1. Central Vietnam Veterinary Institute
Preparation and Evaluation of an Inactivated Multi-Strain PRRS Vaccine Made with Viruses Isolated from Vietnam

2. Introduction
PRRSV causes severe economic loss in swine production
worldwide including Vietnam.
Prepare and Evaluate an Inactivated PRRS Vaccine Made with Viruses Isolated from Vietnam
Swine producers and veterinarians have expressed concerns over current imported vaccines due to immunological variation among PRRS viruses for cross-protection and inherent high mutation rates of the virus.
Review of the PRRS situation in Vietnam over 4 years ( )
Year
PRRS Cases Reported
No. of Pigs
Provinces
Districts
Wards
Infected
Destroyed
2007 18 65
324
70,577
20,366
2008 26
103
956
309,586
300,906
2009 13 69
7,030
5,847
2010 49
268
1,978
812,947
442,699
Central Vietnam Veterinary Institute (CVVI) has reviewed possible options of the PRRS vaccine to control the disease nationwide.

3. Steps 1 2 3 4 5 6 Review PRRS field samples from 2007 to 2011
PRRSV Sequencing to evaluate field samples
Isolate viruses from sequenced samples
Select PRRSVs for vaccine preparation
Produce the trial-batch of vaccine
Evaluate the vaccine in vivo

4. 1. Evaluation of PRRS field samples11 2 5 7 16 24 13 8 18 19 6 4 12 1
1. Evaluation of PRRS field samples 18 21 11 68 8 12 7 5 14
Total
530
- 530 PRRS field samples collected from 2007 to 2011 2 21 3 14 53 7 15 46 9 1

5. 2. PRRSV Sequencing ORF5-Sequencing for 312 field samples Year Region
Total
Northern
Central
Southern
Collected
Sequenced
Sequenced/Collected
2007 17 31 23 3
23/51
2008 35 19 5 9 1
25/63
2009 6 8 2
7/33
2010 78 50
136 75 74 54
179/288
2011 95
78/95
153
194
104
183
133
312/530

6. MJPRRS® Grouping for PRRS Viruses
2. PRRSV Evaluation
MJPRRS® Grouping for PRRS Viruses
PRRS Isolates
N. American strain
European
Group D
Group S S
E-1
E-2
E-3
E-4
E-5
E-7
E-6
E-8 D

7. Total Sequenced Samples Special Comments (Results)
2. PRRSV Evaluation
PRRSV Cases in Vietnam
Year
Total Sequenced Samples
Special Comments (Results)
2007 23
1x D-2
1x S-3
21 x D-1, Typical China strain
2008 25
1x D-6
24 x D-1, Typical China strain
2009 7
6 x D-1, Typical China strain
2010
179
177x D-1, Typical and variant of China strains
2011 78
2x D-1, Typical NA strains
2x D-4, Typical NA strains
2x D-6, Variant of China strain
72x D-1, Typical and variant of China strains

8. 2. PRRSV Evaluation
Different approach compared to the conventional way to make the vaccine
Chasing PRRSVs
Trapping PRRSVs

9. 3. Isolate PRRSV from Field Samples
195 of 312 Sequenced samples were confirmed as VI positive.
Healthy MARC 145 Cell
CPE

10. 4. Select PRRS Viruses for Vaccine Preparation
Thirty out of 195 VI positive samples were initially selected for next steps based on
Year of sampling
Origin of sample
MJPRRS group
Finally, 6 PRRS viruses from the 3 regions of Vietnam were chosen for vaccine preparation.

11. 5. Produce the Trial Batch of Vaccine
Six Selected Viruses for the Trial Batch of Vaccine
No.
Strain
Samples from
date
MJPRRS Groups 1
TB- 3
Khanh Hoa
2007
S3- Unique Vietnam strain 2
TB- 8
Thai Binh
2008
D1- Typical China strain 3
TB- 9
Ha Noi
2009
D2- Unique Vietnam strain 4
TB- 16
Binh Duong
2011
D1- Typical NA strain 5
TB- 28
Ho Chi Minh
2010
D1- Variant of China strain 6
TB- 30
Ninh Binh

12. 5. Produce the Trial Batch of Vaccine
MJPRRS® Technology for PRRS vaccine production
Viral Antigen Concentrate
Vaccine production is focused on harvesting and concentrating viral envelope proteins from the tissue culture infected with PRRS virus before intact virus particles are assembled.
It is common belief that PRRSV envelope proteins are the most important antigens to raise good protective antibodies in a pig.

