RU2585695C1 - Method of inhibiting infectious activity of ebola virus in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to experimental virology, and can be used to inhibit infectious activity of Ebola virus in experiment. Method involves introduction to Guinea pigs preparation of recombinant human interferon alpha-2 before or during penetration agent in animal's body in daily dose sufficient for developing antiviral agent inhibiting activity. For this purpose, preparation “Reaferon-Lipint” in liposomal form is used in daily dose from 200,000 to 300,000 IU/kg. When simulating prevention and treatment of viral infection this preparation is introduced for 12 and 1 hours to virus infection inoculation of Ebola virus, at moment of infection and through 12, 24, 36, 48, 60, 72 hours after infection of Ebola virus.

EFFECT: method provides achievement of inhibitory effect at reduction of preventive and therapeutic dose of recombinant human interferon alpha-2.

2 cl, 3 tbl, 2 dwg

Description

The invention relates to methods for non-specific prophylaxis and emergency prophylaxis of the Ebola virus and can be used in medicine and experimental virology.

Currently, effective means, methods of treatment and prevention of Ebola, which is a serious, especially dangerous disease with a mortality rate of up to 88%, are absent.

The magnitude of the latest outbreak of the Ebola virus disease in Guinea, as well as concerns about bioterrorism and imported cases of hemorrhagic fevers, again and again testify to the relevance of the search and development of new effective preventive and therapeutic drugs against diseases caused by filoviruses (Bausch D., Sprecher A. , Jeffs B., Boumandouki P. Treatment of Marburg and Ebola hemorrhagic fevers: A strategy for testing new drugs and vaccines under outbreak conditions / Antiviral Research 78 (2008) 150-161). All this necessitates the intensive development of studies to understand the pathogenesis of hemorrhagic fevers, their epidemiology, and measures to prevent outbreaks of this group of diseases. However, despite many years of efforts, there are currently no clinically tested vaccines or specific antiviral drugs against hemorrhagic fever caused by the Ebola virus (HLVEV).

In a small number of cases, the effectiveness of treating patients with serum or blood preparations of convalescents was shown (Mupapa, K., Mukundu, W., Bwaka, M.A., Kipasa, M., De Roo, A., Kuvula, K., Kibadi, K., Massamba, M., Ndaberey, D., Colebunders, R., Muyembe-Tamfum, JJ, 1999b. Ebola hemorrhagic fever and pregnancy. J. Infect. Dis. 179 (Suppl. 1), S11-S12 )

It is known to use the blood serum of people who have had Ebola fever in combination with large doses of interferon for treating a patient with Ebola fever that has become infected as a result of a random injection of a finger (Emond RTD, Evans B., Bower ETW et al. // Brit. Med.J. - 1977, vol. 2. N 6086, p. 541-544).

However, this treatment strategy is limited by the number of convalescents themselves and the need to control sera for contamination with hepatitis viruses, HIV, etc.

It is known to use a preparation containing immunoglobulin against Ebola, having neutralizing activity against Ebola virus and used for emergency prevention of Ebola fever (RF patent No. 2130318, IPC A61K 39/42, published on 05/20/1999). The drug is a 9-11% solution of immunoglobulins of hyperimmune blood serum of a horse containing antibodies to the Ebola virus and obtained after 3 or more cycles of immunization of the animal with the native Ebola virus, containing gamma-globulin fraction in an amount of from 0.081 to 0.110 g / ml of the drug, which corresponds to at least 90% of the total protein content, characterized by a virus neutralizing antibody titer of at least 1: 4096, having a pH value of 7.0-7.5, containing albumin, alpha and beta globulin fractions as non-specific impurities in an amount of not more than 10% residual ethyl alcohol in an amount of not more than 4%.

However, the above analogues have insufficient efficiency, high cost of the used immune serum.

Based on the accumulated clinical and laboratory data, it was suggested that if filoviruses are able to suppress the production of endogenous type 1 interferon during the implementation of their propagation strategy, then the administration of exogenous interferon a should be very effective (Kudoyarova-Zubavichene NM, Sergeyev NN, Chepurnov AA, Netesov SV, 1999. Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections. J. Infect. Dis. 179 (Suppl. 1), S218-S223). However, in experiments using various animal models, interferon has shown unequal efficacy. Thus, in the model of white mice infected with the adapted Ebola virus Zaire strain, the effectiveness of interferon α was highest when administered no later than the 2nd day after infection and when administered daily for 5-7 days (Bray M., Paragas J. Experimental therapy of filovirus infections Antiviral Research 54 (2002) 1-17).

