Transcript File - BHS116.3 Physiology III

Aims
• Describe whole organism vaccines and the
advantages of live/attenuated vs. killed vaccines.
• Describe each of the vaccine types given, state given
examples and briefly explain how they are prepared.
• List target populations of vaccines given in lecture
and contraindications of each type of vaccine.
• Describe clinical techniques which utilize antibodies.
Vaccines
• Vaccines induce protection against infections by
stimulating the development of Abs, long-lived
effector cells, and memory cells.
• What characteristics would an ideal vaccine
possess?
– Stable
– Cheap
– Effective - lifelong immunity with one administration
– safe/no side effects in all populations
– prevents carrier state
– does not complicate diagnostic tests
Types of Vaccines
• Whole organism vaccines
– Killed or inactivated
– Live attenuated_
– live heterologous species
• Purified macromolecules
– Toxoid
– Polysaccharides
– Subvirion
– Recombinant
– Naked DNA
Killed/Inactivated Vaccines
• Strategy: pathogen causing a disease is isolated, grown in pure
culture, killed or inactivated by physical or chemical means, then
injected to induce an immune response against that pathogen.
• Examples:
–
–
–
–
–
–
–
–
Pertussis (old whole cell vaccine)
Rabies
Hepatitis A
i.m. poliovirus (a.k.a IPV)
influenza
plague
cholera
paratyphoid fever
Why Can’t we use Killed/Inactivated
Vaccines Against all Pathogens?
• Dead pathogens are not processed by the
immune system like the living pathogen
– Immune responses that develop may not be
protective – dead pathogens usually elicit different
types of responses than live ones…
Characteristics of Killed Whole Organism
Vaccines
• DISADVANTAGE: Killed vaccines are weakly
immunogenic
• DISADVANTAGE: Killed vaccines induce only humoral
immune responses
• DISADVANTAGE: Killed vaccines do not account for
variety of mutations microbes undergo ADVANTAGE:
Killed vaccines are stable
• ADVANTAGE: Antibody responses may be sufficient
• ADVANTAGE: Safe to administer to immune
compromised and pregnant women
Live Attenuated Whole Organism Vaccines
• Strategy: Pathogen is identified and grown in culture in a
way that causes them to lose their virulence (diseaseproducing ability) but retain the ability to undergo limited
replication within the host.
• Examples of live attenuated vaccines
–
–
–
–
–
–
Measles, mumps, rubella (MMR)
Oral poliovirus vaccine (OPV)
Varicella zoster virus (VZV, chickenpox)
Rotavirus vaccine
BCG vaccine (for tuberculosis)
Yellow fever
Attenuation of a Pathogen
• Growth in a heterologous species
• Culture attenuation in artificial medium
– Organisms tend lose characteristics when not needed
– Example, is BCG vaccine for TB
– Temperature sensitive mutants
– Most pathogens grow at 36-37o
• Recombinant DNA technology (genetic knockouts
of pathogen virulence factors) – mostly
experimental.
Characteristics of Live Attenuated
Whole Organism Vaccines
• ADVANTAGE: processed by the immune system like
the actual infection (MHC-II and MHC-I pathways)
• ADVANTAGE: elicit _sustained_ immune responses
similar to that of the actual infection
• DISADVANTAGE: not very stable – may require
“cold chain”
• DISADVANTAGE: may revert to from a virulent form
to virulence – particularly in the immune compromised
Vaccines
Characteristic
Live/attenuated
Inactivated/killed
• production
selection for avirulence
• boosters
few or not needed
physical or chemical
means
multiple
• stability
less stable
more stable
• immunity
endogenously and
exogenously processed
Ab & CMI
exogenously processed
mostly Ab
• reversion to
virulent form
possible
no
• administered to
generally, no
compromised person
yes
Vaccines
• Purified macromolecules
• toxoid (e.g., tetanus, diphtheria)
Pathogenic exotoxin
Harmless preparation (Toxoid)
Chemical
Modification
Pathogenic site
Vaccines
• Purified macromolecules
• polysaccharides (e.g., pneumococcal, Hib, meningococcal)
• Streptococcus pneumoniae produces a slimy capsule
X
Slimy strep bug
with capsule
Sticky strep bug
without capsule
Example of a Virulence Factor Used as
a Vaccine
• Streptococcus pneumoniae produces a slimy capsule
Add Ab and C’ for opsonins
Slimy strep bug
with capsule
Sticky strep bug
without capsule
Example of a Virulence Factor Used as
a Vaccine
• Streptococcus pneumoniae produces a slimy capsule
Slimy strep bug
with capsule
Sticky strep bug
without capsule
Vaccines
• Purified macromolecules
• polysaccharides (e.g., pneumococcal, Hib,
meningococcal)
capsule
Bacterial cell
Grow in pure
culture - remove
capsule
Separate
and purify
capsule
vaccine
Naked DNA Vaccines
OUCH!
plasmid with
promoter only
OUCH!
Cloned gene for
influenza hemagluttinin
into plasmid with
promoter
Inject cloned gene i.m.
Naked DNA Vaccines
plasmid with
promoter only
Infect mouse with
influenza virus
Cloned gene for
influenza hemagluttinin
into plasmid with
promoter
Inject cloned gene i.m.
Infect mouse with
influenza virus
Naked DNA Vaccines
plasmid with
promoter only
X
Dies
Cloned gene for
influenza hemagluttinin
into plasmid with
promoter
Inject cloned gene i.m.
