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

The organism grow throughout the medium instead of as a tough surface pellicle
Clumping - not formed
Reconstitution easy
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

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

Suspended cell culture:

• Cells are suspended – liquid medium

• Aim is to simply to maintain the cell metabolism

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

Scarified - lightly scratched with a comb like device without drawing blood
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

Rabbit spinal cord – rabbit brain (better yield)
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

Inactivated vaccine
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

• 9^thand 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

Attenuated strains - prepared – rapid passages through tissue cultures of monkey kidney cells
No inactivation stage
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

Infectious disease – glanders – Actinobacillus mallei
Immunized to tetanus
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

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