PREPARATION OF VACCINES Vaccine production.pptx

Vaccine preparation

1. Vaccine production

2. Process of bacterial harvest Killing : process in which live bacteria in the culture are killed. Eg : heat or formalin required to kill the cells of bordetella pertusis (whooping cough vaccine). Phenol for vibrio cholera vaccine and salmonella typhi (typhoid vaccine) Separation: process to separate bacterial cells from fermentation medium. Eg : centrifugation or precipitation Fractionation: components extracted from bacterial cells. Eg : pseudomonas aeroginosa extracted with water . Polysaccharide antigen of neissaria meningitidis by treatment with CTAB ( hexadecyltrimethyl ammonium bromide)

3. Detoxification: toxins are converted into harmless toxoids. Eg : formalin for corynebacterium diphtheriae and clostridium tetani . Adsorption: the mineral adjuvants or carrier are used to increase immunogenicity and decrease toxicity. Eg : aluminium hydroxide, calcium phosphate Conjugation: linking of vaccine component (poor immune response) with a vaccine component that induces a good immune response. Eg : the immunogenicity for infants of hemophilus influenzae type b is enhenced by conjugation with diphtheria nad tetanus toxoids

4. Processing of virus harvest

5. Filling and drying Bulk vaccines are dispersed into single dose ampoules or into multiple dose vials. Vaccines that are filled as liquids are sealed and capped in the container. Vaccines that are dispensed as dry preparation are freeze dried before sealing.

6. Storage : most vaccines and immunological preparations- at 2 to 10 ⁰C. Freeze dried at- not more 10 ⁰C and ⁰C.

7. Bacterial vaccines and toxoids Bacillus calmette guerin vaccine (BCG Vaccine) Live bacterial vaccine bearing an attenuated bovine strain of Mycobacterium tuberclosis developed by camette and Guerin (1921). The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a suitable liquid. Preparation: Strain grown on a suitable culture media show not less than 20 million colonies (Seed lot system) After growth separated by filtration form cake. Killed by Heat treatment. Cake is homogenised in grinding flask and suspended in liquid medium designed to Preserve the antigenicity and viability of the vaccine ( determine by plate count) The suspension transferred into final sterile container and freeze dried under aseptic conditions and sealed Available as white pellet or powder , which on constitution yield opalescent and homogenous suspension Storage : in light resistant glass container at 2 to 8 ⁰C. reconstituted vaccine should be Used immediately after preparation. Dose: prophylactic, by intracutaneous injection as single dose, 0.1 ml Use: As immunising agent which provide protection against tuberculosis

8. (2) Cholera vaccine Homogenous suspension of a suitable strain of vibrio cholerae . The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a suitable liquid. Preparation: prepared from equal portion of vibrio cholerae Inaba and Ogawa strain. Each strain grown separately on a solid culture medium for 24 to 48 hrs (Seed lot system) Bacteria are then washed with normal saline solution The suspension of bacteria are killed either by heating the suspension (56 ⁰C for 1 hr ) or by heating with suitable bactericide such as phenol or formaldehyde The suspension transferred into final sterile container and freeze dried under aseptic conditions and sealed The vaccine may contain preservative Storage : stored at 2 to 8 ⁰C without freezing. Dose: prophylactic, for children and adult over 10 years (subcutaneous injection) initial dose, 0.5 ml second dose- 1.0 ml after an interval of 4 to 6 weeks. For children below 10 years initial dose-0.3 ml, second dose-0.3 ml Use: As immunising agent which provide protection against cholera

9. (3) TAB vaccine(Typhoid-paratyphoid A,B) Sterile suspension of strain of salmonella typhi and salmonella paratyphi A and B . Preparation: TAB mixed polyvalent vaccine and prepared by mixing of simple vaccines of salmonella typhi and salmonella paratyphi A and B These strains grown separately in acid digested agar medium and cultivated for 48 hr at 37 ⁰C (Seed lot system) Bacteria Strain then harvested with sterile normal saline solution The strain are diluted to form 3000 million organisms/ml of salmonella typhi and 2250 million organisms/ml of salmonella paratyphi A and B All these strains killed by addition of 0.1% formalin or by heat treatment Bacterial strains incubated at 37 ⁰C for 4 days for detoxification and then tested for sterility Storage : stored at 2 to 8 ⁰C without freezing. Dose: prophylactic, 0.5 ml (subcutaneous injection) 2 to 3 injection at 2 to 4 weeks interval booster doses may be given every 1 to 2 years Use: polyvalent vaccine for enteric infections Bacterial strain mixed together to contain 1000million organisms/ml of salmonella typhi and 750 million organisms/ml of salmonella paratyphi A and B

