Vaccines

Vaccines

Content

       Vaccine – introduction

       Types

       Bacterial vaccine

       Chlorea

       Pertussis

       BCG

       Viral suspension

       Virus cultivation

       Small pox vaccine - preparation

       Rabies vaccine

       Polio

       Diphtheria antitoxin

Objectives

At the end of the lecture the student will be able to

       Classify the types of vaccines

       Explain the sources of vaccine preparation

       Describe the method of preparation of Cholera, pertussis and BCG vaccines

       Explain the cultivation methods of virus for vaccine production

       Describe the various methods of preparing viral vaccine

       Explain the source and method of preparation of small pox vaccine

       Explain the method of preparation of Rabies, polio vaccine

       Describe the production of antisera

Suspension of Microorganisms

       Vaccines – Bacteria, rickettsia or viruses

       Organism – dead or living condition

Simple vaccine: Prepared – one species Eg. Plague vaccine – Pasteurella pestis

Mixed vaccine: Mixture of two or more simple vaccines   Eg. Typhoid – paratyphoid A and B – mixing three simple vaccines – one from salmonella typhi and two from salmonella paratyphi

       Univalent vaccine – Prepared from one strain

             Eg. Yellow fever vaccine – 17D strain of yellow fever virus

       Polyvalent vaccine – prepared from more than one strain

             Eg. 1. Chlorea vaccine – two main serological type of Vibrio cholerae – Inaba and Ogawa

                   2. Poliomyelitis vaccine – Type I, II,III of Polio virus

                   3. TAB vaccine – mixed and polyvalent vaccine made up of A and B strains of Salmonella paratyphi

Bacterial Variation

       Causes – loss of antigens from the cells

       Hence these variants should not be used in vaccine preparation

       Defiency of antigens – unable to stimulate the production of antibodies – if used for immunisation – will produce little or no immunity

       Pharmacopoeia – specifications – use of variants

1. Exact strain or strains should be used

       TAB vaccine – Strains of salmonella typhi, Salmonella paratyphi A    and B

       Plague Vaccine – capsulated form of Pasteurella pestis

       Typhus vaccine – Virulent Rickettsiae

        Yellow fever vaccine – 17D strain

2. Antigen that must be present

        Cholera vaccine – Type O antigen + Inaba and Ogawa

        TABC vaccine – O and H antigen, paratyphi – Vi antigen

3. Time of harvesting

BCG vaccine – NMT 14 days

Plague vaccine - capsule production is maximum

Killed Bacterial Suspensions

       Each strain used for preparation – carefully checked for freedom from variation and contaminating organism

       Inoculated – solid or liquid medium – incubated – optimum conditions – one or three days

       From solid media – cells – washed with sterile saline – centrifuged – to remove pieces of agar

       From liquid media – centrifuged – cells settles down – supernatent removed – cells washed – free from broth content – which may cause reactions on injection  - re suspended in saline

Sterilization of bacteria

Bacteria may be killed in one or more ways

1. By heat

       Low temperature – avoid damage to antigens

       56˚ C  - 1 hr

2. By chemical bactericides

       Heat treatment affects antigenicity – chemical treatment used (plague vaccine)

       Formalin  - 0.5% (pertussis and plague)

       Bactericides like phenol (cholera), thiomersal (alternate for pertussis), 75% alcohol (TAB and TABC)      

Standardization of suspension

       Total number of organism per ml – determined

        Directly – Helber cell

        Indirectly – Opacity method like Brown’s tube or photoelectric nephelometer

       Preparation – diluent – minimizes the loss of antigenicity – suitable bactericide

Cholera Vaccine

       Official Killed Bacterial Vaccine

       Intestinal infection – Spirillum Vibrio cholerae - diarrhoea

       Used – travelers – tropical countries – disease is endemic

       Protection – short lived – six months

       In the Production of vaccine – good antigenicity depends on selection of suitable strain

       A less severe form of cholera become wide spread in far east –Eltor variants – eltor vaccine, Pharmacopoeia – mixed preparation – cholera and eltor vaccine

Pertussis – Whooping cough

       Prepared from killed bacterial suspensions

       Whooping cough – Bordetella pertusis

       Common disease of childhood - babies – don’t receive antibodies from mother

       Form of triple or quadruple antigen – adjuvant effect

Living Bacterial Suspensions

       Manufacturing of dead vaccine – is not always feasible

       Sterilization methods – damages the antigens

       Best way – weaken or attenuate the organism – safe to administer but  still able to stimulate antibody productions

       Called as Live attenuated vaccine (LAV) – virulence is reduced – viable (live) – harmless

