Lecture No' 14 April 1st, 2004

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Lecture No. 14 - April 1st, 2004 Testing
veterinary vaccines Clayton Kelling
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Testing Veterinary Vaccines Vaccinology April
1, 2004 Clayton L. Kelling Dept. of Veterinary
Biomedical Sciences University of
Nebraska-Lincoln
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Testing Veterinary Vaccines A. INTRODUCTION 1.
Expectations for a biological product -
yield expected results.
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Testing Veterinary Vaccines

• 2. Requirements for veterinary biologicals
• - tested pure, safe, potent, and
  efficacious
• -Virus-Serum-Toxin Act, 1913

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B. VACCINE PURITY 1. Test for
contamination -extraneous agents 2. Confirm
identity of fractions
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C. VACCINE SAFETY 1. Most significant risks
associated with the use of vaccines -
residual virulence and toxicity - allergic
effects
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VACCINE SAFETY (cont) 2. Residual virulence
and toxicity - BVDV - mucosal disease -
Endotoxins - abortion - Bluetongue -
fetopathic - Local Reactions -injection
sites - Postvaccinal disease - canine
distemper encephalitis
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VACCINE SAFETY (cont) 3. Hypersensitivity
Reactions - Type I - IgE, mast cells,
basophils - Rapid onset - Associated with
repeat administration of vaccine
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VACCINE SAFETY (cont) 3. Hypersensitivity
Reactions - Type III - Immune complex
formation - Local or generalized
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VACCINE SAFETY (cont) 3. Hypersensitivity
Reactions - Type IV - Cell-mediated, -
Granuloma formation - at site of
injection - water in oil adjuvants
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D. VACCINE POTENCY 1. Potency - relative
strength of a biological product 2. Potency
tests are conducted on each serial (lot) of
product prior to releasing for
marketing.
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. VACCINE POTENCY (cont) 3. Determination of
Potency a. Titration - live viral
product b. Relative potency -
inactivated product - compared to
standard
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3. Determination of Potency - cont
c. Vaccination - challenge efficacy test
eg. Inactivated poultry product d.
Host animal serology - antibody titer
correlated with protection
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VACCINE POTENCY (cont) e. Vaccination and
challenge of lab animals - hamster for
leptospiral bacterins f. Vaccination and
antibody production - clostridial
vaccines
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VACCINE POTENCY (cont)

• g. In vitro potency tests
• - ELISA for E. coli pilus antigens

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E. VACCINE EFFICACY 1. Demonstration that the
product will protect the host animal against
challenge exposure with the
pathogen. 2. Laboratory studies -
Laboratory challenge models - Specific
measures - Number of animals - variable
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E. VACCINE EFFICACY

• 2. Laboratory studies
• - Laboratory challenge models
• - Specific measures
• - Number of animals - variable

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VACCINE EFFICACY (CONT) 3. Post-licensing
clinical field trials. - Field conditions -
Demonstrate field efficacy of the vaccine -
Confirmation of benefits of vaccine for
marketing - Comparison with competition - To
gain endorsements
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F. SUPPLEMENTAL ASSAY METHOD FOR PSEUDORABIES
VIRUS CHALLENGE TEST IN SWINE A.
Introduction - In vivo method - challenge of
swine to determine potency of killed
pseudorabies virus (PRV) vaccines or the
immunogenecity of PRV master seed viruses
(9CFR).
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B. Materials - PRV susceptible swine -
Numbers of animals - PRV challenge, Becker
strain C. Preparation for test - Personnel
must have training and experience D.
Performance of the test - First vaccination,
bleed animals - Second vaccination if label
recommends
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Performance of the test (cont) - Draw blood
14-28 days ,postvaccination - Test serial gt 4
of 5 VS have not seroconverted, challenge. -
Challenge Swine - test serial lt4 of 5 VS not
seroconverted - master seed virus -
intranasal administration - determine and
record rectal temperatures daily (1-7 d
pc) - note and record all clinical signs,
daily
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-Clinical observations - severe central nervous
system signs falling, difficulty or inability
to rise, rise, head tilt, head pressing,
paralysis, tremors, spasms, convulsions,
padding, opisthotonos, circling, and coma. -
plus pruritus, teeth grinding, empty chewing,
persistent or unusual vocalization,
disorientation, ataxia, stumbling, loss of
postural control, and proprioceptive placing
deficits.
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-Clinical observations (cont) Clinical signs of
PRV infection in VS include the CNS signs listed
previously, and - anorexia 2 or more
consecutive DPC - fever gt 106oF(gt 41.1oC) on any
2 or more DPC - stunting, weakness, vomiting,
diarrhea, constipation on any 2 or more DPC -
blindness or other ocular disease, -persistent
sneezing, persistent or deep coughing, labored
breathing, and pneumonia.
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- Clinical observations (cont) The following
clinical signs of PRV infection will not be
included in the evaluations, due to their
ambiguity - transient inappetence, depression,
shivering, - occasional sneezing, occasional
upper respiratory cough - reduction in rate of
weight gain - fever which is transient or lt
106oF (lt 41.1oC).
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• - Interpretation of the test results
• - validity requirements
• Inconclusive gt 4 of the 5 control swine do
  not develop CNS signs or die on one or more
  days.
• Test serial unsatisfactory - gt 2 of VS develop
  clinical signs or die.
• Master seed unsatisfactory - gt 2 of VS develop
  clinical signs or die.
• _______________
• Center for Veterinary Biologies, NVSL Testing
  Protocol

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Experimental Challenge Study van den Hurk, CJVR
2001

• BHV-1 challenge viruses mutant, 108
• MLV BHV-1 vaccine
• Results
• Vaccinated calves less wt. Loss
• less temp rise
• lower nasal
  scores