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11. Viral vaccines Viral vaccines are prepared by using free living animals, fertile egg and tissue cultures. Viruses require a living medium for growth unlike bacteria which can grow on non-living media. Viruses are responsible for many infectious diseases in man, animals and plants. Important viral diseases are influenza, common cold, measles, mumps, polio, smallpox, yellow fever, rabies etc. For preparation of viral vaccine, viruses are usually grown in the chorio-allantoic membrane of incubated fertile hen egg or in whole animals. Some vaccines preparation given below:

12. Dose : For primary immunization, oral polio vaccine is generally given at birth, then at 6,10 and 14 weeks. Booster dose: 15 to 18 months. Oral vaccine is the choice for active immunization. Simultaneously through pulse polio programme vaccination to infants and children upto 5 years age has conducted to eradicate wild virus in many countries.

13. Small pox vaccine Small pox vaccine is a freeze dried suspension of living vaccinia virus, a strain capable to protecting against small pox. For this vaccinia virus is grown on skin of healthy sheep or calves or in the membrane of chick embryo. Method of preparation: (1) Selection of animal: healthy sheep or calves. 10 to 14 day keep in isolated area to remove any communicable diseases. (2) Preparation of animal for scarification : the abdomen and flanks (the side between ribs and ileum) are cleaned, scrubbed and disinfected. Then animal are transferred to special room were abdomen and flanks areas were shaved, cleaned and disinfected. (3) Inoculation: Light Incisions are made in the cleaned skin without drawing blood with the help of scarifler . This process of making light scratches on the skin of the animal is known as scarification. The scarified area is then rubbed with a seed vaccine of known potency

14. (4) Incubation: During the next 4 to 6 days, vesicles containing the virus develop along the lines of scarification. During this period of incubation, every precaution is taken to keep the inoculated areas aseptically clean. (5) Harvesting: The animal is taken on the operation table and killed. A post mortem of the animal is made to check the absence of disease, The abdomen and flanks are then washed with sterile water. The material in the vesicles is withdrawn with the help of a sharp edged spoon under aseptic conditions. (6) Purification: The contents of vesicles are mixed with an equal volume of glycerin, cooled and then finely ground to form homogeneous mixtures. It is then stored for a long time at - 10°C to remove impurities. Now-a-days, more efficient methods are used for purification such as:

15. (1) The lymph is extracted with a protein solvent (protein solvents lowers the efficiency of the bactericidal agent) e.g.trichlorofluoroethane . (2) Brilliant green or any other suitable colouring matter is added to mark the area of application of the vaccine. (3) Phenol (0.4%) is added and the material is incubated at 22°C for 2 days for reduction of bacterial count. (4) A mixture of glycerin (40%) and peptone (1%) is added and then stored at -10°C. The glycerin assists the bactericidal action of the phenol during storage and also gives viscosity to the product. The peptone helps to preserve the viability of the viruses (in freeze-dried product). (7) Filling and storage: Purified vaccine is filled into the final container under aseptic conditions. The liquid vaccine is commonly packed in single-dose capillary tubes of glass or plastic. The freeze-dried material is supplied in multi-dose container together with suitable volumes of reconstituting fluid. Liquid smallpox vaccine retains its potency for a year at -10°C but at higher temperatures its stability reduces. The freeze-dried product is more stable and is kept between 2 to 8°C.

16. Smallpox vaccine is also prepared from virus grown on the chorio-allantoic membrane of the fertile hen's egg. process: The eggs of hen are selected from fertility and incubated for 12 days. A small portion of the shell of the egg is removed and the chorio-allantoic membrane is inoculated with a seed vaccine of known potency. The portion of the shell is replaced and sealed with melted paraffin wax. The eggs are incubated for 72 hours. Then shell removed by using aseptic conditions and chorio-allantoic membrane is separated. It is placed in a sterile saline solution at 0°C. Glycerin (50%) is added and the material is ground to produce a homogeneous suspension. It is then transferred into a final container, freeze- dried and sealed.

