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
•
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
- 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
• 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)