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Shashikala Metri
Asst. Professor
Dept of Pharmacognosy
Shri D.D. Vispute College of Pharmacy, Mumbai.
 Immunization is the process whereby a person is
made immune or resistant to an infectious
disease, typically by the admi...
 Types of Vaccines
Active Immunization
 Live attenuated vaccines
 Inactivated or killed vaccines
 Toxoids
 Cellular f...
Classification of vaccines
I. Live attenuated
Eg. Measles, Mumps, Rubella, Varicella, Rotavirus,
Tuberculosis (BCG)
II. In...
I. Live attenuated vaccines
 To produce a live vaccine, the wild or disease-
causing virus is attenuated (weakened),
trad...
II. Killed and inactivated vaccines
The term ‘killed’ is generally used for bacterial
vaccines and the term ‘inactivated’ ...
 Toxoid vaccines
 In some bacterial infections (eg, diphtheria,
tetanus), the clinical manifestations of
disease are cau...
Recombinant vaccines
 Recombinant vaccines, such as those used against hepatitis B and HPV,
are made using a gene from th...
VACCINE
 Vaccine is an antigenic substance
prepared from the causative agent of a
disease or a synthetic substitute, used...
Polio vaccines are vaccines used to prevent poliomyelitis (polio), 2
types
1. Inactivated poliovirus and is given by injec...
Salk vaccine Sabin vaccine
 Jonas Salk developed
the vaccine
 An inoculation of
dead polio virus
 It is administered as...
• The original Salk polio vaccine is an example of an inactivated
(killed) vaccine. The Salk vaccine, or inactivated polio...
• The Sabin oral polio vaccine is attenuated
(weakened) vaccines
• To make an attenuated vaccine, the pathogen is
grown in...
 The attenuated Sabin poliovirus vaccine
replicates very efficiently in the gut, the
primary site of infection and replic...
 Based on EP and WHO guidelines following tests are used for the
evaluation (standardization) of OPV and IPV products.
Ge...
 MAPREC test Molecular biological test, Mutant Analysis by
PCR and Restriction Enzyme Cleavage: The MAPREC assay is
a mol...
 Batch release for IPV:
 ELISA test
 D-antigen test
 Rat potency test
 An improved ELISA test for determination of po...
 Potency of inactivated poliovirus vaccine (IPV) is tested either by
injecting experimental animals and then measuring ne...
 Vaccine:
 1 - contains dead bacteria or weak bacteria
or toxins.
 2 - stimulates the body to make antioxidants.
 3 - ...
The production of a vaccine can be divided in the following
steps:
 1. Generation of the antigen
 The first step in orde...
 2. Release and isolation of the antigen
 After the antigen is generated, vaccines are
isolated from the cells, used to ...
3. Addition of adjuvants, stabilizers and
preservatives
 Once the antigen is developed the vaccine is
formulated by addin...
 Is a formal toxoid prepared from the toxins
produced by corynebacterium diphtheriae
 A suitable strain of corynebacteri...
 Preparation of diphtheria toxoid
i. Formal toxoid (FT) in the diphtheria toxin 0.5%
of formalin is added and the mixture...
iii. Alum precipitated toxoid:
 It produces more antibodies than formal toxoid and TAF
 Slow absorption of precipitated ...
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Vaccines and sera

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Vaccines and sera

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Vaccines and sera

  1. 1. Shashikala Metri Asst. Professor Dept of Pharmacognosy Shri D.D. Vispute College of Pharmacy, Mumbai.
