Life Sciences /5. Molecular Biology

Kovalenko T., student

National university of food technologies, Ukraine

Using of rabies virus strains for anti-rabies vaccine production

Rabies (hydrophobia) - acute infectious disease of humans and warm-blooded animals, which is manifested in defeat of the central nervous system. The causative agent of this disease is a virus that belongs to the family Rhabdoviridae, genus Lyssavirus. Thus, the main and the only producer of vaccines that prevent the development of this disease is that virus and its strains [1].

Today we know that it is necessary to use different cell lines for the production of viral vaccines:

• avian embryos derived from eggs without specific pathogens;

• human diploid cells (it passes control on presence of retroviruses, B and C hepatitis viruses nucleic acids, human immunodeficiency virus, also chromosomal characterization is controlled on various animal species: mice, guinea pigs, rabbits). Rabies vaccine, mumps and rubella are received on them;

• Rabbit’s kidney (it passes inspection for presence of myxomatosis, mycobacteria, leptospirosis, toxoplasmosis and other infectious agents).

These cells have to be grown on such nutrient media:

Ø 199 medium: L-form amino acids, vitamins, inorganic salts, Tween 80, uracil, phenol red and others. Serum components are added from 2% to 10% under cells cultivation.

Ø Basal Medium Eagle, BME: medium contains the minimum quantity of amino acids and vitamins. It was provided for different types of cells cultivation. Medium doesn’t contain glycine and serine;

Ø Minimal Eagle Medium, MEM: compared to BME, the content of arginine is increased in 5 times, in 4 times - histidine, 2 times - all other amino acids except of glutamine. Does not contain biotin. It allows to maintain the culture for a long time without further feeding. It uses for cells cultivation under production of viral vaccines, such as rabies and rubella;

Ø Dulbecco's Modified Eagle Medium, DMEM: compared to BME content of arginine is increased in 5 times, in 2 times – glutamine, also glycine and serine were added, the amount of other vitamins and amino acids was increased in 2 times;

Ø RPMI 1640 medium: synthetic medium enriched with serum will be able to grow cell cultures;

Ø Leibovitz L 115 medium: glucose is replaced by galactose. The buffer system is not added by sodium bicarbonate;

Ø Minimal Eagle Medium for suspension cultures, MEMS: unlike MEM it contains MgSO₄ in 10 times more, doesn’t contain CaCl₂, increased content of NaH₂PO₄ [2, 3].

The method of preparing a rabies vaccine, containing a rabies virus surface glycoprotein, expressed in eukaryotic cells is known. A DNA sequence encoding the rabies virus glycoprotein prepared by reaction of reverse transcription of virus RNA template or artificially synthesized, after which it can be inserted into a commercially expressed vector [4].

For example, the specific commercial plasmid vector pYES2 was developed for expression of the rabies virus glycoprotein in Saccharomyces cerevisiae cells. Preparation of the rabies virus glycoprotein in insect cells is provided by baculovirus expression system, for example dial of МахВас. [4, 6].

The high expression level of the interest gene is provided by the presence of the promoter sequence, an enhancer, a polyadenylation signal. Thus, preparation of the surface glycoprotein of rabies virus is possible in eukaryotic and prokaryotic cells, but it is more expedient to use eukaryotic cells as they provide the necessary modifications which bring to occurrence of a functionally active product. Glycoprotein of rabies virus enters into the body, communicates with the epithelium of the oral cavity, which leads to the top of immune responses under oral immunization of this method [4].

But this method has disadvantages:

• short disintegration period of foreign protein in the body (2-3 hours);

• necessity of introducing into the body a large doses of the drug to achieve a positive effect;

• necessity of adjuvant application;

• the high cost of the process of drug obtaining from these glycoproteins of the virus [4], [5].

CVS and SAD strain, which are derived from strains SAD, such as the SAD Berne and SAD B19, are the most commonly used strains of rabies virus. The amino acid sequence of the SAD Berne strain glycoprotein is not fully established. But it can be established that the antigenic site III of the SAD Berne strain glycoprotein is identical to that site of strain CVS glycoprotein [5].

Strain SAD Berne, used for vaccines producing, is a danger to wildlife and humans, and the same is about strain SAD B19. It anti-viral rabies vaccine is proposed to remove this disadvantage, which described in patent application EP 2128519, it contains avirulent SAD strain mutant of rabies virus, characterized by containing of amino acid that differs from the lysine, for example, glycine, isoleucine or serine, instead of arginine 333 of glycoprotein. This mutant is prepared by replacement of one nucleotide in arginine 333 codon [5].

The disadvantage is that he returns to the parent form of the strain under reverse mutation. Therefore, this vaccine is not safe for animals of other species, except the foxes [6, 7].

Literature:

1. Petersen Brett W. , Rupprecht Charies E.. Human rabies epidemiology and Diagnosis // J. Biotechnol № 63. – 2010. – P. 247 – 253.

2. Jackson Alan C. Update on rabies // Review. № 12 – 2011. – P.31 – 43.

3.Краснопольский Ю.М, Борщевская М.И. Биотехнология иммунобиологических препаратов. – К: Харьков, 2008. – 312 c.

4. Патент № 2432963. Способ получения пероральной вакцини против вируса бешенства. Шмаров М. М.,Грибова И. Ю., Тутыхина И. Л.  и др. – 2011.

5. Патент №2128519. Антиверулентная вакцина против бешенства. Штамм вируса бешенства используемый для изготовления вакцины против бешенства и способ его получении. Жаклин Бенжан, Анн Фламан, Мари – Кристин Тюфферо, Патрис Кулон, Флоранс Лафэй. – 1999.

6. Bourhy H., Cowley J.A., Larrous F. at all. Phylogenetic relationships among rhabdovruses inferred using the L polymerase gene // J. General Virology. – № 6 2005. – Р. 145 – 159.

7. Xi Jin, Guo Huancheng, Feng Ye at all. Differentiation of the seven majo lyssavirus species by oligonucleotide microarray // J. Clinical Microbiol. № 11 – 2011. – Р. 619 – 624.