1. Preparation and Evaluation of an Inactivated Multi-Strain PRRS Vaccine Made with Viruses Isolated from Vietnam Central Vietnam Veterinary Institute This study is presented by Hung Vu Khac, Ph.D. of the Central Vietnam Veterinarian Institute in Nha Trang, Vietnam.

2. PRRSV causes severe economic loss in swine production worldwide including Vietnam. Central Vietnam Veterinary Institute (CVVI) has reviewed possible options of the PRRS vaccine to control the disease nationwide. 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. Prepare and Evaluate an Inactivated PRRS Vaccine Made with Viruses Isolated from Vietnam Introduction Year PRRS Cases ReportedNo. of Pigs ProvincesDistrict s Wards InfectedDestroyed 2007186532470,57720,366 200826103956309,586300,906 20091326697,0305,847 2010492681,978812,947442,699 Review of the PRRS situation in Vietnam over 4 years (2007-2010)

3. Steps 1234 Review PRRS field samples from 2007 to 2011 PRRSV Sequencing to evaluate field samples Select PRRSVs for vaccine preparation 5 Isolate viruses from sequenced samples 6 Produce the trial-batch of vaccine Evaluate the vaccine in vivo

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

5. ORF5-Sequencing for 312 field samples Year Region Total NorthernCentralSouthern CollectedSequencedCollectedSequencedCollectedSequencedSequenced/Collected 200717031233023/51 20083519 59125/63 200923681207/33 20107850136757454179/288 20110000957878/95 Total15375194104183133312/530 2. PRRSV Sequencing

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

7. PRRSV Cases in Vietnam Year Total Sequenced Samples Special Comments (Results) 200723 1x D-2 1x S-3 21 x D-1, Typical China strain 200825 1x D-6 24 x D-1, Typical China strain 20097 1x D-2 6 x D-1, Typical China strain 2010179 1x S-3 1x D-6 177x D-1, Typical and variant of China strains 201178 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 2. PRRSV Evaluation

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

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

10. PRRS Ag-Test Kit (MJ-Biologics) to confirm PRRSV culture (X+1) 4. Select PRRS Viruses for Vaccine Preparation

11. RT- PCR to confirm PRRSV culture (X+2) TB-3 6 8 9 10 16 19 20 27 28 29 30 5 8 11 13 16 11 of 15 cultures advanced to the next passage. 4. Select PRRS Viruses for Vaccine Preparation

12. TB-3 TB- 8 TB- 9 TB- 16 TB- 28 TB- 29 TB- 30 (+) M - Amplify ORF-5 gene by RT-PCR  Re-sequencing Re-Sequencing Samples from Virus Cultures (X+3) - PRRV-Ag Test Kit 4. Select PRRS Viruses for Vaccine Preparation

13. 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. www.mjbio.com

14. S 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 S 1 2 3 1 2 3 1 2 3 1 2 3 TB-3 TB-8 TB-9 TB-10 TB-16 TB-27 TB-28 TB-29 TB-30 Finding Right Harvesting Time for Each Virus by using Western blot Envelope proteins N-Protein 5. Produce the Trial Batch of Vaccine

15. Six Selected Viruses for the Trial Batch of Vaccine No.StrainSamples fromdateMJPRRS Groups 1TB- 3Khanh Hoa2007S3- Unique Vietnam strain 2TB- 8Thai Binh2008D1- Typical China strain 3TB- 9Ha Noi2009D2- Unique Vietnam strain 4TB- 16Binh Duong2011D1- Typical NA strain 5TB- 28Ho Chi Minh2010D1- Variant of China strain 6TB- 30Ninh Binh2010D1- Variant of China strain

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

17. 6.1. Sterility Test - Bacterial contamination: I.Blood Agar II.Yeast agar III.Liver-meat anaerobic broth IV.Meat broth. - Viral contamination: i. In vivo test; a.Inject 2ml of the vaccine to pigs b.Observe for 10 days. c.RT-PCR for blood samples taken at 7 dpi. ii. In vitro test: Culture the vaccine sample on MARC-145 6. Evaluate the Trial Batch of Vaccine Results; All tests were negative  The vaccine passed all sterility tests. Results; All tests were negative  The vaccine passed all sterility tests.

18. 6.2. Safety test Results; All pigs were healthy and PCR-negative → The vaccine passed 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. 6. Evaluate the Trial Batch of Vaccine

19. 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 6. Evaluate the Trial Batch of Vaccine

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

21. Body Temperature ( o C) Vaccinated Unvaccinated 6. Evaluate the Trial Batch of Vaccine Body Temp. ( o C)

22. ELISA 6. Evaluate the Trial Batch of Vaccine

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

24. Western Blots S Pig #1 Pig #2 Pig #7 Pig #3 Pig #4 MJ S Pig #5 Pig #6 Pig #8 Pig #9 Pig #10 MJ S Pig #1 Pig #10 Pig #2 Pig #3 Pig #4 MJ S Pig #5 Pig #9 Pig #6 Pig #7 Pig #8 MJ dpi 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15 dpi 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15 Representing anti-PRRSV envelop proteins antibodies in pig serum, protective antibodies Representing anti-PRRSV N-proteins antibodies in pig serum, non-protective antibodies 6. Evaluate the Trial Batch of Vaccine

25. Summary 1.A trial batch of vaccine was made with 6 antigen extracts from 6 PRRSV strains based on MJPRRS ® Technology. 2.The trial batch met the requirements of sterility and safety tests. 3.The vaccinated group showed 1)Faster response on ELISA and body temperature after challenged. 2)10 times lower virus titer compared to the unvaccinated group. 3)Higher and faster protective antibody formation shown by Western blot. 4)These are indications of the early on-set of an immune response due to “memory cell effect” developed from the vaccine. 4.The lack of weight difference in the trials may be due to the lack of secondary infections as in a field environment.

26. 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”

27. Thank you for your attention Central Vietnam Veterinary Institute