Viral vaccines

VIRAL VACCINES
Since viruses are intracellular parasites they will grow only within other living cells.
Methods of viral vaccine production:
Cultivation of virus using free living animals
Fertile eggs
Tissue cultures

1. SJM College of Pharmacy, Chitradurga Prepare By, Adarsh Patil Ass Professor(Pharmacognosy) SJM College of Pharmacy 1 PHARMACEUTICAL BIOTECHNOLOGY

2. VIRAL VACCINES Since viruses are intracellular parasites they will grow only within other living cells. Methods of viral vaccine production: Cultivation of virus using free living animals Fertile eggs Tissue cultures

3. Cultivation of virus using free living animals: Now a day very few vaccines are made from viruses grown in free living animals. The products are good antigens but the method is inconvenient and costly and adventitious Contamination is difficult to prevent. In some parts of the world typhus vaccine is prepared from rickettsiae grown in the lungs of small rodents or the peritoneal cavity of gerbils, and rabies vaccine may be produced from the brain of sheep or rabbit, but in the UK, the most important prophylactic prepared from free living animals is vaccine lymph(small pox vaccine). There are alternative sources of this vaccine but the products are less thermostable and no more effective than the material of free living animals . Example: Polio vaccine

4. POLIO VACCINE: Polio vaccine is a live oral aqueous suspension This is obtained from attenuated strains of polio myelitis virus. ( sabin ), type 1,2,3. This polio myelitis virus can grow on a suitable approved cell cultures. The vaccine which is administered parenterally , which stimulates the production of antibodies in the blood and gives protection by neutralizing the virus as it passes through blood stream on the way from the alimentary canal to central system .

5. Preparation: There are three distinct antigenic types of polio myelitis virus types I, II and III and each has much greater antigenic stability. There three types of viruses are gram antigenic in suspended (or) fixed cell culture of monkey kidney tissue. The cell culture is maintained not more than at 35 C during growth of virus. The harvesting is done within 4 days of inoculation and virus suspicions is tested for identity, sterility and free from adventitious viruses.

6. Description: which is a freeze dried power (or) pellets, which goes readily into solution with the diluents which is supplied by manufacturer, this diluent produce a clear ( or) slightly opalescent liquid. Storage: store in a "sealed tight resistant containers" at temperature between 2-8ºC when the stored under these prescribed conditions the vaccines may be expected to retained its potency for not less than two years from the date the potency test was started.

7. 2. Fertile Eggs: Many virus can be grown in some part of chick embryo. Example: Influenza virus growth in eggs Before the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. 

8. vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. 

9. For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe.

10.  Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum . After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle.

11. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid . The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours . During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by  budding , they are released into the allantoic fluid.

12. To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg . When the handle of this tool is squeezed, it makes a neat crack around the top of the egg.  It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg.

13. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine . 3. Tissue culture method : It has been known for many years that vaccinia virus will grow in certain minced animal tissues kept alive in suitable solutions of salts but the method remained undeveloped as means of growing viruses because of the success of chick embryo techniques and the difficulty of preventing contamination of the cultures.

14. Steps involved in production of viral vaccines using tissue culture technique Selection of suitable tissue : A large number of tissues can be successfully cultivated outside the animal Body but because certain viruses will grow only in primate cells, monkey kidney have become the most widely used in the manufacture of poliomyelitis and measles vaccine), Chick embryo more satisfactory in some cases e.g., for attenuated measles virus and cow pox, has also been successfully grown in calf embryo skin .

15. Different Methods used: 1. Primary cells: Primary cells cultures (PCCS) can be established from safe animals or embryos, or from Selected tissues from embryos, new born animals, or adult animals of almost any species. Primary cells do not expand much, but can be dissociated with protease and expanded in a New container for multiplication. IPV (Inactivated polio vaccine) derived from the kidney of clinicly healthy monkeys .

16. Other Methods, Diploid cell lines, Stem culture etc