  2. 2.  Immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine.  Vaccines : vaccines are preparations of antigenic materials, which are administered with the objective of inducing in the recipient specific and active immunity against infectious microorganisms or toxins produced by them  They contain living or killed microorganisms, bacterial toxoids or antigenic material from the particular parts of bacterum, rickettsia, or virus
  3. 3.  Types of Vaccines Active Immunization  Live attenuated vaccines  Inactivated or killed vaccines  Toxoids  Cellular fractions  combinations Passive Immunization  Immunoglobulins  Antisera
  4. 4. Classification of vaccines I. Live attenuated Eg. Measles, Mumps, Rubella, Varicella, Rotavirus, Tuberculosis (BCG) II. Inactivated or whole killed Eg. Hepatitis A, Some influenza vaccines III. Subunit: sub classified as a) Toxoid: Diphtheria, tetanus b) Polysaccharide: Pneumococcal (23- valent), meningococcal ACYW-135 c) Conjugate: Pneumococcal (10- and 13-valent), Haemophilus nfluenzae type b,meningococcal C and ACYW-135 d) Recombinant: Hepatitis B, human papillomavirus e) Other subunit: Pertussis, acellular influenza
  5. 5. I. Live attenuated vaccines  To produce a live vaccine, the wild or disease- causing virus is attenuated (weakened), traditionally by repeated culture in the laboratory,  The virulence properties of the virus are reduced so that it does not cause disease in healthy individuals. The attenuated vaccine virus multiplies to a limited extent in host tissue and induces an immune response similar to wild virus infection in the majority of subjects. Live vaccines are generally very effective and induce long-lived immunity.  Eg. Measles, Mumps, Rubella, Varicella, Rotavirus, Tuberculosis (BCG)
  6. 6. II. Killed and inactivated vaccines The term ‘killed’ is generally used for bacterial vaccines and the term ‘inactivated’ for viral vaccines. These vaccines are prepared by treating the whole cell or virus with chemicals that cause inactivation. Generally these organisms remain intact and whole. They generate an immune response (to a broad range of antigens) but cannot cause an infection because they are dead and so cannot reproduce. Eg. Hepatitis A, Some influenza vaccines III. Subunit vaccines Subunit vaccines are developed using only the antigens known to elicit protective immunity. They can be further categorised as follows.
  7. 7.  Toxoid vaccines  In some bacterial infections (eg, diphtheria, tetanus), the clinical manifestations of disease are caused not by the bacteria themselves but by the toxins they secrete. Toxoid vaccines are produced by harvesting a toxin and altering it chemically (usually with formaldehyde) to convert the toxin to a toxoid. The toxoid is then purified. Toxoid vaccines induce antibodies that neutralise the harmful exotoxins released from these bacteria.
  8. 8. Recombinant vaccines  Recombinant vaccines, such as those used against hepatitis B and HPV, are made using a gene from the (disease-causing) pathogen as an antigen, which generates a protective immune response. The gene is inserted into a cell system capable of producing large amounts of the protein of interest.  There are four genetically-engineered vaccines are currently available:  the gene for the hepatitis B surface antigen is inserted into yeast cells, which replicate and produce large amounts of the hepatitis B surface antigen. This is purified and used to make vaccine. The advantage of this approach is that it results in a very pure vaccine that is efficient to produce.  Human papillomavirus vaccines are produced by inserting genes for a viral coat protein into either yeast (as the hepatitis B vaccines) or into insect cell lines. Viral-like particles are produced and these indice a protective immune response.  Live typhoid vaccine (Ty21a) is Salmonella typhi bacteria that has been genetically modified to not cause illness.  Live attenuated influenza vaccine (LAIV) has been engineered to replicate effectively in the mucosa of the nasopharynx but not in the lungs.
  9. 9. VACCINE  Vaccine is an antigenic substance prepared from the causative agent of a disease or a synthetic substitute, used to provide immunity against one or several diseases.  Contains dead bacteria or weak bacteria or toxins.  stimulates the body to make antioxidants.  Gaining immunity after a period.  Remain immune for long.  Use a series of complex methods to separate and purify the virus and extract the required part, reduce the low virulent or inactivated and produced vaccine.  A vaccine consists of normally a single type of antibodies( usually monoclonal) or may be for a particular disease SERA  The clear, pale yellow liquid that separates from the clot in the coagulation of blood  does not contain bacteria or toxins.  contains anti formed in another animal.  acquired immune immediately.  remain a short time.  specific animal by immunization, whole blood collected in, but the serum is a nonspecific mixture obtained after centrifugation.