A known method for the prevention of Ebola infection in monkeys infected by intramuscular injection, which includes the introduction of human interferon (intramuscularly) 2 days before infection or 1 hour after infection, followed by continued prophylactic treatment (Bowen ETW Baskerville A. // Ebola virus haemorragic fever. Ed. by Pattyn SR North Holland, Amsterdam, NY. Elsevier, 1978, p. 245 251).

However, as a result of these experiments, there were no differences in the clinic and disease outcome between the experimental and control groups of monkeys.

The closest analogue (prototype) is a method for the prevention of an aerogenic infection of the Ebola virus, including the intramuscular injection of a drug containing interferon into the body of guinea pigs before or during the possible exposure of the pathogen to the body by an aerogenous route (RF patent No. 2105565, IPC A61K 38/21 , published on 02.27.1998). Simultaneously with the intramuscular injection of an interferon preparation into the body, intranasal administration of the same preparation is additionally carried out in the same doses, and reaferon is used as a preparation containing recombinant human interferon alpha-2, which is introduced into the body no more than twice a day in total doses: reaferon (1.0-100.0) × 10 ⁶ IU / kg.

However, to achieve inhibition of the Ebola virus, high doses of reaferon (up to 100 × 10 ⁶ IU / kg) are required, which has a toxic effect on the body of guinea pigs.

The technical result is to reduce the prophylactic and therapeutic dose of recombinant human interferon alpha-2 to achieve an inhibitory effect in the prevention and early stages of infection.

The specified technical result is achieved by the fact that in the method of inhibiting the infectious activity of the Ebola virus in an experiment on guinea pigs, comprising administering to the human body a preparation of recombinant human interferon alpha-2 before the start or during the period when the pathogen enters the animal’s body in a daily dose sufficient for the antiviral to manifest a preparation of inhibitory activity, according to the invention, as a preparation of recombinant human interferon alpha-2, the drug in liposome form is used Reaferon-Lipint in a daily dose of 200,000 to 300,000 IU / kg. When modeling the prophylaxis and treatment of a viral infection, the Reaferon-Lipint drug in the liposomal form is administered 12 and 1 hour before infection with the Ebola virus, at the time of infection (within 1 hour after infection) and 12, 24, 36, 48, 60, 72 hours after infection, and when modeling the treatment of viral infection, the drug is administered at the time of infection (within 1 hour after infection) and 12, 24, 36, 48, 60, 72 hours after infection with the Ebola virus. To assess the inhibitory effect, the animals were infected intraperitoneally with Ebola virus in a volume of 0.5 ml with a dose of 10-30 MID ₅₀ .

The invention is illustrated by the following graphic materials.

In FIG. Figure 1 shows a graph of the accumulation of Ebola virus in the plasma of guinea pigs at various times. For group 1, the preventive and therapeutic regimen of the drug was used; for group 2, only the therapeutic regimen was used. The control group did not receive the drug. In FIG. Figure 2 shows a graph of the accumulation of the Ebola virus in the liver of guinea pigs at various times. For group 1, the preventive and therapeutic regimen of the drug was used; for group 2, only the therapeutic regimen was used. The control group did not receive the drug.

All infectious work with the virus was carried out on the basis of the laboratory of the collection of viruses of the I-th pathogenicity group of the Federal State Budget Scientific Center of the World Bank “Vector”. The laboratory has a license and a sanitary-epidemiological permit for work with this viral agent. The work was carried out in accordance with WHO recommendations and the requirements of the Sanitary Rules for working with pathogens of I-II pathogenicity groups with the required level of biosafety - BSL4. Virus. Ebola virus strain MS 8 was obtained by eight serial passages of the strain Zaire 76 (Maynga) of the Ebola virus in guinea pigs. The strain MS 8 is deposited in the State collection of the Federal State Budgetary Institution Scientific Center of the World Bank "Vector".

