GB2238049A

GB2238049A - Method for preparing inhibitor for influenza virus neuraminidase

Info

Publication number: GB2238049A
Application number: GB8925499A
Authority: GB
Inventors: Arkady Fedorovich Frolov; Anatoly Veniaminovich Shapiro; Svetlana Leontievna Rybalko; Igor Leonidovich Marichev; Jury Petrovich Galaguza
Original assignee: KI NII EPIDEMIOLOGII I INFEKTS
Current assignee: KI NII EPIDEMIOLOGII I INFEKTS
Priority date: 1989-11-22
Filing date: 1989-11-10
Publication date: 1991-05-22
Also published as: DE3938003A1; FR2654623A1; GB8925499D0

Abstract

The method for preparing an inhibitor of influenza virus neuraminidase comprises growing Staphylococcus aureus on a nutrient medium containing sources of carbon, nitrogen, mineral salts, growth stimulants, followed by separation of the culture liquid and isolation and purification of the desired product. The thus-produced inhibitor of influenza virus neuraminidase can be useful for diagnosis of influenza, for studying into antigenous structure of viruses and for the production of vaccines.

Description

- 1 c) fa=:g METHOD FOR PREPARING INHIBITOR FOR INFLUENZA VIRUS

NEURAMINIDASE The present invention relates to the art of medicine and, more particularly, to a method for preparing an inhibitor of influenza virus neuraminidase which can be useful for diagnosis of influenza, research into the antigenous structure of viruses and for the production of vaccines.

Known in the art is a method for preparing an of influenza virus neuraminidase (W. Lin, K.Oishi, "Agricultural and Biological Chemistry", 1975, 39, 759-765).

The method resides in that a producer of an inhibitor of neuraminidase is grown on a nutrient medium containing a source of carbon, a source of nitrogen, mineral salts and growth stimulants. As the producer use is made of actinomyces. The cultural liquid after growing is separated and repeatedly treated with ethanol. The resulting residue is dissolved in a phosphate buffer and dialyzed against a phosphate buffer. The duration of the process of the recovery and purification of the preparation is 12 days without taking into consideration the time required for the preparation and identification of an active inhibitor of neuraminidase. The yield of the desired product is 33 mg per litre of the cultural liquid. The thus-obtained desired inhibitor a Ko Aida No. 3, pp.

product ensures 80% of inhibition of influenza virus neuraminidase of the A2 type.

This method features a long duration of the process, an insufficient activity and a low yield of the produced neuraminidase inhibitor.

It is the main object of the present invention to improve the yield and activity of the obtained neuraminidase inhibitor.

It is another object of the present invention to shorten the process duration.

The main and other objects of the present invention are accomplished by that in a method for preparing an inhibitor of influenza virus neuraminidase on a nutrient medium incorporating sources of carbon, nitrogen, mineral salts and growth stimulants, separation of the cultural liquid, treatment thereof with ethanol, followed by separation and purification of the desired product, according to the present invention, use is made, as the producer of the inhibitor of neuraminidase, of a microorganism of the species Staphylococcus aureus.

The novel producer of the species Staphylococcus has a high antineuraminidase activity.

The method according to the present invention is simple, features a high yield and a high activity of the desired product. The resulting inhibitor of influenza virus neuraminidase ensures a 100% inhibition thereof and it is by 10 times more active that the inhibitor produced by the known method. The resulting inhibitor is heat-resistant and does not change its activity upon the effect of acids and alkalis.

The inhibitor according to the present invention increases reproduction of influenza viruses which can be used for acceleration of diagnosis and improvement of accuracy thereof, as well as for a fast preparation of vaccines against circulating strains of influenza viruses 1 - 3 and a monovaccine based thereon for prophylaxis of influenza viruses.

In order to simplify the process, it is advisable to carry out purification by the method of gel-filtration. The method according to the present invention makes it possible to shorten the duration of the process for the production of an inhibitor of neuraminidase.

The method according to the present invention is performed in the following manner- As the producer of an inhibitor of influenza virus neuraminidase use is made of any known strain StalDhylococcus aureus. The producer is cultured on a nutrient medium incorporating sources of carbon, nitrogen, mineral salts and growth stimulants. The starting strain Staphylococcus aureus is grown, for accumulation of the inoculation culture, in 5 ml of the nutrient medium for 24 hours and then in 100 ml of the nutrient medium for additional 24 hours. The thus-obtained inoculation culture is introduced into an enzymatic nutrient medium and is grown therein for 24 hours under continuous stirring. Then the cultural liquid is separated and treated with ethanol. The precipitate is separated and the supernatant liquid is discarded. The precipitate is washed with ethanol, then dissolved in a phosphate buffer and again precipitated with ethanol. The resulting precipitate is dissolved, the solution is heated and the formed residue is removed.

The desired product is obtained from the supernatant liquid by purification thereof by gel-filtration through Sephadex, followed by lyophilic drying.

The duration of the recovery of the desired product by the method according to the present invention is 5 days. The yield of the desired product is 67-70 mg per litre of the cultural medium.

1 The obtained substance is characterized by a high inhibiting activity; its 1% solution ensures a 90-100% inhibition of neuraminidase of influenza viruses A.

The high activity of the resulting substance is not decreased after a long-time (10-60 minutes) boiling and incubation for 24 hours at a pH value of from 1.0 to 10.0.

For a better understanding of the present invention some specific examples illustrating the method for the preparation of an inhibitor of neuraminidase are given hereinbelow.