Live attenuated vaccines

Bacteria – Tuberculosis, BCG, Typhoid vaccine

Virus - Oral Polio vaccine (OPV), Measles, Rotavirus, Yellow fever, Influenza vaccine (H1N1 flu nasal spray)

Advantages - LAV

       Immunity  - stronger and more lasting – virus multiplies in the tissues

       Multiplication – smaller dose can be used

       Administration – normal route of infection is possible – which makes injection unnecessary

            Eg. Attenuated poliomyelitis vaccine – oral route – sugar lump

BCG vaccine

History of BCG vaccine

       Albert Calmette and Camille Guerin – french scientists – 1905 – developing a vaccine for – TB – living cells of Mycobacterium tuberculosis

       BCG – Bacillus Calmette-Guerin – Bacilli of Calmette and Guerin

       Cultured – bacillus - successive culturing weakened – bacillus

       Produced - more weakened strains of the bacillus - successive sub culturing every three weeks

       Research – stop - first world war- resumed in 1918 - by 1921 - tubercle bacillus - sub cultured 230 times -  so weakened - believed that it could confer immunity without causing disease in humans

       First used -  humans in 1921 - child -  Paris by Dr Weil-Hale

       Baby’s mother- had tuberculosis -  died just after the baby was born – baby also had tuberculosis - 6 mg – orally – normal - till 1927 – 969 children - vaccinated

Lubeck Disaster

       German city of Lubeck 252 infants - BCG - Pasteur Institute in Paris,

       Seventy two children developed TB and died - year as a result of the disease

       A subsequent investigation carried out by German TB experts, revealed that the vaccine had become contaminated with the distinct virulent human strain during its preparation at a local laboratory

       Two people who had worked in the local laboratory were sent to prison in 1932 for “bodily injury due to negligence

       Its use declined for several years afterwards

       Resurgence -  TB - second world war -  BCG vaccine - again used on a massive scale and public confidence in its safety was restored

       Then each country maintained its own supply

       Next few decades - each of these laboratories developed its own sub strains or “daughter strain” of BCG

       laboratory, country or person’s name with which they were associated -  Moscow and Gothenburg strains

BCG Vaccine

       Live attenuated bacterial vaccine – strain of Mycobacterium tuberculosis

       Treatment of Tuberculosis

       Orally – poor absorption in gut – intracutaneous route is used

       Preparation – preventing and detecting contamination of the product with virulent strains

       The method used to prepare killed and live vaccine is same except for live vaccine preparation

                i. No sterilization stage

                ii. Viability of the cells must be maintained

                iii. Standardization – viable count

Preparation of BCG Vaccine

       Strain – checked – antigenicity and free from pathogenicity

       Grown – liquid medium – NMT 14 days – older culture has less efficient antigens

       Organisms are separated – centrifugation – washed – suspended – vehicles – preserve its antigenicity and viability for long period of time - Freeze dried .The solution form has disadvantages

1. Even when stored – ideal condition (2-10˚) – rapidly detoriates

2. Vital test for virulence – six weeks – short life of bacillus –cannot be finished before – issue of vaccine – solve – stop further use of the batch as soon as failure of any test become known

       Freeze dried product – stored for 1 year – all tests completed before issue

       Freeze dried product – difficulties

       Material may be so fluffy – part of content is lost – vacuum is released – drying chamber

       When reconstituted with sterile saline or water - clump free homogenous suspension – not obtained

       Earlier – grinding clumps – steel balls – small sterile mill

       Non-ionic surfactant  - polyoxyethylene, dextran – added growth medium – clumping avoided

Advantages

  1. The organism grow throughout the medium instead of as a tough surface pellicle
  2. Clumping  - not formed
  3. Reconstitution easy
  4. Improved the appearance of the product, fluffiness was reduced

       Glucose – added to medium – prevents excessive drying and allows retention of optimum amount of moisture

Viral Suspension

       Immunity after viral infection – long lasting

       Eg. Measles, mumps, small pox and yellow fever

       Reason – how they infect

       Enter – mucous membrane – transported to all parts of reticulo endothelial cells – phacocytosis – ingest viruses – can destroy those which have low virulence

       Long incubation period of virus – 2-3 weeks – characteristics of viral disease – during which it provides continuous and strong antigenic stimulus in our body  - actually produce antibodies

Viral Vaccine

Cultivation of viruses

       Intracellular parasites – grow only within other living cells

                       Free- living animals

                       Fertile eggs

                       Tissue culture

Free Living Animals

       Very few vaccines – free living animals

       Product – good antigens

       Method – inconvenient, costly, contamination is difficult to prevent

       Eg. Typhus vaccine – Rickettsiae – lungs of small rodents, Peritoneal cavities of gebrils

