RU2743300C1 - Method of human culture rotavirus inactivation - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to the field of pharmaceuticals, namely, to inactivating human cultural rotavirus method. The method consists in the fact that the suspension of human rotavirus, obtained by successive passages on the culture of transplanted animal cells Cell Growth&Differentiation (CG&D), is subjected to freezing-thawing, centrifuged, then the resulting supernatant virus containing liquid is exposed to UV-C radiation with a wavelength of 253.7 nm, reaching a damaging biological dose of 178 J/m2.EFFECT: invention provides 100% inactivation of rotavirus infectious properties, while maintaining its protein structure and immunogenic properties.1 cl, 4 tbl, 3 ex, 2 dwg

Description

The invention relates to medical virology and biotechnology and relates to a method for inactivating a cultured human rotavirus of group A for use in obtaining diagnostic and vaccine preparations.

Rotavirus gastroenteritis is an acute infectious disease widespread in all countries of the world, including the Russian Federation, and causes more than half of all intestinal disorders in children in the first two years of life, therefore, the development of vaccines against rotavirus infection is a priority.

Live rotavirus vaccines are known, but they retain some level of residual pathogenicity, albeit very low, and in children, especially those with defective immune systems, they can cause severe disease. Biological instability during storage can also be a serious problem with live vaccines.

The difficulties encountered in obtaining and using live vaccines can be overcome by using inactivated (killed) rotavirus vaccines. There is practically no risk of infection when vaccinating with an inactivated vaccine. Inactivated vaccines meet the requirements of infectious safety, the absence of post-vaccination consequences ensure the stability of the immunogenic principle, the preservation of the usefulness of antigenic proteins and the chemical purity of the vaccine preparation. In this case, inactivation should be directed to the viral genome and, if possible, not affect the protein framework of the viral particle.

In the development of an inactivated rotavirus vaccine, a method of inactivation of a rotavirus is of great importance, which should ensure irreversible damage to its replicative mechanism while fully preserving the original antigenic structure. Particularly important is the selection of a method for inactivating human rotavirus, which is difficult to cultivate and more sensitive to destruction.

There are chemical and physical ways to inactivate a viral antigen.

In the chemical inactivation of viruses, various chemical preparations are used, including formalin (RU 2078816), β-propiolactone (RU 2537183), ethyleneimine dimer (SU 594771), and others. However, reliable suppression of infectivity is often accompanied by a significant decrease in other types of biological activity, including immunogenicity. In some cases, neutralization of inactivating agents is required. In addition, all inactivating agents, to one degree or another, cause changes both in the nucleic acid and in the protein coat (Sergeev V.A., Sergeev E.A., Nepoklonov V.A., Aliper T.I. and viral vaccines.M .: Biblionika, 2007. - S.206-216, http://bookre.org/reader?file=1483804&pg=207).

Physical methods of inactivation of viral antigens include exposure to high temperatures (warming up) (Zemskov M.V., Sokolov M.I., Zemskov V.M., Fundamentals of general microbiology, virology and immunology. - M., Kolos. - 1972.- p. . 117), gamma rays (Sergeev V.A., Sergeev E.A., Nepoklonov V.A., Aliper T.I. Viruses and viral vaccines. M .: Biblionika, 2007. - P.216-217, http://bookre.org/reader?file=1483804&pg=216).

Heat treatment and exposure to infrared rays on vaccinated liquid do not always lead to complete inactivation of viral pathogens, while partial inactivation of immunogenic proteins also occurs.

Gamma rays have a high penetrating power. Infectiousness of viruses when exposed to gamma rays is lost faster than antigenicity due to various damage to nucleic acids, but at the same time the possibility of damage to the protein envelope remains (Sergeev V.A., Sergeev E.A., Nepoklonov V.A., Aliper T.I. Viruses and Viral Vaccines. 2007. - C.216, http://bookre.org/reader?file=1483804&pg=216).

The physical methods of inactivation of viral antigens also include exposure to ultraviolet rays (Sergeev V.A., Sergeev E.A., Nepoklonov V.A., Aliper T.I. Viruses and viral vaccines. M .: Bibionika, 2007. - S. 217, http://bookre.org/reader?file=1483804&pg=217.

The effect of ultraviolet irradiation on the infectivity of the monkey rotavirus SA11 9 was studied (Smirnov Yu.A. The effect of ultraviolet irradiation on RNA-containing viruses. Abstract of the thesis for the degree of Doctor of Medical Sciences - Moscow, 1993. - P. 4 , 40 http://earthpapers.net/preview/424125/a#?page=1). It was shown that short-wave ultraviolet irradiation (UV) has an irreversible damaging effect on the genomic apparatus and the mildest effect on the SA11 monkey rotavirus capsid proteins without changing the primary and secondary structure of the protein.