  10. 10. Polio vaccines are vaccines used to prevent poliomyelitis (polio), 2 types 1. Inactivated poliovirus and is given by injection (IPV) The inactivated polio vaccines are very safe. Mild redness or pain may occur at the site of injection. 2. Weakened poliovirus and is given by mouth (OPV). Oral polio vaccines result in vaccine-associated paralytic poliomyelitis in about three per million doses.[ The World Health Organization recommends all children be vaccinated against polio. The two vaccines have eliminated polio from most of the world, and reduced the number of cases each year from an estimated 350,000 in 1988 to 74 in 2015.  The first polio vaccine was the inactivated polio vaccine. It was developed by Jonas Salk and came into use in 1955. The oral polio vaccine was developed by Albert Sabin and came into commercial use in 1961
  11. 11. Salk vaccine Sabin vaccine  Jonas Salk developed the vaccine  An inoculation of dead polio virus  It is administered as an injection  Complete dose includes a satisfactory immune response  Systemic antibody response  Albert Sabin developed the vaccine  An inoculation of attenuated live polio viruses  It is administered oraly  1 or 2 doses of oral vaccine gives 90%-100% result  Local immunity in gut
  12. 12. • The original Salk polio vaccine is an example of an inactivated (killed) vaccine. The Salk vaccine, or inactivated poliovirus vaccine (IPV), is based on three wild, virulent reference strains, Mahoney (type 1 poliovirus), MEF-1 (type 2 poliovirus), and Saukett (type 3 poliovirus), • It is made by growing virulent polio virus in tissue culture [monkey kidney tissue culture (Vero cell line)] , by the microcarrier technique • Then the viruses are concentrated, purified and inactivated by treating the virus with formaldehyde so that it cannot reproduce in the person who receives the vaccine. • Each 0.5 ml dose of trivalent vaccine is formulated to contain 40 D antigen units of Type 1. 8 D antigen units of Type 2. and 32 D antigen units of Type 3 poliovirus. 0.5% phenoxyethanol and a maximum of O.02X formaldehyde. Neomycin. streptomycin·and polymyxin B are used in vaccine production • Neutralizing antibody produced to polio virus is very efficient at blocking the ability of the virus to infect host cells and offers good protection from infection.
  13. 13. • The Sabin oral polio vaccine is attenuated (weakened) vaccines • To make an attenuated vaccine, the pathogen is grown in animals or tissue culture under conditions that make it less virulent. Steps involved are-  The pathogenic virus is isolated from a patient and grown in human cultured cells  The cultured virus is used to infect monkey cells  The virus acquires many mutations that allow it to grow well in monkey cells  The virus no longer grows well in human cells(it is attenuated) and can be used as vaccine
  14. 14.  The attenuated Sabin poliovirus vaccine replicates very efficiently in the gut, the primary site of infection and replication, but is unable to replicate efficiently within nervous system tissue.  In 1961, type 1 and 2monovalent oral poliovirus vaccine (MOPV) was licensed, and in 1962, type 3 MOPV was licensed. In 1963, trivalent OPV (TOPV) was licensed, and became the vaccine of choice in the United States and most other countries of the world, largely replacing the inactivated polio vaccine.
  15. 15.  Based on EP and WHO guidelines following tests are used for the evaluation (standardization) of OPV and IPV products. General assays: e.g. free formalin test, Sterility test etc.  Free formalin test: inactivation may lead to the presencer of free formalinmay not exceed 0.02% which is estimated by colour development with acetylacetone  Sterility test: vaccines must be bacteriologically and mycologicaly sterile. In each batch of product the number of containers to be tested depends on the batch size and the subject of pharmacopoeial regulation. Membrane filtration is commonly used for sterility testing. Specific assays :  Batch release for OPV:  potency test in cell culture: the potency of the live viral vaccines is estimated by using substrates of living cells. Dilutions of vaccines are inoculated on tissue culture monolayers in petriplates and the live count of vaccines are calculated from the infectivity of dilutions and dilution factor involved
  16. 16.  MAPREC test Molecular biological test, Mutant Analysis by PCR and Restriction Enzyme Cleavage: The MAPREC assay is a molecular biological method used to determine the proportion of a 5 single base mutation at a given point within the viral RNA. If the calculated value of the 6 mutation at this site is greater than acceptable the vaccine will fail the MAPREC test.  transgenic mouse neurovirulence test (TgmNVT) TgPVR mice developed by introducing into genome the human gene encoding the cellular receptor of poliovirus When infected with poliovirus, TgPVr mice develop flaccid paralysis and then death in some cases with histological lesions in central nervous system similar to those observed in monkey
  17. 17.  Batch release for IPV:  ELISA test  D-antigen test  Rat potency test  An improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) is proposed. The method is based on the use of IgG purified from immune rabbit serum conjugated with biotin.  The assay is based on direct ("sandwich") ELISA scheme, in which antigens are captured on ELISA plates coated with purified rabbit polyclonal D- antigen specific IgG raised against wild polioviruses of three serotypes. D-antigen specificity of the IgG was at least 10 times higher than to H-antigen (heat- inactivated virus).