Example 1

The strain Staphvlococcus aureus No. 392, phagogroup III, Pillet serotype 18, is grown on a nutrient medium of the following composition, per cent by mass; enzymatic peptone lactose 1.0 1.0 yeast extract 0.3 MgS04.7H20 0.05 phosphate buffer the balance.

The pH of the medium is 7.2.

For accumulation of the inoculation culture the starting strain is grown in 5 ml of a nutrient medium of the above mentioned composition for one day, then in 100 ml for additional day. The growing is effected at the temperature of 370C. The resulting inoculation culture is introduced into 3 1 of a nutrient medium and grown for 1 day under continuous stirring. Then the cultural liquid is separated by centrifugation at 8,000 r.p.m. for 15 minutes. The obtained supernatant liquid is precipitated with ethanol in the ratio of 1:1.5, kept for 12 hours at the temperature of + 40C and centrifugated for 15 minutes at 6,000-8,000 r.p.m. The supernatant liquid is removed. The precipitate is washed with a 60% ethanol in the ratio of 1:1.5 and subjected to centrifugation for 15-30 minutes at 6,000-8,000 r.p.m. The resulting precipitate is again dissolved in a 1 1 1,1 phosphate buffer with the pH of 7.2, the solution is heated for 10 minutes at the temperature of 1000C, subjected to centrifugation for 15 minutes at 6,000-8,000 r.p.m. and the resulting precipitate is removed. The supernatant liquid is subjected to gel-filtration through a column of 2.Ox3O.O cm size with Sephadex G 200.

The resulting eluate is lyophilically dried to a uniform powder-like dry mass. The yield of the desired product is 67 mg per litre of the initial volume of the cultural medium.

The thus-obtained desired product has a molecular mass of from 94,000 to 120,000, contains proteins and carbohydrates in the ratio of 1:10, fatty acids containing 11 to 20 carbon atoms. The aminoacid composition of the proteins is as follows: phenylalanine, thyrosine, isoleucine, leucine, methionine, valine, alanine, glycine, proline, glutamine, serine, threonine, asparagine, histidine, lysine.

Carbohydrates of the inhibitor are presented by neutral monosugars mannose and glucose in the ratio of 24:1, by two hexosamines galactosamine and glucosamine in the ratio of 1:3.5.

The peak of the optical density of the thusobtained substance is detected at the wavelength of 210 nm.

The resulting substance comprises, as regards its chemical composition, a glycolipoprotein complex similar to the inhibitor of influenza virus neuraminidase. The inhibiting activity of the obtained substance was subjected to laboratory tests in respect of neuraminidase of viruses of influenza A01 Al, A2. To 0.2 ml of a suspension of virus of influenza Ao, Al, A2 (Hong Kong)68 (H3N2) in the concentration of 1:1,025 lg 7.5 EID50 0.2 ml of a 10% aqueous solution of the obtained substance is added. The mixture is subjected to incubation for 30 minutes in a water bath at the temperature of 370C. The control suspension of the virus of influenza of a similar character 0.2 ml of a physiological solution is added. In all samples the neuraminidase activity of influenza viruses is tested following a conventional procedure. The tests are conducted in comparison with the activity of an inhibitor produced by the known method. The results of the tests are shown in Table hereinbelow.

Example 2

The process is conducted in a manner similar to that described in Example 1 hereinbefore. As the producer use is made of the strain Staphylococcus aureus No.1025. The yield of the desired product is 70.3 mg per litre of the cultural medium. The chemical composition of the resulting product is similar to that described in the foregoing Example 1.

The inhibitor activity of the obtained product in respect of neuraminidase of viruses of influenza AO, Al and A2 is tested in a manner similar to that of Example 1.

The test results are shown in Table hereinbelow.

Example 3

The procedure is carried out in a manner similar to that described in Example 1 hereinbefore. Use is made of the strain Staphylococcus aureus 1431. The yield of the desired product is 66.7 mg per litre of the cultural medium. The resulting product has a chemical composition similar to that obtained in Example 1 hereinbefore. Testing of the inhibition activity of the resulting product is carried out in a manner similar to that described in Example 1.

The test results are shown in the following Table. Table Inhibition activity of the substance (in per cent) isolated from different strains of Staphylococcus aureus in respect of neuraminidase of influenza viruses A 7 Strains of influenza viruses Producer of inhibitor of influenza virus neuraminidase according to Example 1 Example 2 Example 3 known method Strain AO Strain Al Strain A2 100 100 100 100 100 100 -------------- 80 80 The method according to the present invention is simple, features a high yield and a high activity of the desired product, it makes possible to shorten the duration of the process for the preparation of the inhibitor. The obtained inhibitor of influenza virus neuraminidase ensures a 100% inhibition thereof which is by 10 times higher than the activity of the inhibitor produced by a known method.

- 8

Claims

CLAIMS: 1. A method of preparing an inhibitor for influenza virus neuraminidase comprising the steps of growing an inhibitor of neuraminidase of the species Staphylococcus aureus on a nutrient medium containing sources of carbon, nitrogen, mineral salts and growth stimulants; separation of the culture liquid; treatment thereof with ethanol; followed by isolation and purification of the desired product.

1

2. A method according to claim 1, wherein the purification of the desired product is carried out by the method of gel-filtration.

3. A method according to claims 1 and 2, substantially as described herein with reference to the Examples.

Published 1991 at'Me Patent OMce. State House. 66/71 High Holborn. london%VC) R 47P. Further copies rnav be obtained from Sales Branch. Unit 6. Nine Mile point, Cwmfelinfach. Cross Keys. Newport. NPI 7HZ. Printed by Multiplex techniques lid. St Mary Cray. Kent-