of these vaccines can be used in the European regions of Russia, as it does not provide protection against the Puumala virus, the causative agent of HFRS in these regions.

For the manufacture and control of the vaccine against the Puumala virus, it is necessary to solve the problem of developing effective methods for controlling the content of specific antigens - inducers of the immune response in vaccine preparations, including at the technological stages of their production. One of the methods for the immunochemical determination of viral proteins - antigens is an enzyme-linked immunosorbent assay.

Currently, FSUE PIPVE named after MP Chumakova produces the Khantagnost immunoenzyme test system, with which it is possible to detect antibodies to various epitopes of the nucleocapsid protein. However, protective proteins are of greatest importance in the design of the vaccine preparation, in this case the surface protein G _N : G _C , certain epitopes of which induce the production of virus-neutralizing antibodies.

The aim of the invention is the construction of an enzyme-linked immunosorbent assay system for determining the enveloped protein of the Puumala virus, which is an inducer of the humoral immune response associated with the virus-neutralizing properties of antibodies.

As the basis of this method, murine monoclonal antibodies having neutralizing activity against the Puumala virus were prepared.

The principle of action of the invention is based on the specific immunochemical interaction of monoclonal antibodies with neutralizing activity with the corresponding epitopes of the enveloped protein protein of Puumala virus, polyprotein G _N : G _C. Visualization of the antigen bound to monoclonal antibodies is carried out using horseradish peroxidase-labeled highly avid IgG to Puumala virus isolated from serum HFRS convalescence.

Briefly, the process of constructing the KHANTA-PUU immunoenzyme test system includes: obtaining purified monoclonal antibodies (mAbs) to the Puumala hantavirus, selection of antibody clones with virus-neutralizing activity, production and purification of neutralizing mAbs, isolation of IgG from serum HFRS convalescents with high titers of specific antibodies to the PUU virus and preparation of the conjugate with horseradish peroxidase (hereinafter referred to as peroxidase conjugate); complete set of test system.

In more detail, obtaining a test system is as follows.

I. Obtaining monoclonal antibodies. BALB / c mice are immunized with a purified preparation of inactivated Puumala virus (strain K-27 / Ufa-85) at a dose of 100 μg / mouse, immunization is carried out in the pads of the hind legs twice with a two-week interval. Cells of the popliteal lymph nodes of immune mice are hybridized with murine myeloma cells of the SP2 / 0 line according to the standard procedure using polyethylene glycol 4000. Hybridoma cells producing specific antibodies are subjected to 2-4-fold cloning by the method of limiting dilutions. Clones of hybridomas with stable antibody production are produced in culture in DMEM (HyClone) supplemented with 5% fetal calf serum. To generate antibodies, hybridoma cells are injected into the peritoneal cavity of mice; after 7-14 days, the development of ascites tumors is recorded.

Ascitic fluids are checked for the content of specific antibodies by enzyme-linked immunosorbent assay. For this, samples containing purified hantavirus antigens at a concentration of 100 ng / ml in phosphate-buffered saline (PBS) are added to the immunopanel wells (Costar, 3018), followed by incubation at 6 ± 2 ° C for 18 hours. Next, 3% bovine serum albumin (BSA) (Sigma, 8159) was added to the wells and the panels were left for 1 h at room temperature (22 ± 3 ° C). After washing the panel (hereinafter: 5 times for 5 minutes with a solution of 0.01 M PBS + 0.05% Tween-20), hybridoma culture fluid is added to the wells. After 1 h of contact at 37 ° C, the wells are washed and peroxidase-labeled with horseradish IgG against mouse immunoglobulins (Sigma, A4416). As an indicator system use a substrate-indicator mixture (TMB, Sigma, T8665). Antibodies (IgG) are isolated from ascites liquids by affinity chromatography on a HiTeap ProteinG column (GE Healthcare, 17-0405-03). Purified monoclonal antibodies are standardized for protein (up to 1 mg / ml) and then their specific activity is determined by neutralization.

II. The selection of clones that produce neutralizing antibodies to the virus is carried out by the method of neutralization reaction (PH). Samples of monoclonal antibodies, as well as control sera in 2-fold dilutions, are mixed in equal volumes with a culture liquid containing 50-100 IU of the viruses of Puumala, Dobrava, Hantaan and Seoul. After incubation at 6 ± 2 ° C for 18-20 hours, 0.2 ml virus-serum mixtures are added to a monolayer of VERO-E6 cells grown in 24 well panels (Costar, 3524) and left to contact at 37 ° C within 1 hour, after which the inoculum is removed and methylcellulose coating is applied. The surviving virus is determined by the method of indicating FOE. The titer of virus-neutralizing antibodies in the sample is determined as the reciprocal of its dilution, which suppresses 80% of the CFU. As a result of the studies, the PUU / G10 clone was selected, which is characterized by the ability to neutralize 50-80 IU of Puumala virus at a concentration of ≥1 μg / ml in the absence of neutralizing activity against other hantaviruses at a concentration of 1000 μg / ml.