       Rabies vaccine – brains of sheep or rabbits

Fertile Eggs

       Viruses can be grown  - part of chick embryo

Advantages over free living animals

       Easy to keep the product free from contamination

Regions of egg used in preparation of official Vaccine

Precautions – Fertile eggs

       Strict aseptic techniques should be maintained – prevent bacterial contamination

       Yolk sac – excellent medium – bacterial growth – even though amniotic and allantoic fluids – antibacterial – cannot cope with heavy infection

       Repeated passage from egg to egg – avoided – virus – less virulent to host tissue

       To ensure adequate supply  - virus of virulence – grown in one batch – freeze dried – stored at low temperature – used for many future batches

       Viruses grown – yolk sac or embryo – separated by grinding – traces of egg protein – vaccine – reactions like serum protein

       When these two regions – used – harvesting time – eggs should not be more than 10-11 days old – proteins are not sufficiently developed  - hypersensitivity reactions

       Eggs – candled – confirm – embryos are alive

       Eggs – bright light – spontaneous movement of blood vessels – living embryo

Tissue Culture

  1. Selection of suitable tissue

       A large number of tissues can be successfully cultivated outside the animal body bur cetain virus will grow – only in primate cells – monkey kidneys

       Tissue – free from living microorganism

       TB – monkeys – confirm – quarantine – post mortem before use of kidneys

       Many years – believed – chick embryo – safe – but - carrier of virus – avian leucosis – present

       Avian virus – tumors in birds – no evidence of transmission to humans - leucosis free flocks are used for measles vaccine

       Monkey – carry – more viruses – most are non pathogenic – prevented – long quarantine – strong and constant vigilance

Establishment of Growth

       Organ or tissue – removed – surgical procedures

       Cut or minced – trypsin  added to disperse the cells

       Result is a suspension of cells or small aggregates

  1. Suspended cell culture:

       Cells are suspended – liquid medium

       Aim is to simply to maintain the cell metabolism

  1. Fixed cell culture:

       Fewer cells are added to medium

       Allow to settle on one side  - large flat sided bottle

       Incubation – attached to bottom glass and multiply into uniform layer of one cell thick

       When the cells spread over the lower side of the bottle the medium changed – from the one which support growth to medium that maintain cell metabolism

       Fixed cell culture gives higher yield of virus per cell – since multiplication – viability of cells are more

Media Composition

       Extremely complex media – required – grow and maintain these cultures

       Balanced salt solution – optimum PH and osmotic pressure

       Nutrients added – complex materials like serum and proteins – excluded – reactions when vaccine is administered

       Essential amino acids, growth factors, dextrose, purines, pyrimidines and inorganic salts

       PH indicator – phenol red – state of cell metabolism – PH falls – indicate change in medium

       Antibiotics – antibacterial and antifungal

Cultivation of Virus in the Cells

       After the suspended cells have become adjusted to the medium or monolayer is formed – seed virus added – culture – incubator – slowly rocked – prevent  - accumulation of harmful metabolites – to ensure free exchange of oxygen and CO2

       Virus – invade cells – multiply – released into medium

       Suspended cells allowed to settle down – removed aseptically

Smallpox Vaccine

       Free living animals

       Initially living cowpox virus – good immunity to small pox – both are closely related species

       Vaccine IS OBTAINED FROM lesions produced on the skin of suitable living mammals – calves or sheep

Selection of animals

       Healthy calves or sheep – quarantined – examined for communicable diseases

Inoculation

       Flanks (bet rib and hip) and abdomen – scrubbed, disinfected, shaved, rescrubbed and redisinfected. Then in special room the shaved areas are

  1. Scarified  - lightly scratched with a comb like device without drawing blood
  2. Inoculated –by rubbing seed virus of known potency into scratches

Incubation

       Next four to five days – vesicles containing virus develop along the lines of scarification – after this period – every precaution – taken – keep the inoculated areas aseptically clean

Harvesting

       Animals – killed – exsanguinated, washed

       Contents of vesicles – lymph – removed – curettage (scraping with special spoon – very sharp edge)

       Pooled material - homogenized

       Post mortem – animals – carcase – confirm absence of infectious diseases

Purification

       Lymph – grinded – equal volume of glycerin and stored -10˚C – to kill and reduce the number of residual bacteria

More efficient methods are

       Lymph extracted – protein solvent (trichlorofluroethane) – presence of protein lowers the efficiency of bactericidal agent

       0.4% phenol – added – incubated - 22˚C – 2 days – until bacterial count low -Virus has more resistance to phenol