17. Hepatitis vaccine: Hepatitis A vaccine is prepared from hepatitis 'A' virus grown in human diploid cell cultures by inactivating with formaldehyde. A single 0.5 ml injection is generally used for protection from hepatitis A. Hapatitis 'B' vaccine is prepared in yeast cells by recombinant DNA technique and contains aluminium hydroxide adsorbed hepatitis 'B' virus surface antigen 20 µg in-1 ml suspension Three 1 ml injections (at 0, 1 and 6 months) are given to produce protective antibody titers. Children below 10 years are given 0.5 ml doses in the thigh.

18. ANTITOXIC ANTISERA/ IMMUNE SERA Antisera or immune sera are preparations which contains antibodies. If the blood of an immune person or animal is withdrawn and allowed to clot, a large number of antibodies are found in the serum that separates. A serum may contain antitoxic, antibacterial or antiviral serum respectively. Antitoxic sera are more effective than antibacterial or antiviral sera. Diphtheria antitoxin Diphtheria antitoxin is a sterile, non-pyrogenic solution containing the specific antitoxic antibodies obtained from the serum of healthy horses and have the power of neutralizing the toxin formed by Corynebacterium diphtheriae . Method of preparation: The method of preparation of the Diphtheria antitoxin is divided into the following steps:

19. preparation of toxin : A pure culture of Corynebacterium diphtheriae is grown in suitable culture media at 37 ⁰ C for 4 to 5 days. After incubation, 0.5% phenol is added and the culture media is filtered through bacteria proof filters. The filtrate (crude toxin) is converted into a toxoid by detoxification method. Selection of horse : Horses are selected for production of Diphtheria antitoxin because they are easy to handle and readily produce antitoxins. Considerable quantity of blood can be withdrawn at a time without any ill effects. The horses selected must be free from disease, Horses are kept in an isolated place for 7 days and then carefully examined for Infectious diseases. Active Immunization of the horse : Diphtheria toxoid is given to the selected healthy horses for active Immunization. The toxoid is injected into the muscles of the neck by Intramuscular injection. The first dose of the toxoid is usually not more than 5 ml which is gradually Increased daily or after 1 to 2 days for about 2 months and as much as 600 ml is injected for the final dose. (4) Separation of serum from the horse : After about 10 days of the injection of the final dose, about eight litres of blood is withdrawn aseptically into bottles containing an anticoagulant solution. Similarly two more collections each about eight liters of blood are made within the next eight days, after which the horse is given about 15 days rest. Then another course of the toxoid is repeated in similar doses to stimulate further antibody production. Blood is collected in three batches each about eight litres . Further courses of administration of the toxoid and bleeding are continued for 4 to 5 times or till the animal stops producing satisfactory antitoxins.

20. After the collection of the blood, it is allowed to clot to separate the serum. The serum contains antitoxin along with other proteins, such as beta-globulins, gamma globulins and albumins. Antitoxins are largely associated with beta-globulin. (5) Concentration and refinement: Horse serum contains a high concentration of several other proteins which may cause undesirable reactions, such as anaphylactic shock or serum sickness. So these undesirable proteins are separated by fractional precipitation or by fractional proteolytic digestion method. Diphtheria antitoxin has a potency of not less than 1000 IU/ml, in the case of antitoxins obtained from horse serum and not less than 500 IU/ml for antitoxins obtained from other animals. Storage: stored in containers protected from light at temperature between 2 to 8°C. It should not be allowed to freeze. Dose: Diphtheria antitoxin is administered by subcutaneous or intramuscular injection. For prophylactic use , the dose is not less than 1500 IU and for therapeutic use, the dose is not less than 50,000 IU. Use: Passive immunizing agent for diphtheria.

21. Tetanus antitoxin Tetanus antitoxin is a preparation containing antibodies that have the power of specifically neutralizing the toxins formed by Clostridium tetani . It is obtained by fractionation from the serum of horses or other mammals, that have been immunized against Tetanus toxins. Method of preparation, storage and dose are similar to diphtheria antitoxin.

22. Antiviral sera These are used in passive immunization against certain viruses. It acts differently as viruses are intracellular parasites while antibodies can not penetrate the cells. Inactivation of viruses takes place in body fluids or on surfaces at which invasion occurs. The main source of antiviral antibodies is human serum. The horse is mainly used for antiviral sera production, because the horse is not susceptible to many viruses against which protection may be required e.g. measles, rubella, poliomyelitis, thus official antiviral sera (e.g. rabies antiserum) is prepared in horses.