The human rotavirus is more sensitive to destructive effects than the monkey. To inactivate it, a milder method of inactivation is required, which should ensure irreversible damage to its replicative mechanism while fully preserving the original antigenic structure.

The aim of the present invention is a method for inactivation of human cultured rotavirus, providing 100% inactivation of the infectious properties of rotavirus, not changing the protein structure and preserving its immunogenic properties.

This goal is achieved by exposure to ultraviolet radiation of a certain wavelength and exposure to group A human rotavirus with preliminary high-speed centrifugation of the rotavirus suspension to remove cellular debris components and ensure the efficiency of viral RNA inactivation.

A suspension of group A human rotavirus is obtained by successive passages on a culture of transplanted cells SPEV (pig embryonic kidney cell culture) according to the generally accepted method (Virology. Methods / ed. B. Meikhi. - M., Mir; 1988. - P.207). A monolayer of SPEV cells is grown on medium 199 with the addition of 10% bovine serum (cattle), washed 3 times with Hanks solution. Cultural human rotavirus of group A in a separate vial is treated with trypsin solution (20 μg / ml). Trypsin-activated human rotavirus is layered on the washed cell monolayer for 10 minutes. The supporting medium is added to the vials to the original volume and kept at 37 ° C for 10 days. The accumulation of viruses in the cell culture is assessed by the cytopathogenic effect (CPE) - the number of destroyed cells of the monolayer to the total initial number of cells, which in percentage terms should be equal to 100% in 24-36 hours from the beginning of infection. Freezing is carried out three times, followed by thawing of the rotavirus suspension and centrifuged at 5000 g for 30 minutes.

The resulting suspension is monitored by an electron microscopic method to confirm the typical morphology of rotavirus particles and by direct counting of particles in 1 ml of culture fluid.

In electron microscopic preparations, icosahedral particles 60-75 nm in size are visible, separate and in the form of clusters, with a structure unique for rotavirus: the outer capsid is in the form of a thin film, the inner one has a pattern similar to the spokes of a wheel and a core of 30-40 nm. The capsomeres forming the inner capsid form a clear structure of penta- and hexomers.

When directly counting rotavirus virions in the preparation, calculated in 1 ml, the number of virions is 10 ⁸ - 10 ⁹ particles.

The rotavirus suspension obtained by the above method contains residual components of cell detritus, which makes it difficult to carry out a complete inactivation of rotaviruses.

To remove cell debris and impurities, the rotavirus suspension is additionally subjected to high-speed centrifugation at 40,000 g for 5 min. For further work, a supernatant fluid containing a virus is used.

The virus-containing liquid is placed in a quartz glass cuvette with a size of 20 mm × 200 mm. The cuvette is sequentially placed at distances from 1 m to 0.01 m from the surface of the lamp of the OBN-75 UHL4 bactericidal irradiator generating a wavelength of 253.7 nm.

Virusosoderzhaschie sample number 28 samples to culture human rotavirus, were exposed to UVB radiation with different doses of 0.04 J / m ² to 355 J / m ^2. The final dose received by each sample with cultural rotavirus was calculated using the formula:

H = E * _{ist efficiency.} * To _{tank .} * To _{absor .} * S _{about . n .} * t , where

H is the radiation dose over time t per unit area;

E is the power of the radiation source W / m ² ,

Efficiency _ist. - the efficiency of the radiation source,

To the _tank. - coefficient of bactericidal flow,

To _absor. - coefficient of absorption of rays by the material of the cuvette,

S _o.p. - the area of the irradiated surface,

t - exposure of the action,

at the same time E (power of the radiation source W / m ² ) changes in accordance with the Law of inverse squares when the distance from the radiation source to the irradiated sample changes (table 1).

The effect of UV irradiation on human rotavirus is tested on SPEV cells using the universal biological property of viruses, the so-called. "Cultural" - to induce CPP on transplanted mammalian cells sensitive to it. An inactivated virus loses this property as a result of damage to the genetic apparatus. A monolayer of transplanted SPEV cells is infected with an irradiated vaccinated liquid and cultured according to the above method. At the same time, control 1 is set - the SPEV cells are infected with a cultural rotavirus using the same technique; control 2 - culture of SPEV cells in a supporting medium, previously washed three times with Hanks solution and grown under the same conditions.

