Current status - State of the Union - PRRS vaccine research - Dr. Mike Roof, Boehringer Ingelheim Vetmedica, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting

1. THE EVOLUTION OF PRRS CONTROL EFFORTS
AND NEW MODELS OF VACCINE ASSESSMENT
Mike Roof, PhD
Executive Director Bio R&D

2. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 2
THE GOOD, THE BAD, AND THE UGLY….
Wrong/Unsuccessful PRRS Control
• Industry wasted a lot of time and $$
trying to solve PRRS problems with
vaccines and antibiotics without
correcting pig management and bio
security. “Solution in a bottle”
• Rambo vaccine programs
• Many “PRRS”problems were caused by
Secondary agents
• SEW,MEW,3 Site, AIAO alone were not
a PRRS solution
• Did not use proper Dx and surveillance
tools
• Live viral exposure
Correct/Successful PRRS Control
• Proper Dx and surveillance
• Have a clear and defined target
• Target PRRS control as a POPULATION
• Control programs must be at the whole
herd level and must include the growing
pig
• Proper gilt isolation / acclimation
• Where, when, and how to most
effectively use vaccines
• PRRS vaccine alone is not the solution
and MUST be used with stringent
biosecurity and management
Content Provided by Dr Dale Polson

3. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 3
THREE DECADES OF PRRS CONTROL
EFFORTS
(DECADE 1)
Decade 1 – Virus characterization
• Viral genetics and diversity
• Differences between virus
(phenotype)
• Mixed infections
• Host response to PRRS
• Focus was on the individual virus
and individual pig response
1994 Swenson PRRS inboar semen
1995 Yoon PRRS antigenic diversity androle inDx
1995 Yoon PRRS humoral response
1995 Christopher-HenningsPersistence of PRRS inserum andsemen
1996 Mengeling Comparisonbetweenstrains onreproductive failure
1996 Mengeling Aleolar macrophages for PRRS detection
1997 Will PRRS route of excretion
1997 Lager Durationof homologous immunity ingilts
1997 Addreyev Genetic variationbasedonORF 5 sequence
1999 Yoon Effect of dose androute onPRRS
1999 Mengeling Dx implicationof a mixedPRRS infection
1999 Mengeling Protective immunity ingilts withNADC 8
1999 Wesley Use of RFLP to differentiate PRRS isolates
1999 Mengeling Diagnostic implications of multiple isolate PRRS exposure

4. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 4
THREE DECADES OF PRRS CONTROL
(DECADE 2)
Decade 2 – Aerosols, Fomites,
Transmission
• Viral stability both genetically as well as
in the environment
• Fomites
• Mechanical transmission
• Initial methods for area surveillance
• Forms of PRRS control
• Disinfection
• Filters
• More defined PRRS virus
characterization
• Homo/hetero protection
• Duration of…..
• Age, route, dose
2001 Cho Influence of isolate on pathogenicity (aerosol)
2001 Yoon Genetic and antigenic stability of PRRS
2002 Otake Mosquitoes
2002 Batista Duration of persistance and shedding
2002 Dee Mechanical transmission
2002 Chang Evolution of PRRS during sequential passage in pigs
2003 Otake Biological vectors for PRRS
2003 Otake Housefly and PRRS survival
2003 Lager Strain predominance following multi-strain exposure
2003 Mengeling Safety and efficacy of PRRS strains
2004 Dee Intervention strategies to prevent mechanical spread
2004 Johnson Pathology and humoral response is dose dependent in acute
2005 Dee Disinectants for transport vehicles
2005 Dee Thermo-assisted drying for vehicles
2005 Schurrer PRRS in house fly
2006 Cho Impact of animal age, bacteria, and isolate on shedding
2006 Dee Alternate air-filtration system
2006 Dee Filtration to reduce viral transmission
2006 Fang PRRS infectious clone
2007 Cano Vaccination on homologous viral infection
2007 Cano Vaccination on heterologous viral infection
2007 Hermann Effect of Temp and humidity on PRRS
2008 Prickett Oral fluids
2009 Pitkin Regional models for aerosol spread
2009 Pitkin Fomites and person
2009 Pitkin House fly and PRRS
2009 Dee Alternate strategies to Merv 16 filters
2009 Hermann Dose response estimates of airborne PRRS
2009 Klinge PRRS replication is pig age dependent

5. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 5
THREE DECADES OF PRRS CONTROL
(DECADE 3)
Decade 3 – The Regional/Pop.
Approach
• Regional Control programs
• Disease Bio-Portal
• Oral fluid detection and Dx for
groups of pigs
• Regional Diversity of PRRS
• Group assessment (not pig)
• PRRS shedding/Control of shedding
of groups of pigs
• Focus on regions, Group dynamics,
and population methods
2010 Dee Regional models andfiltrationefficacy
2010 Prickett Oral basedfluiddiagnostics
2010 Ellingson PRRSvaccine chimera as a vaccine
2011 Dee UV light to inactivate PRRS
2011 Holtkamp Classificationof PRRSstatus swine herds
2012 Dee Longtermeffect of airfiltrationonnew PRRSinfect
2012 Linhares Effect of PRRSMLV onsheddingof wt virus
2012 Ramirez Efficient surveillance of pigs populations ugins oral fluid
2013 Olsen Probability of PRRSdetectionvia penbasedoral fluids
2014 Brito Genetic diversity of PRRScollectedinairfromdiff regions
2014 Alonso Epidemiological study of airfiltrationinlarge herds
2017 Rotolo Samplingmethods forgrouphousedanimals

6. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 6
AREA COORDINATED DISEASE CONTROL:
TOOLS, METHODS & COLLABORATIONS
TARGETING PRRS
“1,000 foot view…”

7. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 7
AREA COORDINATED DISEASE CONTROL:
TOOLS, METHODS & COLLABORATIONS
TARGETING PRRS
“100,000 foot view…”

8. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 8
AREA COORDINATED DISEASE CONTROL:
TOOLS, METHODS & COLLABORATIONS
TARGETING PRRS
“1,000 mile view…”

9. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 9
VACCINE LICENSURE REQUIREMENTS
USDA – Memorandum 800.202
Memorandum addresses basic
principles for conducting efficacy
studies for biological products
• Statistically significant (USDA method)
• Prevent CLINICAL disease or reduce
clinical severity
• Do not accept serology
• Reduction in the magnitude of shedding
are typically not sufficient to support
efficacy.
• Lab based studies carry most weight
EMEA - Europe
EU memorandum for conducting
efficacy studies
• Statistically significant
• Prevent clinical disease or reduce
clinical severity
• Will accept shedding or reduced
magnitude of shedding due to a vaccine
effect for some pathogens.
• Will accept production data (ADG)
• Heavy weight on field efficacy supported
by lab studies

10. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 10
VACCINE ASSESSMENT – CLINICAL FOCUS!!

11. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 11
ROLE OF VACCINE FOR PRRS CONTROL
Direct benefit – proven reduced clinical disease following wild type exposure in
heterologous challenge studies
• Pig model – % lung lesions
• Sow Model – reproductive performance
With a POPULATION and REGIONAL focus we need to consider different models
to evaluate vaccines to supplement the clinical picture

12. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 12
MODEL #1 – PRRS “SHIELD” MODEL (I.E.
ROGER MAIN MODEL)
What is the effect of challenge dose in vaccinated pigs?
1. What challenge dose of virulent PRRSV is required to cause infection and
consequences of infection in a vaccinated animal?
• viremia, fever, reduced ADWG
• How does vaccination impact the host PRRS status to various dose exposures?
2. Is there a challenge dose where vaccination prevents consequences of
infection?
• Does vaccine “blunt” or “shield” a pig from PRRS infection at any level?
• Aerosols, fomites, and transmission with lower levels of PRRS exposure (vs pig to pig)
• Applications to regional control programs

13. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 13
STUDY DESIGN
Randomized, blinded vaccination-challenge study
Pigs used for the study were 3 weeks of age and PRRSV naïve; confirmed
PCR negative for PRRSV
Statistics
• Results summarized via descriptive statistics by day, challenge dose and group
• For number days pyrexic and ADWG post-challenge
• P-value < 0.05 was used to indicate statistical significance

14. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 14
STUDY DESIGN
Group
No. Inglevac
PRRS® MLV
Vaccinated
Pigs
(2ml IM)
No. Non-
vaccinated
Challenge
Control
Pigs
PRRSV
SDSU-73
Challenge
Dosage
(Log10TCID50/
ml)
(2ml IN)
Study
Termination
Day 0 Day 0 Day 28 Day 70
1 10 10 4 logs
2 10 10 3 logs
3 10 10 2 logs
4 10 10 1 log
5 10 - None

15. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 15
STUDY DESIGN
Parameter Day
Viremia PCR (+/-) 0, 7,14, 21, 28, 31, 33, 35, 38, 42, and
weekly thereafter until day 70
Temperature
(Pyrexia defined as a rectal temp >
40.0°C)
Day 27
Daily for 14 days until Day 42
ADWG 0, 28, 70

16. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 16
RESULTS – INTERPRETATION AND
CONSIDERATIONS
• Viremia is measured by PCR which was the most conservative evaluation and
measure of vaccination impact.
• At high CT values that are PCR positive, we expect many of these
samples would be virus isolation negative or very low levels of virus
• Current PCR results did not differentiate between Ingelvac and SDSU 73
and so data needs assessed by GROUP comparison of Ingelvac only vs
Vacc/Exposed.
• Future opportunities that are being considered
• Complete virus isolation
• Assess via differential PCR

17. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 17
RESULTS – VIREMIA 4LOG CHALLENGE
0%
20%
40%
60%
80%
100%
120%
28 31 33 35 38 42 49 56 63 70
%PCRPositive
Days
Ingelvac
PRRS® MLV
(challenge)
Challenge
Control (non-
vaccinated)
Ingelvac
PRRS® MLV
(no challenge)
Figure 1. Percentage of viremic pigs per treatment group challenged with 4 logs
Reduction in post-
challenge viremia
in vaccinates

18. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 18
RESULTS – VIREMIA 2LOG CHALLENGE
0%
20%
40%
60%
80%
100%
120%
28 31 33 35 38 42 49 56 63 70
%PCRPositive
Days
Ingelvac PRRS®
MLV (challenge)
Challenge
Control (non-
vaccinated)
Ingelvac PRRS®
MLV (no
challenge)
Figure 2. Percentage of viremic pigs per treatment group challenged with 2 logsVaccinates
similar to non-
challenged pigs

19. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 19
RESULTS – VIREMIA FOLLOWING 2 LOG VIRUS
CHALLENGE
Average CT Values per Group Challenged w/ 2 Logs of Virus

20. 20
RESULTS – PYREXIA/FEVER (40 C)
Mean Number Days Pyrexic Post-Challenge
Treatment Group
4 log
challenge
3 log
challenge
2 log
challenge
1 log
challenge
No
challenge
Ingelvac PRRS®
MLV
4.41 4.21 1.01 1.41 1.8
Challenge Control
(non-vaccinated)
11.2 8.8 10.0 6.0 -
Vaccinated groups had significant decrease
in fever days and maintained lower
average temperature at all challenge doses
Measurable negative impact in
non-vaccinated challenged groups
at all challenge doses
Vaccinates
not different
than non-
challenge

21. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 21
ROLE OF VACCINE - QUESTIONS
Is there a challenge dose where vaccination prevents consequences of
infection?
 At a challenge of 2 logs or less, the consequences of challenge in
vaccinated pigs were similar to non-challenged pigs
 At all challenge doses, Ingelvac® PRRS MLV mitigated the consequences of
infection as compared to challenge controls using the SDSU 73 wt virus
ADWG Viremia Temperature
↑ ↓ ↓

22. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 22
USING THIS MODEL – CAN WE MOVE VACCINE
EFFICACY FROM 2 LOGS OF RESISTANCE TO
SOMETHING HIGHER ( 3 LOGS!?)
Group Treatment #Pigs Vaccination
(Inglevac)
Challenge* Necropsy
1 PBS Control 36 None Day 56 Day 126
2 Ingelvac
PRRS
36 Day 0 Day 56 Day 126
3 Ingelvac
PRRS
36 Day 0, 28 Day 56 Day 126
4 Ingelvac
PRRS +
Immunostimulant
36 Day 0 Day 56 Day 126
• Challenge with 3 logs/dose of PRRS SDSU 73 wt virus (1 ml IN/1ml IM)

23. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 23
PCR ASSESSMENT OF THE POST-
VACCINATION EXPOSURE ( CT VALUES
REPORTED)
0.00
2.50
5.00
7.50
10.00
12.50
15.00
17.50
20.00
22.50
25.00
27.50
30.00
32.50
35.00
D56 D57 D59 D61 D63 D70 D77 D84
Control
PRRS 1ds
PRRS 2ds
PRRS + Imm

24. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 24
LOW DOSE WT EXPOSURE/VACCINE
BLUNTING EFFECT
Conclusions:
• In contrast to a model focused on clinical disease (6 logs wt chall), the 3 log model
(SDSU73) provided consistent result.
• Model may benefit from later exposure (day 56) so most animals are PCR Neg at
the time of wt exposure (reduced complexity)
• In the current test system, neither 2 dose of vaccine nor an immunostimulant
changed the post-challenge exposure PCR compared to a single dose of Ingelvac
PRRS vaccine.
• A single dose of Ingelvac PRRS remains the benchmark for efficacy and provided a
significant reduction on post-challenge viremia reduction.
• Model can/will be used to evaluate further treatments relative to their ability to
increase the “shielding” effect of vaccine when used in regional control programs.

25. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 25
MODEL #2 – BIO FILTER MODEL
Background:
• Zimmerman/Bio-assay – The real truth lies in the pig!
• Mengeling and the PRRS cocktail
• There currently is no definitive in vitro tool to define a “protected herd”
• Methods and tools have evolved – Bio Portal and NGS

26. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 26
BIO FILTER – THE HYPOTHESIS
• Hypothesis – Using the immune pig as the test system, we can expose small groups of
animals to a defined pools of PRRS isolates, and then use the pig to biologically filter
the isolates most likely to evade the current immune status.
• Implications:
• Better regional decisions/risk management
• Information is relevant to herd and region based on field based pigs and regional
PRRS isolates
• A tool that could be used by our larger integrated systems for on going herd
monitoring
• Use of new tools such Next Gen Sequencing
• If the tool is predictive of isolates that lead to a break, can we model herd
treatments to reduce the impact or stop the break?
• Does vaccine selection impact the result of the Bio-filter and PRRS response?

27. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 27
NGS PRRSV MIXED INFECTION STUDY DESIGN
• Experimentally mixed PRRSV at
approximately equal inputs at varied
ratios
• NextGen sequencing performed on
mixed samples to determine level of
sensitivity to detect mixed infection.
• Dilutions performed in both media and
PRRSV free media to approximate both
field and laboratory conditions.
• Samples also submitted to diagnostic
labs for detection; including a 1:1 mix not
analyzed by NextGen

28. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 28
NGS: EXAMPLE OF GRAPHICAL ALIGNMENT OF
MIXED INFECTION
• Example: 10 to 1 mixture in serum
• Snap-shot look at positions 10,132 – 10,181 (ORF1B) with percentages of sequences corresponding to
MLV and Field strain by sequence
• Across entire genome using data from 789 positions reveal a mixed infection of:
• MLV = 78.21%
• Field strain = 20.31%

29. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 29
SUMMARY OF RESULTS
Conclusions:
• Theoretical maximum limit of detection of mixed infection as low as 0.5%.
• Estimated level of high confidence detection of recombination as low as 1%
• Additional benefits of using NGS include:
• Generation of full genome sequence
• Detection of both presence and position of recombination
• Identical samples submitted to 2 independent diagnostic labs failed to detect mixed infection (all returned as
“MLV”) for all dilutions shown above
• Submission of 1:1 experimentally mixed sample was returned as “Mixed infection” from both labs
Sample Diluent % MLV %Field
MLV:Field 10:1 Media 78.69 20.66
MLV:Field 10:1 Serum 78.21 20.31
MLV:Field 100:1 Media 95.45 3.93
MLV:Field 100:1 Serum 95.09 4.41
MLV: Field 1,000:1 Media 99.06 0.86
MLV:Field 1,000:1 Serum 99.18 0.67
MLV:Field 10,000:1 Media 99.09 0.84
MLV:Field 10,000:1 Serum 99.03 0.89
Base line N/A 99.19 0.39

30. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 30
BIO-FILTER – STUDY DESIGN TO TEST THE
CONCEPT
Testing
• Focus is NOT on clinical disease
• Focus on relative viral load over time and compare between treatments (PCR)
• Next Gen Sequencing to characterize the viral pool in vivo
• Are there differences in the viral pool dynamic based on previous immune treatment
Results – Stay Tuned…In Progress
Group Treatment (Day 0) Challenge ( D=28) End of Study
1 Ingelvac PRRS ATP 3 isolate pool Day 42
2 Ingelvac PRRS 3 isolate pool Day 42
3 Experimental PRRS
Vaccine (MLV)
3 isolate pool Day 42
4 Neg Controls (PBS) 3 isolate pool Day 42

31. Presentation title, date, author 31
HOW DOES THIS HELP!?

32. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 32
TAKE HOME MESSAGES
Relevance in the field
For pigs or populations of pigs at risk of challenge/infection vaccine derived immunity
matters!
• Direct benefit – Reduced clinical impact in vaccinated pigs following wt exposure
• Indirect benefit
• Reduced viral load in pig and viral shedding (including aerosol)
• “Shielding” of low level exposure to further reduce risk
• Focus on reduced viral levels may be important in area control programs
• New models may improve risk management and new approach's for incremental gain
• in vitro results vs Bio assay
• Does knowing the isolate of highest risk allow better decisions?
• Can changes in vaccine use/immune status impact the results of a regional PRRS pool exposure?

33. The Evolution of PRRS Control Efforts and New Models of Vaccine Assessment , Mike Roof 33
INGELVAC® PRRS
Mitigate Consequences +
Generate Uniform Immunity +
Reduce Circulating Virus =
Improved Health & Performance
Thank you!!!!
SPECIAL THANKS TO THE FOLLOWING CONTRIBUTORS TO THIS PRESENTATION
DALE POLSON • GREG HAIWICK • ABBY PATTERSON • JOE VICTORIA • WES JOHNSON

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