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Effect of Vaccination on Weight Gain van den
Hurk, 2001
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Effect of Vaccination on Rectal Temperature van
den Hurk, 2001
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Effect of Vaccination on Nasal Scores van den
Hurk, 2001
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Outcome Calves were protected against
challenge with either virus by vaccination
with MLV
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G. VACCINE FAILURE 1. Wrong strain of
organisms or the wrong antigens - Method
of production destroy protective epitopes
2. Failure of an effective vaccine to
stimulate protective immunity - Maybe due
to 1. Unsatisfactory administration
2. A live vaccine may have died 3. Timing
- Vaccination Schedules
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G. VACCINE FAILURE (cont) - Principles
underlying decisions on vaccination
schedules A. Requirements are variable B.
Optimal age for vaccination - - Disease risk is
greatest in young animals -Maternal antibody
interference
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G. VACCINE FAILURE (cont) 3. Animal
fails to develop immune response -
Protection is never absolute -
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H. FACTORS THAT INFLUENCE VACCINE USE 1.
Diagnosis - - Absolute identification of
cause 2. Establish that the immune
response can protect against disease. -
Immune response is detrimental in some
diseases - Immune response is not protective
for some diseases
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H. FACTORS THAT INFLUENCE VACCINE USE (cont)
3. Assess risks of vaccination. - Pneumonic
pasteurellosis - Bovine viral diarrhea virus
4. Economic Considerations - Cost benefit
factors - Many vaccines available
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H. FACTORS THAT INFLUENCE VACCINE USE (cont)
5. Decision to use a vaccine - Based on
expected extra profit vs. costs - Example
bovine respiratory disease
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Laboratory clinical study vs. field clinical study

• Difference Natural operational conditions in a
  livestock production system
• Multifactorial diseases
• Respiratory tract diseases
• Digestive tract diseases

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Bovine Respiratory Disease

• Multifactorial
• Complex interactions
• Difficult to reproduce severe disease in
• experimental challenge models

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Experimental BVDV/BRSV Co-infections

• Animals
• Controls
• BVDV-infected
• BRSV-infected
• BVDV/BRSV-infected

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STUDY NO.1

• CLINICAL SCORES
• LESIONS

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STUDY 2

• CLINICAL SIGNS
• LESIONS

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I. POST-LICENSING CLINICAL FIELD STUDIES 1.
Description- Controlled comparative study under
field conditions 2. Advantages - Natural
conditions are critical for evaluating vaccines
for multi-etiological diseases. - Most valid
efficacy data 3. Limitations- Limitations
imposed by the management system
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CLINICAL FIELD TRIALREQUIREMENTS

• 1. Randomization
• 2. Experimental unit
• 3. Sample size
• 4. Blind
• 5. Clustering of data

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4. Requirements A. Randomization -
Allocation of animals to treatment and
control groups - Animals are assigned to
treatments so that all the animals are
equally likely to be assigned to any of the
treatments. - Eliminates bias
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4. Requirements (cont) B.
Clustering - Animals are maintained in groups
(pens or herds) - Pen is the experimental unit
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C. Experimental Unit - Smallest unit with a
unique set of important characteristics. -
Field trials in veterinary medicine, using food
animals, often the experimental unit is a
complete herd.
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C. Experimental Unit (cont) -Herd Effect -
Two biological factors important in trials
dealing with vaccination against an infectious
disease, two biological factors 1. Herd
Immunity Infectious diseases - lack of
independence among the individuals in a
group. - Occurs when unprotected animals are
protected because of the presence of protected
animals in the herd.
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Unprotected animals are protected because of the
presence of protected animals in the herd.

• Mechanisms
• Vaccinated are less susceptible to infection
• Protected animals shed less virus, if they do
  become infected

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Unprotected animals are protected because of the
presence of protected animals in the herd.

• Outcome
• Underestimation of the effectiveness of vaccine
• due to early die out of virus in the herd

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• Experimental C. Unit (cont)
• 2. Spread of vaccine virus protects controls
• - Occurs when a herd is divided in half
  (half vaccinated, half controls)
• counteract by separation of experimental
  units

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C. Experimental Unit (cont) . Number of
Experimental Units - Sample size - Depends
on 1. Level of confidence - Probability
that when no difference exists between
treatments, results show no
difference. Type 1 error when its
concluded that there is a difference,
when there is actually no difference
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3. Number of Experimental Units (cont) 2.
Level of power - Probability that a
significant difference between 2 treatments
is observed. - complement of a type II error-
when it is concluded that no difference exists
between treatments when a treatment between
treatments does exist.
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3. Number of Experimental Units (cont) 3.
Expected difference - -Size of difference
between two treatments affects the number
of experimental units required.
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3. Number of Experimental Units (cont) 4.
Expected variance - Pilot studies used to
estimate variance. -when expeected variance is
high its difficult to detect differences
between treatments -estimate from pilot
studies
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K. Measuring Responses eg. Reduction of bovine
resp disease Clinical signs are shared by many
infections. Measure of success of a
respiratory vaccine reduce the overall level
of respiratory disease. eg. Severity of
sickness Ave no. of days treated Overall
morbidity mortality
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K. Measuring Responses A case of bovine
respiratory disease - 1. Increase in body
temperature 2. Depression 3. Specific signs
(clinical disease signs body
temperature, respiratory rate,pathogen
shedding). 4. Necropsies
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- Single blind study - treatment group not
known by person making measurements. -
Double blind study - treatment not known by
person caring for animal or by person making
measurements.
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SUMMARY 1. Vaccine safety and
efficacy -Clinical studies -Lab -Field 2.
Experimental design and data analysis are
critical