According to the ability or lack of the ability of rotavirus to induce CPP, the completeness of inactivation is estimated, expressed as a percentage of the area of the destroyed monolayer in relation to the original monolayer before infection. The observation period was 10 days. When infected with an inactivated virus, the monolayer is not disturbed, the cells continue to divide until a dense monolayer is formed. The absence of CPP (0% of the destroyed cells of the monolayer of the total number of cells) during the observation period of 36 hours from the beginning of infection and the subsequent observation of the cell culture for 10 days confirms the completeness of the inactivation of the rotavirus and the loss of its reproductive properties in the cell culture. The dynamics of changes in the cultural properties of the irradiated rotavirus is presented in Table 1. To avoid cluttering the table, the tabular material does not include data on intermediate exposure indicators (3 min, 7 min) within each distance group, since these data fit into the general logic of events.

Table 1.

Dependence of the cultural properties of rotavirus in samples on the doses received, depending on the variable irradiation parameters (distance to the radiation source and treatment time).

Distance from the UV source to the cuvette with the virus, m one 0.5 0.25 0.125 0.06 0.03 0.015 0.01 UV exposure, min five ten five ten five ten five ten five ten five ten five ten five ten Received dose, J / m ² 0.03 0.1 0.17 0.35 0.7 1.4 2.8 5.6 11.1 22.4 44.5 89 178 355 712 1423 CPD value,% of the destroyed monolayer area one hundred one hundred one hundred one hundred 80 60 40 thirty 15 ten 3 one 0 0 0 0

It is found that the effect of UV radiation wavelengths of 253.7 nm for the rotavirus in the preparation of a dose of 178 J / m ² (provided by this dose quantities corresponding time and distance) irreversibly deprives human rotavirus ability to multiply in culture transplanted cells and the destruction of the cell monolayer SPEV cells ...

The minimum dose of damaging biological study cultural properties 178 J / m ² calculated as follows (see. Above).

This minimum damaging biological dose physical factor UVC radiation may also be achieved by changes in the parameters: distance from the UV source, exposure time, and should be at least 178 J / m ² (Table 2).

Table 2.

Cultural properties of UFS-treated rotavirus
178 J / m ² , obtained in different ways.

Distance from the UV source to the cuvette with the virus, m 0.06 0.03 0.015 UV exposure, min 79.4 20 five Received dose, J / m ² 178 178 178 The value of the CPD,% of the area of the destroyed monolayer 0 0 0

In order to confirm a persistent violation and the absence of reversion of pathogenic properties in relation to the cell culture of inactivated rotavirus, three consecutive infections of SPEV cells are carried out according to the above technique with inactivated virus and culture (control 1) with control of cell culture in a supporting medium, preliminarily washed with Hanks solution (control 2 ) under the same growing conditions. The results of monitoring the absence of manifestations of infectivity of inactivated human rotaviruses in relation to cell culture are shown in Table 3. The absence of reversal of infectious properties is demonstrated by the example of a rotavirus sample that received a minimum damaging biological dose of 178 J / m ² .

Table 3.

Cytopathogenic effect on SPEV cell culture infected with inactivated and cultured rotavirus

Consecutive
rotavirus infection Cytopathogenic effect on SPEV cell culture
(in %) UV-inactivated rotavirus cultural rotavirus
( control 1 ) Culture of SPEV cells in a supporting medium
( control 2 ) 1 infection 0 one hundred 0 2 infection 0 one hundred 0 3 infection 0 one hundred 0

In successive (1,2,3) infections of the cell culture with an inactivated rotavirus, there are no signs of damage to the cell monolayer. SPEV cells after infection with inactivated rotavirus continue to divide until a dense monolayer is formed during the entire observation period (10 days), as in control 2 (uninfected cell culture), which indicates complete irreversible inactivation of viral particles.

Thus, rotavirus, inactivated with a dose of 178 J / m2 ^of UV radiation, does not exhibit infectious properties on the SPEV cell culture for several infections (does not replicate, does not destroy the cell, does not change the structure of the cell monolayer), which indicates a persistent irreversible loss of reproductive capacity by the rotavirus ...

The ability of a human rotavirus strain inactivated by UV radiation to form neutralizing antibodies in mammals was tested in an experiment on white laboratory mice (9 heads) with 5-fold immunization with an inactivated virus at a dose of 0.2 ml, alternating intramuscular and oral infection, followed by blood sampling from immunized animals. In the control group (5 animals), immunization in the same mode was carried out with the maintenance medium 199 without serum. The level of antibodies to human rotavirus was determined as follows:

- in the reaction of indirect hemagglutination - RNGA - erythrocyte diagnosticum was used to determine the working dose of antigen in AE (agglutinating units);

- in the reaction of inhibition of indirect hemagglutination - RTNGA - the established working dose was determined by the titer of antibodies of the sera of immunized animals (Immunological diagnosis of viral infections / edited by TV Peradze, P. Halonen. - M .: Medicine, 1985. - P. 108; Rotavirus disease surveillance. Methodical recommendations. Approved by Gokomsanepidnadzor of Russia 28.11.1994).