  18. 18.  Potency of inactivated poliovirus vaccine (IPV) is tested either by injecting experimental animals and then measuring neutralizing antibodies in their sera or by estimating content of D antigen (complete viral particles) in the vaccine.  The recent derivation of transgenic mice expressing the human poliovirus receptor (Tg PVR mice) and susceptible to poliomyelitis allowed us to attempt to develop a new potency test for IPV based on protection of mice against lethal challenge with polioviruses of all three antigenic types.  Mice were vaccinated with IPV and then evaluated for induced immunity against poliomyelitis by intraperitoneal injection with 25 lethal doses of wild-type virus. A single injection of monovalent type-3 IPV elicited protective immunity, while 2 injections of trivalent IPV were required to protect mice against challenge with the same dose of virus. Both neutralizing antibody response and protection against challenge were vaccine dose-dependent.  Tg PVR mouse-protection test may be useful in evaluating existing potency tests of IPV and in attempts to improve formulations of trivalent IPV or of IPV combined with other vaccines suitable for simplified childhood immunization schedules.
  19. 19.  Vaccine:  1 - contains dead bacteria or weak bacteria or toxins.  2 - stimulates the body to make antioxidants.  3 - gaining immunity after a period.  4 - remain immune for long.  Serum:  1 - does not contain bacteria or toxins.  2 - contains anti formed in another animal.  3 - acquired immune immediately.  4 - remain a short time.
  20. 20. The production of a vaccine can be divided in the following steps:  1. Generation of the antigen  The first step in order to produce a vaccine is generating the antigen that will trigger the immune response. For this purpose the pathogen’s proteins or DNA need to be grown and harvested using the following mechanisms:  Viruses are grown on primary cells such as cells from chicken embryos or using fertilised eggs (e.g. influenza vaccine) or cell lines that reproduce repeatedly (e.g. hepatitis A)  Bacteria are grown in bioreactors which are devices that use a particular growth medium that optimises the production of the antigens  Recombinant proteins derived from the pathogen can be generated either in yeast, bacteria or cell cultures.
  21. 21.  2. Release and isolation of the antigen  After the antigen is generated, vaccines are isolated from the cells, used to generate it. For weakened or attenuated viruses no further purification may be required.  The aim of this second step is to release as much virus or bacteria as possible. To achieve this, the antigen will be separated from the cells and isolated from the proteins and other parts of the growth medium that are still present.  Recombinant proteins need many operations involving ultrafiltration and column chromatography for purification before they are ready for administration.
  22. 22. 3. Addition of adjuvants, stabilizers and preservatives  Once the antigen is developed the vaccine is formulated by adding adjuvants, stabilizers, and preservatives.  The role of the adjuvant is to enhance the immune response of the antigen.  The stabilizers increase the storage life.  Preservatives allow the use of multi dose vials.  Finally, all components that constitute the final vaccine are combined and mixed uniformly in a single vial or syringe.  4. Packaging  Once the vaccine is put in recipient vessel (either a vial or a syringe), it is sealed with sterile stoppers.
  23. 23.  Is a formal toxoid prepared from the toxins produced by corynebacterium diphtheriae  A suitable strain of corynebacterium diphtheriae is grown on liquid medium at 350C for 7 days  After maximum toxin production, the bulk of the organisms are separated and filtered and filtrate is sterilized
  24. 24.  Preparation of diphtheria toxoid i. Formal toxoid (FT) in the diphtheria toxin 0.5% of formalin is added and the mixture is incubated at 370C for 3-4 weeks to remove the toxicity ii. Toxin-antitoxin floccules (TAF) suitable quantity of toxoids and antitoxins are mixed to form the floccules which contain the good antigenic activity  Floocules are separated and washed to remove the contaminations and are resuspended in saline solution containing a bactericide
  25. 25. iii. Alum precipitated toxoid:  It produces more antibodies than formal toxoid and TAF  Slow absorption of precipitated toxoids from the site of injection and slow excretion from the body increased its antigenicity  In the procedure high quality toxoids are treated with charcoal to remove the colouring matter and filtered to remove the charcoal and other impurities  Than suitable concentration of alum is added  This reacts with bicarbonate, phosphate and proteins in the toxoid to produce a precipitate of aluminium hydroxide and phosphate.  The precipitate is washed and suspended in saline containing bactericide iv. Purified toxoid aluminium phosphate: different purification steps are involved using magnesium hydroxide to precipitate colour, ammonium sulphate, cadimum chloride, and some protiens

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