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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