Table 1 The neutralizing activity of mAbs obtained for the Puumala virus mat Subiso type MFA with antigens PH PUU KHAN SEOUL ADD Vero-e6 PUU KHAN SEOUL ADD PUU / G10 IgG2a 2048 * <64 <64 <64 <64 1280 <20 <20 <20 PUU / D21 IgG1 4096 1024 4096 4096 <64 <20 <20 <20 <20 PUU / H6 IgG1 4096 4096 4096 4096 <64 <20 <20 <20 <20 Control polyclonal sera PUU number 421 8192 256 512 512 <64 2560 <20 <20 <20 HTN No. 6851 512 4096 2048 4096 <64 <20 1280 40 <20 SEOUL No. 11 256 512 1024 512 <64 <20 40 640 <20 ADD No. 68 256 2048 1024 4096 <64 <20 80 40 2560 Note: * - antibody titers, expressed in terms of the reciprocal dilution of the sample

III. Conjugation of immunoglobulins from serum of the convalescence of HFRS-PUU with horseradish peroxidase.

Dissolve 5 mg of horseradish peroxidase with RZ 3.0 in 1 ml of 0.3 M bicarbonate solution and after complete dissolution of peroxidase add 0.025 ml of 0.32% formaldehyde solution (1: 100). Gently stirred on a magnetic stirrer for 30 minutes at a temperature of 15 to 25 ° C, then 1 ml of a 0.04 M sodium periodate solution was added to the mixture. After 30 minutes of mixing, the color of the mixture (brown) takes on a greenish tint. Add 1 ml of 0.16 M ethylene glycol solution, mix for 1 hour. The mixture is dialyzed against 1 l of 0.01 M carbonate-bicarbonate buffer solution (KBB) for 16-20 hours at a temperature of (6 ± 2) ° C. In parallel, dialysis of 1.5 ml of IgG PUC at a concentration of 10 mg / ml under the same conditions. After dialysis, activated peroxidase is transferred into a vial, 1 ml of IgG PUC dialyzed against CBB is added, the mixture is gently stirred at a temperature of 18-25 ° C for 2 hours. Add 5 mg NaBH4 and leave for 2 hours at a temperature of (6 ± 2) ° C. The resulting conjugate is dialyzed against 1 l of 0.01 M FSB for 16-20 hours at a temperature of 18-25 ° C. After dialysis, the conjugate is removed from the bag and applied to a column (1.6 × 90 cm) with Sephadex G-100 balanced with 1M FSB. Elute with the same buffer (free elution rate) at a temperature of (6 ± 2) ° C, collecting 3 ml fractions. The colored fractions are photometri at 280 nm and 403 nm. The ratio of the optical density (OD) of each fraction at 403 nm to 280 nm is determined (RZ = OD 403 / OD280) and 1-2 peak fractions with RZ 0.45-0.6 (optimum 0.5) are selected. The conjugate is diluted 1:10 in a 1% solution of bovine serum albumin to 0.1 M FSB. For storage in liquid form, dilute 1: 2 in glycerol and pour into insulin vials.

The enzyme immunoassay system "HANTA-PUU", designed according to the proposed method, has the following advantages:

1) species specificity, manifested in the fact that the “HANTA-PUU” test system exclusively detects the Puumala virus;

2) antigenic specificity, manifested in the fact that the HANTA-PUU test system exclusively identifies epitopes of a protein with protective activity, in particular, the ability to activate the humoral immunity, inducing the production of neutralizing antibodies;

3) antigens of both live and formalin inactivated virus are detected with equal sensitivity.

The fundamental difference between the proposed test system "HANTA-PUU" from the enzyme immunoassay system "Khantagnost" produced by FSUE PIPVE named after MP Chumakova according to TU 9388-016-01895045-2010, is the ability to detect exclusively epitopes of the surface protein of the Puumala virus, which are an inducer of neutralizing antibodies. The determining technological solution to the problem is to obtain specific virus-neutralizing mAbs. An additional control of the specificity of detecting Puumala virus antigens is the use of immunoglobulins from serum HFRS-PUU as blood serum (secondary) antibodies to this virus. The HANTA-PUU test system allows identifying antigens of both live and formalin inactivated viruses with the same sensitivity, which is important from the point of view of assessing the specific activity of inactivated vaccine preparations at the technological stages of their production.

Example 1

To determine the protective protein of the Puumala virus in vaccine semi-finished samples (concentrate of virus-containing culture fluid, purified concentrate, formalin inactivated concentrate), the HANTA-PUU test system is used. Into the immunopanel wells, MAT PUU / G10 was added at a concentration of 1 μg / ml in KBB. After contact for 12-18 hours at a temperature of (6 ± 2) ° C, 3% BSA is added to the wells and the panels are left for 1 h at room temperature. In the auxiliary panel, double dilutions of the test samples on the FSB are prepared. After washing the panel (as described above), the test samples are introduced into the wells. After 2 hours of contact at 37 ° C, the wells are washed, the peroxidase conjugate is added and the panels are incubated for 1 hour at 37 ° C. After washing, TMB is introduced into the wells of the panel (Sigma, T8665) and after 20 min the experiment is measured by determining the optical density in the panel wells using a spectrophotometer at a wavelength of 450. The final dilution of the sample for which the optical density ratio in the well is specific immunoglobulin exceeds the optical density with a normal immunoglobulin of 2.1.