13. Western Blots of Ag-Extracts for the Trial Batch of Vaccine
Virus #1 Virus #2 Virus # Virus # Virus #5 Virus #6
S 2x 3x 4x v 2x 3x 4x v 2x 3x 4x v S 2x 3x 4x v 2x 3x 4x v 2x 3x 4x v
Envelope Proteins
N-Protein
Unique antigen preparation of the concentrated PRRSV envelope proteins in free forms

14. 6. Evaluate the Trial Batch of Vaccine
6.1. Sterility Test
- Bacterial contamination:
Blood Agar
Yeast agar
Liver-meat anaerobic broth
Meat broth.
- Viral contamination:
i. In vivo test;
Inject 2ml of the vaccine to pigs
Observe for 10 days.  
RT-PCR for blood samples taken at 7 dpi.
ii. In vitro test:
Culture the vaccine sample on MARC-145
Results; All tests were negative
The vaccine passed all sterility tests.

15. 6. Evaluate the Trial Batch of Vaccine
Safety test
Test in 4 healthy naïve pigs, 4 weeks old, PCR & ELISA negative
- Inject 4-ml ( 2 doses) of the vaccine per pig.
Observe for 21 days.
RT-PCR for blood samples taken at 7 dpi.
Results; All pigs were healthy and PCR-negative
→ The vaccine passed safety test.

16. 6. Evaluate the Trial Batch of Vaccine
6.3. Efficacy test
Ten healthy naïve pigs; 4 weeks old, PCR & ELISA negative
Inject 2-ml (1 dose) of the vaccine to 6 pigs.
Inject 2-ml of saline to 4 pigs as negative control
Challenge all pigs 28 day later via IN and IM.
Blood samples were taken at 0, 5, 10, and 15 dpi* for ELISA, PCR and Western blot analysis.
Body temperature was measured everyday after challenged.
Body weight was measured at 0, 5, 10 and 15 dpi.
* Days Post Injection of challenge virus

17. 6. Evaluate the Trial Batch of Vaccine
Weight (kg)
Average Weight (kg)

18. 6. Evaluate the Trial Batch of Vaccine
Body Temperature (oC)
Body Temp. (oC)
Vaccinated
Unvaccinated

19. 6. Evaluate the Trial Batch of Vaccine
ELISA

20. 6. Evaluate the Trial Batch of Vaccine
Quantitative - PCR
Q-PCR, CT Values
Days post challenge

21. 6. Evaluate the Trial Batch of Vaccine
Western Blots
V U V V V V U V U U
S Pig # Pig # Pig #2 Pig #3 Pig # MJ S Pig # Pig # Pig #6 Pig #7 Pig # MJ
dpi
S Pig # Pig # Pig # Pig #3 Pig #4 MJ S Pig #5 Pig # Pig # Pig #9 Pig #10 MJ
dpi
Representing anti-PRRSV envelop proteins antibodies in pig serum, protective antibodies
Representing anti-PRRSV N-proteins antibodies in pig serum, non-protective antibodies

22. Summary
A trial batch of vaccine was made with 6 antigen extracts from 6 PRRSV strains based on MJPRRS® Technology.
The trial batch met the requirements of sterility and safety tests.
The vaccinated group showed
Faster response on ELISA and body temperature after challenged.
10 times lower virus titer compared to the unvaccinated group.
Higher and faster protective antibody formation shown by Western blot.
These are indications of the early on-set of an immune response due to “memory cell effect” developed from the vaccine.
Diminished weight gain for the vaccinated group between 5 and 10 dpi may be related to lack of secondary infection that may be present in a field environment.

23. Comments
Efficacy test results are quite well matched to the study presented at AASV-2012 by Dr. Mark Wagner, “Evaluation of the efficacy of one dose of autogenous MJPRRS ® vaccine in nursery pigs”

24. Thank you for your attention
Central Vietnam Veterinary Institute
Thank you for your attention