Preparations. Reaferon-Lipint (liposomal form of interferon) in the form of a pharmaceutical dosage form - capsules, activity - 500,000 IU / ml. Producer of CJSC Vector-Medica. Reaferon-Lipint and Lipint-Placebo were obtained directly from the manufacturer.

Before use, the drug was diluted in distilled water to the desired concentration.

Animals. Guinea pigs free of pathogenic microflora weighing 200-250 g were obtained from the laboratory animal nursery FBUN SSC VB "Vector" (Koltsovo, Russia), for this study.

They were kept on a standard diet. Animal studies were conducted in a certified laboratory with a BSL4 biological safety level.

Infection and treatment of animals. To determine the infectious dose of MID ₅₀ , guinea pigs were infected i.v. in a volume of 0.5 ml with a series of 10-fold dilutions of the virus. The number of doses tested was 5 and 8 pigs were taken for each dose of infection. On the 6th day of the delivery, blood was taken under aseptic conditions, then the animals were euthanized with chloroform, an autopsy was performed, and the lungs were taken. The lungs were weighed, homogenized, and a 10% suspension was prepared on DMEM supplemented with antibiotics. Centrifuged at 5 thousand rpm for 10 minutes to get rid of cell debris of lung tissue. 2 aliquots of each sample were stored at -80 ° C. The presence of virus in the lungs of mice was determined by titration on a Vero cell culture, as described above. The study of the antiviral effect of the drug in vivo was carried out in several groups of animals. The drug was administered both as a prophylactic and therapeutic regimen. In this study, animals were challenged with i.v. br in a volume of 0.5 ml with a dose of 10-30 MID ₅₀ .

1st group. Prevention and treatment. The drug is administered 12 and 1 hour before infection with the virus, at the time of infection (within 1 hour after infection) after 12, 24, 36, 48, 60, 72 hours p / s.

2nd group. Treatment. The drug is administered at the time of infection (within 1 hour after infection) after 12, 24, 36, 48, 60, 72 hours p / s.

3rd group. Control group without infection (herd control). The drug (saline solution) is administered 12 hours and 1 hour before the start of the experiment and 0, 12, 24, 36, 48, 60, 72 after the start of the experiment without infection.

4th group. Control group with infection. The drug "Lipint-Placebo" is administered 12 hours and 1 hour before infection and after 0, 12, 24, 36, 48, 60, 72 hours p / s.

In all groups, daily, starting from the 2nd day and the 6th day inclusively, in 3 pigs, blood and lungs were taken to determine the concentration of the virus.

Control of the used infectious dose in this experiment was carried out using 20 pigs.

The concentration of the virus in plasma and lungs was determined by titration on a culture of Vero cells, as described above. Used 10-fold dilutions from 10 ^-1 to 10 ^{-6 in} at least 4 repetitions for each dilution of the sample. The concentration was estimated at a ₅₀ / ml CPD.

The antiviral effect of the drug was assessed by the degree of suppression of reproduction of the Ebola virus on days 2, 3, 4, 5, and 6 p / s.

Data analysis. The value of a 50% infectious dose (ID ₅₀ ) of Ebola virus for guinea pigs with iv infection and 50% tissue cytopathic infectious dose (CPD ₅₀ ) was calculated by the Reed-Mench method and expressed as the average lg CPD ₅₀ / ml ± standard deviation (SD) . The determination of the significance of differences in the values of virus titers between the experimental groups and / or control was carried out using the Student criterion.

Study of the antiviral effect of Reaferon-Lipint (liposomal form of interferon) in in vivo experiments on guinea pigs

To determine the value of the infectious dose (ID ₅₀ ), five groups of animals of 8 guinea pigs each were infected with a series of 10-fold dilutions. The volume of the inoculum is 0.5 ml. The infectious titer of the working runoff of the virus-containing suspension, determined on a Vero cell culture, was 4.8 lg CPD ₅₀ / ml. The experimental results are presented in table. one.