       Glycerin  40 % and peptone 1 %  - mixture

       Glycerin – assist bactericidal action of phenol – also provide viscosity

       Peptone – preserve the viability of virus – freeze dried

       Tests – confirm the absence of E.coli, aerobic pathogens and anaerobic pathogens

       Number of living extraneous microorganism – NMT – 500 per ml

Smallpox Vaccine - Alternative methods of preparation – Fertile eggs

       Chorioallantoic membrane  - hen’s eggs

Inoculation:

       Eggs that have been incubated for 12 days are candled – lamp – air sac – marked

       A triangle (10mm) – drawn – where Chorioallantoic membrane  is well developed

       Triangle – cut – carborundum disc – driven – dental motor – tiny groove is cut over the air space

       Triangle lifted – separated from shell membrane – drop of saline pipetted – split with blunt needle

Fertile Eggs

       Gentle suction applied – hole –over the air sac

       Air is removed – contents are drawn towards the hole

       Chorioallantaic membrane falls away – shell membrane below the triangular opening – new sac

       Split – shell membrane – widened – virus inoculated – site covered

       Sealing –hard or soft paraffin – covered with strip of transparent adhesive tape

       Eggs – incubated – care – keep – inoculation site uppermost

       Fertile eggs – vaccine – advantage – sterile than from living animals

       Product or vaccine  - living animals – called as Vaccine Lymph

Freeze dried smallpox vaccine

       Liquid vaccine – potency – a year at 10˚C – higher temp – stability is lower – protected from light

       Freeze dried product – more stable – below 10˚C – a year

       37 ˚C – month

       After reconstitution – potency – week – stored below 10˚C

Packaging

       Liquid vaccine – single dose capillary tubes – glass or plastic

       Freeze dried vaccine – multi dose container – together with suitable volumes of reconstituting fluid

Rabies Vaccine

       Louis Pasteur – First rabies vaccine

       Proved – virulence of natural (street) virus – saliva of mad dogs – increased – passage through series of several dozen rabbits – until stable- fixed virus

       Attenuated – drying the infected spinal cord of rabbits

       Degree of attenuation – length of drying

       Protection after infection – possible – rabies virus is unique – very long incubation period – 60 days (leg bite) and 30 days (bite in the region of head)

       Enough time – to stimulate adequate antibody response before the virulent virus enters the blood stream

       Later Pasteur's method – modified in two ways

  1. Rabbit spinal cord – rabbit brain (better yield)
  2. Attenuation  by drying – Inactivation with chemicals

       Rabbits or sheep – injected intracerebrally – fixed rabies virus

       Become completely paralyzed – 24 hrs – killed – brains are harvested

       Homogenized in sodium chloride injection

       Viruses – inactivated – phenol – formaldehyde, beta-propiolactone or ultraviolet light

Poliomyelitis or Polio

       Infectious disease – polio virus

       Generally cause muscular weakness

       Infection -  minor symptoms; upper respiratory tract infection (sore throat and fever), gastrointestinal disturbances (nausea, vomiting, abdominal pain, constipation or, rarely, diarrhea), and influenza-like illness

       About one to five in 1000 cases progress to paralytic disease, in - muscles become weak, floppy and poorly controlled, and, finally, completely paralyzed -  acute flaccid paralysis

       Highly contagious – fecal-oral route

       Contaminated water and food

Poliomyelitis Vaccine

       Three distinct antigenic types of poliomyelitis virus – type I, II and III

       Infection by one type gives protection against the other strains

       Include important strain of each type – polyvalent vaccine – satisfactory and long lasting immunity

Preparation

       Three  types – grown separately – suspended or fixed cell cultures – monkey kidney cells

       Rhesus monkey – quarantined – checked for TB and other communicable diseases – before and after death

       Monkey kidney cells – obtained – continuous line of cells

       Serum – not included – media – Used for maintaining the cell growth during virus propagation

       May be included – media – initiate the growth of tissue cells

       Parenteral vaccine -  NMT one part per million of serum in the final product

  1. Inactivated vaccine
  2. Attenuated (oral) vaccine

Poliomyelitis Vaccine – Inactivated

       Salk type vaccine

       Virus suspension harvested – passed through – filters – increasing fitness – remnants of tissues and bacteria

       Inactivation – 0.01 % formaldehyde – under controlled temperature and PH

       Completed in six days – twice checked for no active virus remains

       9th and 12th day – large samples – tested for absence of infective virus

       Suspension not used unless both are sterile

       Univalent vaccines – blended – trivalent product – large samples are tested

       Formaldehyde – neutralized – sodium metabisulphite  - thiomersal – added as bactericide