table 2 Determination of the surface (G _N : G _C ) Puumal virus antigen using the HANTA-PUU enzyme-linked immunosorbent assay Virus Total Lowry Protein in μg / ml Virus titer in lg CFU / ml Antigen titer before formalin is added formalin inactivated PUU-K * 10800 7.2 8192 8192 PUF F-1 ** 45 5,6 1024 1024 PUF F-2 63 6.2 4096 4096 PUF F-3 94 5,4 1024 1024 PUF F-4 111 3.8 128 128 ADD F-2 89 6.8 <64 <64 XTH F-2 92 7.2 <64 <64 SEOUL F-2 69 6.1 <64 <64 Vero F-2 120 0 <64 <64 Note. * - crude concentrate of virus-containing culture fluid; ** - purified by gel filtration fractions of concentrates of culture fluid

In order to determine the sensitivity of the method, data on the content of live virus in the samples are presented. To control the specificity of the KHANTA-PUU test system, we used a VeroE6 cell culture fluid concentrate, which is a substrate for virus propagation, as well as peak fractions of purified virus concentrates DOB, CEU, and CTN on the target protein. The results are shown in table 1, indicate:

1) the enzyme immunoassay system “HANTA-PUU” identifies exclusively the antigens of the Puumala virus in titers proportional to the titer of the virus and does not detect the antigens of the viruses Dobrava, Khantaan and Seoul;

2) inactivation of the virus with formalin (0.25%) does not affect the sensitivity and specificity of the determination of antigens of enveloped protein G _N : G _C of the Puumala virus.

The closest analogue

Hantavirus Antigens RU 200010888 5 IPC G01N 33/53 1, A61K 39/12 Research Institute of Epidemiology and Microbiology SB RAMS, authors Kushnareva T.V., Slonova R.A., Kompanets G.G., Application: 2000108885/13, 04/10/2002, publication date: 01/27/2002 METHOD FOR OBTAINING DIAGNOSTIC MA HANTAVIR
OWL Hantavirus Antigens RU 2180754, IPC G01N 33/53 1, A61K 39/12 Applicant Research Institute of Epidemiology and Microbiology SB RAMS, authors: Kushnareva T.V., Slonova R.A., Kompanets G.G., Application: Patentee: Research Institute of Epidemiology and Microbiology SB RAMS, application: 2000108885/13 , 10.04.2000, Published: 03/20/2002 METHOD FOR OBTAINING DIAGNOSIS OF MA HANTAVIRUSES Patent expiration date: 04/10/2000 Hantavirus Antigens RU 99103436, IPC G01N 33/56 9 Applicant Bashkir State Medical University, NGO "Bashbiomed", authors: Batyrshin R.A., Plechev V.V., Timerbulatov V.M. application: 99103436/14, 03/01/1999, date of publication of the application: 02/20/2001 DIAGNOSIS METHOD FOR HEMORRHAGIC FEVER WITH RENAL SYNDROME Hantavirus Antigens RU 2159439, IPC G01N 33/569 Applicant Bashkir State Medical University, NGO "Bashbiomed", authors: Batyrshin R.A., Plechev V.V., Timerbulatov V.M. Application: 99103436/14, 03/01/1999 Published: 11/20/2000 DIAGNOSIS METHOD FOR HEMORRHAGIC FEVER WITH RENAL SYNDROME Date of commencement of the term of validity of the patent: 03/01/1999 Search subject Name of source of information Author, company (holder) of technical documentation Year, place and authority of the publication (approval, deposit of the source) one 2 3 four Hantavirus Antigens GPF Research Institute of Epidemiology Date of publication: 01/27/2002

and microbiology SB RAMS, authors Kushnareva T.V., Slonova R.A., Kompanets G.G. Hantavirus Antigens GPF Research Institute of Epidemiology and Microbiology SB RAMS, authors Kushnareva T.V., Slonova R.A., Kompanets G.G. Date of commencement of the term of validity of the patent: 10.04.2000 Hantavirus Antigens GPF Bashkir State Medical University, NPO "Bashbiomed", authors: Batyrshin R.A., Plechev V.V., Timerbulatov V.M. Application publication date: 02/20/2001 Hantavirus Antigens GPF Bashkir State Medical University, NPO "Bashbiomed", authors: Batyrshin R.A., Plechev V.V., Timerbulatov V.M. Date of commencement of the term of validity of the patent: 03/01/1999 Hantavirus Antigens GPF University of Connecticut Health Center, Srivastava P.K. Date of commencement of the term of validity of the patent: 03/01/1999

Claims (1)

A method for producing an enzyme-linked immunosorbent assay system for the determination of epitopes of enveloped protein of the Puumala virus protective layer in vaccines against hemorrhagic fever with renal syndrome (HFRS), including the production of murine monoclonal antibodies with neutralizing activity against the Puumala virus, their sorption on the surface IgG from serum HFRS convalescents with high titers of specific antibodies to the Puumala virus and preparation of a specific Ig conjugate G with horseradish peroxidase.