To clarify the quantitative characteristics of the humoral link of the body's immune response to the introduction of the virus, laboratory animals were immunized with three antigenic drugs:

IQR 1 - cultural rotavirus treated with a minimum damaging biological dose of UV radiation of 178 J / m ² , 5 min;

IQR 2 - inactivated cultural rotavirus treated with a dose of 355 J / m ² , 10 min;

IFR 3 - inactivated rotavirus culture treated dose 712 J / m ^2, 5 min.

Each group of animals underwent 5-fold immunization with a dose of 0.2 ml of the corresponding antigen, alternating intramuscular and oral administration, followed by blood sampling. Control group 1 animals were immunized in the same mode with non-inactivated cultural rotavirus. Control group 2 was immunized with maintenance medium 199 without serum.

The immunization results are shown in Table 4.

Table 4.

Average titers of anti-rotavirus antibodies of sera of laboratory animals immunized with inactivated rotavirus, inactivated with different doses of UV radiation, and non-inactivated cultural rotavirus.

Antibody titers to human rotavirus group A
in immunized animals Multiplicity of introduction 1-fold 2x 3x 4x 5x Group immunized with ICR-1 1 / 20-1 / 40 1/80 1 / 80-1 / 160 1 / 160-1 / 320 1/320 Group immunized with ICR-2 1/10 1/10 1/20 1/20 1/20 Group immunized with ICR-3 1/10 1/10 1/10 1/20 1/10 Control group 1 1/40 1/80 1 / 80-1 / 160 1/320 1 / 320-1 / 640 Control group 2 0 0 0 0 0

Established that rotavirus, treated with minimal UV studying biological damaging dose of 178 J / m ² for immunization of laboratory animals is able to form in the body protivorotavirusnye neutralizing antibodies at levels approaching those of noninactivating immunization with rotavirus by culture.

It has been established that immunization of laboratory animals by culture rotavirus treated with high doses of UV radiation (355 J / m ² - 712 J / m ²⁾ cause the absence of stress and transient immune response compared with the immune response to rotavirus with processing the minimum dose that is connected likely with deep destruction of the viral particle, and the possible formation of toxic endogenous proteins.

It is found that the radiation dose of UVB rays 178 J / m ² dose inactivation the culture is Rotavirus group A, which resulted in loss of infectivity of rotavirus against sensitive thereto culture cells with preservation of antigenic and immunogenic properties.

Morphology, genomic identity of inactivated human rotavirus is checked by electrophoresis of the virus RNA and electron microscopy.

RNA electrophoresis. Electrophoresis of RNA strains of rotavirus is carried out in an 8% separating polyacrylamide gel (EF in PAGE) in the Lemli system for 10 hours, the current strength is 11 mA, followed by staining with a 0.2% solution of silver nitrate.

Electron microscopy. Electron microscopy is performed by negative contrasting using 0.5% uranyl acetate. The grids with the preparation are viewed at an instrumental magnification of 85,000 times.

List of figures with brief explanations

FIG. 1. Electropherogram of human rotavirus group A RNA before UV irradiation (A) and after (B). The result of polyacrylamide gel electrophoresis of human rotavirus RNA of group A (EP in PAGE) is presented. The inactivated rotavirus (B), as compared to the cultured one (A), did not change the molecular weight of RNA fragments, the speed of movement of the fragments, and the distribution pattern of 11 RNA fragments of group A rotavirus: 4-2-3-2 was not altered within the limits of strain variability.

FIG. 2. Group A human rotavirus before UV irradiation (A) and after (B). Electron microscopy. Negative contrasting. An increase of 430,000 times. The morphology of the virions of the UV-inactivated rotavirus strain (B) corresponds to the morphology of the rotavirus virions before irradiation (A), belonging to the genus Rotavirus of the Reoviridae family (thin outer capsid, inner capsid in the form of a wheel, core 30-40 nm, capsomeres are arranged in the form of hexomers).

The proposed method inactivates 3 strains of human rotavirus of group A, adapted to growth on cell culture, deposited in the State collection of viruses of the V.I. DI. Ivanovsky "of the Ministry of Health and Social Development of Russia (numbers of GKV depositors No. 2727, 2728, 2729).

Claims (1)

The method of inactivation of human cultural rotavirus, which consists in the fact that a suspension of human rotavirus is obtained by successive passages on the culture of transplanted animal cells with SPEV, threefold freezing-thawing, centrifugation at 5000 g for 30 minutes, then the resulting rotavirus suspension is additionally centrifuged at 40,000 g in within 5 minutes; the obtained supernatant vaccinated liquid is exposed to UV-C radiation with a wavelength of 253.7 nm, reaching a damaging biological dose of 178 J / m ² .

Images