23. Rabies Antiserum or Antirabies Serum (ARS) Antirabies Serum (equine rabies immunoglobulin, ERIG) is a refined, concentrated and lyophilized serum from horses hyper-immunized by repeated injections of fixed rabies virus. Horses are injected with dead virus and when a good level of immunity has developed, are injected with live virus. The methods of protein purification used to refine the serum are designated to separate the gamma-globulin fraction that contains the antiviral antibodies. Antirabies Serum is indicated promptly after suspected exposure and is given simultaneously with Rabies vaccine to non-immunized individuals.

24. MISCELLANEOUS VACCINES: DNA Vaccine: In 1990, a new approach for vaccination was invented involving injection of plasmid DNA, which elicits an immune response to the encoded protein. DNA vaccines can overcome the most of the limitations of conventional vaccines and has potential for vaccines of the future. The injection of DNA into animals does not generate an immune response against the DNA molecule but that DNA is expressed to yield a protein that can stimulate the immune system. Many DNA vaccines have been used to target viruses such as measles, HIV, Ebola and polio etc. DNA vaccine is composed of a bacterial expression plasmid. Expression plasmid used in DNA based vaccination contains two unit as the antigen expression unit ( promotor enhencer sequence+ antigen encoding region+polyadenylation sequences) and the production unit (bacterial sequence for plasmid amplification and production)

25. Mechanism of action of DNA Vaccine

26. Preparation method of DNA vaccine: Most DNA vaccines carry promoters and enhancer regions from pathogenic viruses such as cytomegalo virus (CMV), simian virus 40 or murine leukaemià virus. Once constructed by recombinant DNA technology, the vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies. The plasmid DNA is then purified by separating the circular plasmid from larger bacterial DNA and other bacterial impurities. This purified DNA acts as the vaccine and is administered through intramuscular, intradermal and intravenous injection. The skin and mucous membranes are the best site for immunization due to the high concentrations of lymphocytes, macrophages and dendritic cells.

27. Mechanism of action of DNA Vaccines: Plasmid vector expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter which is Injected into the skin or muscle of the host. The protein Is produced and processed into small antigenic peptides by the host proteases. The peptides then enter the lumen of the endoplasmic reticulum by membrane associated transporters. In the endoplasmic reticulum (ER), peptides bind to MHC Class - I molecules. There peptides are present on the cell surface in the context of the MHC Class -I. Cytotoxic (CD8) T cells are stimulated and they evoke cell mediated immunity. Cytotoxic T cells inhibit viruses through both cytolysis of infected cells and cytokine production. The foreign protein can also be presented by the MHC Class II pathway by APCS which elicit helper T cells (CD4) responses. These CD4 cells are able to recognize the peptides formed from exogenous proteins that are endocytosed or phagocytosed by APC, then degraded to peptide fragments and load into MHC Class II molecules. Depending on the type of CD4 cell that binds to the complex and stimulated B cells for antibody production

28. Advantages of DNA vaccines: DNA vaccines stimulate both humoral and mediated responses. simple vaccine having wide applicability. DNA vaccines are easily constructed with available tools of molecular biology. Rapid and large scale production is possible at low cost. DNA vaccination can be used to treat diseases that are already established as a chronic infection. It may provide an important tool for stimulating an immune response in HCV, HBV and HV patients. DNA vaccines are heat stable and non-virulent. DNA vaccines encoding several antigens or proteins can be delivered to the host in a single dose. Limitations of DNA vaccines are as follows: DNA vaccines may produce anti-DNA antibodies causing auto-immunity in the host. Naked DNA is not very immunogenic in animal studies. It is not exactly known, how long the DNA persists in the host cells. These vaccines do not produce strong immune response in higher animals like primates. Safety is an important factor in DNA vaccines and to improve its safety can be extremely costly and time-consuming.

29. Advantages of recombinant vaccines are as follows: 1.Large amount of antigen can be produced in less cost. 2. Genetic manipulation of the antigen itself is possible after availability of gene sequence. 3. Viral recombinant vector vaccines produce strong humoral and cell-mediated immune responses, resulting in immunological memory. 4. It may be possible to encode for several antigens from different pathogens, Introducing the possibility of a single vaccine for several diseases. Limitations of recombinant vaccineş are as follows: 1. Immune response generated by recombinant antigen vaccines is primarily humoral. 2. In viral vector vaccines, live virus being used is an attenuated form of a human pathogen. 3. There is always a risk of reversion to virulence.

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