Poliomyelitis Vaccine – Attenuated

       Sabin type vaccine

       Same method of production except

  1. Attenuated strains - prepared – rapid passages through tissue cultures of monkey kidney cells
  2. No inactivation stage
  3. In addition to testing the freedom from other viruses, bacteria and moulds – confirm the absence of virulent poliomyelitis virus

Anti-toxins - Antibody containing Preparations

       Plasma – immune person or animal – antibodies

       Blood – collected – allow to clot – serum separated ( antibodies)

       A serum may contain antitoxic or antibacterial or antiviral antibodies -  accordingly called as antitoxic, antibacterial and antiviral serum

       Antitoxic sera – called as antitoxins

       Antisera – prepared – by artificially stimulating active immunity in animals

Diphtheria antitoxin

1. Immunization of Horses

       Horses – large, more volumes of blood withdrawn, easy to handle

       RBC – settle quickly, pack tightly, easy separation of serum

       Other animals (goats) – used – sensitive to horse serum

       Horses – isolated – 7 days

  1. Infectious disease – glanders – Actinobacillus mallei
  2. Immunized to tetanus
  3. Blood examined for existing antibodies

       Increasing amount – toxoid injected – horse neck muscles – every few days – several months

       First dose – 5 ml – 600 ml – satisfactory antibody titre attained

       8 lts blood withdrawn aseptically – jugular vein – bottles anticoagulant solution

       Bleeding – twice daily – next eight days – 10 days rest

       Short course – antigen administered – stimulate further antibody productions – 3 bleedings

       Continued – until animal stops producing satisfactory antitoxin titre – after 4-5 courses

       Blood stored – refrigerated – cells settled

       Plasma is siphoned off and calcium chloride added – clotting

       Clot – serum – filtration

2. Refinement of the serum

       Serum contains high concentration –proteins – albumin, beta-globulin, gamma-globulin

       Similar to human proteins – species variation – act as antigen – hypersensitivity reaction in humans

                             Severe anaphylactic shock

                             Serum sickness

       To reduce protein content – 2 methods used

  1. Concentration -  fractional precipitation

       Salt precipitation  - ammonium sulphate is added to serum – to form 1/3 saturated solution

       ɤ globulin fraction – precipitates – separated and discarded

       More salt added – to give half saturation – ß globulin fraction with its associated antitoxin slowly precipitated

       Liquid portion – albumin – filter press – removed

       Precipitate – removed – filter cloth – sheets of cellophane bags – suspended – tank chlorinated running water

       Dialysis – ammonium sulphate – passes out cellophane – removed

       Antitoxic globulin remains in bag

       Process – one or two days – chlorine – prevent microbial contamination

       Solution – isotonicity – blood plasma – preservative added – passed – pyrogen removing and sterilizing filters

       Serum sickness – crude serum – 50 % - reduced by half

2.       Concentration by proteolytic digestion

       Serum is diluted and pepsin added – PH  4 – optimum for enzyme activity

       Incubate – 37˚C – 2 days – following changes occurs

Ø  Albumin – completely digested – product passed – dialyzing membrane

Ø  ɤ globulin fraction – partly digested and precipitated at this pH

Ø  Beta globulin – split – 2 fragments – one have antitoxic activity

Ø  Filtered – ppt gamma globulin – filtrate – ultra filtration – dialyzable products of digestion, inorganic salts and bulk of water – removed

Ø  Concentrate + ammonium sulphate – heated 55 ˚C /1 hr – Inactive fragment of ß globulin denatured – precipitated

Ø  Filtered off – more salt added – precipitate – active fragment – Separated, dialyzed, isotonicity – preserved

Ø  Serum sickness – only 5 %

Summary

       Vaccines – Bacteria, rickettsia or viruses

       Organism – dead or living condition

       Cholera - Official Killed Bacterial Vaccine

       Intestinal infection – Spirillum Vibrio cholerae

       Used – travellers – tropical countries – disease is endemic

       BCG - Live attenuated bacterial vaccine – strain of Mycobacterium tuberculosis

       Treatment of Tuberculosis

       Intracellular parasites – grow only within other living cells - Free- living animals, fertile eggs and tissue culture

       Easy to keep the product free from contamination

       At the age of use embryo cannot produce antiviral antibodies – affects the yield

       Louis Pasteur – First rabies vaccine

       Proved – virulence of natural (street) virus – saliva of mad dogs – increased – passage through series of several dozen rabbits – until stable- fixed virus

       Attenuated – drying the infected spinal cord of rabbits

       Degree of attenuation – length of drying

       Protection after infection – possible – rabies virus is unique – very long incubation period – 60 days (leg bite) and 30 days (bite in the region of head)

 

 

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