Survives
Vaccine Production
Monvalent vs. Multivalent vaccines
– refers to the number of strains targeted by the vaccine (e.g.,
heptavalent, trivalent, 23-valent)
– if the vaccine targets a single pathogen, it could refer to the
number of antigenic components of that pathogen contained in
the vaccine
– examples are:
•
•
•
•
•
hepatitis B - monovalent
hepatitis A and B - divalent vaccine now available
MMR - measles, mumps and rubella - trivalent
acellular pertussis - quadrivalent - four antigens of one pathogen
pneumococcal vaccine - 23-valent - capsules from the 23 most common
strains or serotypes of Streptococcus pneumoniae
Vaccine Production
• Formulations
• Adjuvants needed?
– Determined through use of animal models and human vaccine trials
– Alum (alum hydroxide salts) is the only one currently approved for
human use
– Cause depot effect of antigen
– Irritants
• Killed and toxoid
– chemical means - formalin or -propiolactone
– physical means - heat, UV, or -irradiation
• Preservatives:
– Benzalkonium chloride, formalin, mercuric salts
Vaccination Considerations
• Population considerations
– Global elimination - smallpox, polio, measles (??)
– Control in a population - Haemophilus influenzae
• Target groups
• Age - infants and children, adults, elderly
• Immunocompromised individuals
• Occupational or lifestyle risks - military, veterinarians, lab
workers, daycare providers, IVDU, travelers, etc.
• Special at-risk populations - prison inmates, travelers
Contraindications
• Contraindications - depending on the
vaccine:
•
•
•
•
•
immunocompromised
severe asthma, allergies
_pregnancy_
infection/disease or febrile illnesses
recent immune globulin administration
Vaccine-Preventable Diseases - still a major
health problem
• Negative attitudes concerning safety and efficacy
of vaccines
• Fear of side effects
• Physician attitudes
•
•
•
•
uncertainty about recommendations
concern about liability
inadequate reimbursement
uncertainty about safety and efficacy
Future Vaccines
• Naked DNA immunization for various diseases
including malaria, TB, HIV
• HPV – human papillomavirus
• New generation smallpox vaccine
• New generation anthrax vaccine
• An HIV vaccine?
Antibody Based Diagnostics
Abs are being used as a very important tool in the
diagnosis of diseases – not only in detecting them in
patients – but also many tests utilize the interactions
between Ag:Ab
Primary Binding Tests
• Enzyme-Linked ImmunoSorbent Assay (ELISA)
or Enzyme ImmunoAssay (EIA)
• Immunofluorescence Assays (Direct & Indirect)
• Immunoblotting or Western blotting
ELISA
• The principles of ELISAs are commonly used
clinically:
– Home pregnancy kits, rapid Strep tests, etc.
• In order to develop a specific ELISA either the Ag
or Ab must be known.
ELISA Plate
1. Add known antigens to a 96 well plastic plate
- wash away free antigen
- block unbound sites
Antigen A
Antigen B
2. Add diluted patient serum
3. Wash away unbound antibody
4. Add enzyme conjugated 2o antibody
5. Wash away unbound conjugated 2o antibody
6.
Add enzyme substrate (chromogen), develop,
then read spectrophotmetrically.
The amount of antibody bound is directly proportional to
the amount of light absorbance at the specified wavelength
A 96 Well ELISA Plate
Titer (inverse serum dilution):
2
4
8
16 32
Patient
sera
1
2
3
4
5
6
7
8
64
128
256
512 1024 +C
-C
Immunofluorescence
• Method for localizing an Ag by the use of a
fluorescently labeled Ab
• Utilizes a fluorochrome labeled Ab
ie. fluorescein isothiocyanate (FITC), phycoerythrin
(PE), rhodamine
• Requires a microscope equipped with a UV light source
• Can be used to detect antigen in tissue or Ab in patient
serum
The Direct Fluorescent Ab Test (DFA)
1 Prepare tissue specimen appropriately
2 Add fluorescein-conjugated (FITC) Ab against suspect
antigen
3 Wash away unbound Ab and view specimen under a
microscope with a UV light source.
UV light
Microscope slide
Fixed tissue specimen
Immunoblotting (aka “Western Blot)
• We will use the example of Western blotting of
patient serum to confirm a positive HIV serology
screening by ELISA
1. Dissociate proteins with detergent and a
reducing agent
SDS + 2-ME
Lab strain of HIV
2. Separate proteins on a gel based on size (molecular
weight) - polyacrylamide gel electorphoresis (PAGE)
If you stain all proteins
+
160 kD
120 kD
66 kD
55 kD
51 kD
41 kD
31 kD
24 kD
17 kD
-
3. Electrophoretically transfer separated proteins to
nitrocellulose paper (aka western blotting)
+
-
Western Blotting
4. Block excess protein
binding sites and overlay
the Western blot with
patient serum
5. Wash away unbound Ab
6. Develop as an ELISA (2o
enzyme conjugated Ab,
etc.)
Next Time
• Intro to Renal Physiology
Objectives
1. Describe whole organism vaccines and the
advantages of live/attenuated vs. killed vaccines.
2. Describe each of the vaccine types given, state
given examples and briefly explain how they are
prepared.
3. List target populations of vaccines given in lecture
and contraindications of each type of vaccine.
4. Describe clinical techniques which utilize
antibodies.