To determine the ID ₅₀ , the Spearman-Kerber formula was used [26]. As a result of the calculation, the value obtained is Lg ID ₅₀ = -2.63 ± 0.25. Knowing the BCC infectious titer on a Vero cell culture, the values of the infectious dose (ID ₅₀ ) for i / v infection of guinea pigs can be expressed in the CPD ₅₀ , i.e. 1 ID ₅₀ is approximately 74 CPD ₅₀ . Value infecting dose of 30 ID ₅₀ / animal, which we intend to use in the study of the antiviral activity "IFN-Lipint" preparation in experiments in vivo, will correspond to CPE 2224 ₅₀ 10 ^3,35 or CPD _50.

As can be seen from the table. 1, 0.5 ml of 10 ^-1 dilution of the working flow of the virus will correspond approximately to a dose of infection of 30 ID ₅₀ / animal. This breeding was used to infect guinea pigs when studying the antiviral activity of Reaferon-Lipint.

The prophylactic and / or therapeutic administration schedule and the time of administration of the drug have been described in detail above. The preparations were administered orally to animals in a volume of 1 ml / animal. The dose of Reaferon-Lipint was 50,000 IU / animal (200,000-300,000 IU / kg).

Animals from each group were kept in separate cages with a lid equipped with a HEPA filter to avoid postexposure transmission of the virus.

The concentration of the virus in the blood and liver of pigs was determined by titration on a culture of Vero cells. The experimental results are presented in table. 2 and 3, FIG. 1 and 2.

The results presented in table. 2, 3 and FIG. 1 and 2 show that Reaferon-Lipint has the ability to effectively delay and inhibit the replication of the Ebola virus in guinea pigs.

In the prophylactic-treatment regimen, the virus is detected two days later than in the group with control animals (placebo). On the 4th day after infection, the viral load (titer) in this group is 100 times lower than in the group with control animals (placebo). On the next day, this trend persists, but to a lesser extent - a decrease in the titer of the virus by about 10 times. This allows you to make a good predictive assessment. It is possible that animals from the group "prevention-treatment" could survive.

It should be noted that in the “treatment” group, the infection develops 1 day later, and on the next day of the disease, a lower viral load is observed in animals than in the group with control animals. But starting from the 3rd day there are no differences in viral load compared with the control group.

The absence of the virus in the control group without infection indicates that when animals are kept in the infectious vivarium, there is no uncontrolled transmission of the virus and there is no “re-infection" of animals.

Thus, in vivo experiments on guinea pigs with an adapted Ebola virus MS 8 strain showed that Reaferon-Lipint (a liposomal form of interferon) has the ability to effectively delay and suppress Ebola virus replication in experimental animals. In the prophylactic-treatment regimen, the virus is detected two days later than in the group with control animals (placebo). On the 4th day after infection, the viral load (titer) in this group is 100 times lower than in the group with control animals (placebo). On the next day, this trend persists, but to a lesser extent - a decrease in the titer of the virus by about 10 times. This allows you to make a good predictive assessment.

Effective treatment of Ebola may require a combination of drugs that will inhibit virus replication in target cells (monocytes / macrophage-like cells), thereby reducing pathological effects, such as coagulopathy.

The use of Reaferon-Lipint (a liposomal form of interferon) in the early stages of infection or for prophylactic purposes in contact with sick people (with confirmed close contact) can prevent the disease or improve the clinical outcome. In addition, a reduction in viral load is likely to reduce environmental contamination and thus reduce the risk of secondary transmission.

Claims (2)

1. A method of inhibiting the infectious activity of the Ebola virus in an experiment on guinea pigs, comprising administering to the human body a recombinant human interferon alpha-2 preparation prior to or during the period when the pathogen enters the animal’s body in a daily dose sufficient for the antiviral drug to exhibit inhibitory activity, characterized in that as a preparation of recombinant human interferon alpha-2, the drug in the liposomal Reaferon-Lipint form is used in a daily dose of 200,000 to 300,000 IU / kg, and when modeling the prevention and treatment of viral infection, the Reaferon-Lipint drug in the liposomal form is administered 12 and 1 hour before infection with the Ebola virus, at the time of infection and 12, 24, 36, 48, 60, 72 hours after infection with the Ebola virus. 2. The method according to p. 1, characterized in that the animals are infected with Ebola virus intraperitoneally in a volume of 0.5 ml with a dose of 10-30 MID ₅₀ .

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