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Central Vietnam V eterinary Institute

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

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Central Vietnam V eterinary Institute

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  1. Central Vietnam Veterinary Institute Preparation and Evaluation of an Inactivated Multi-Strain PRRS Vaccine Made with Viruses Isolated from Vietnam This study is presented by Hung Vu Khac, Ph.D. of the Central Vietnam Veterinarian Institute in NhaTrang, 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 (2007-2010) 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 Select PRRSVs for vaccine preparation Evaluate the vaccine in vivo Produce the trial-batch of vaccine PRRSV Sequencing to evaluate field samples Isolate viruses from sequenced samples

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

  5. 2. PRRSV Sequencing • ORF5-Sequencing for 312 field samples

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

  7. 2. PRRSV Evaluation PRRSV Cases in Vietnam

  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?

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

  11. 4. Select PRRS Viruses for Vaccine Preparation 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.

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

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

  15. 5. Produce the Trial Batch of Vaccine Six Selected Viruses for the Trial Batch of Vaccine

  16. Western Blots of Ag-Extracts for the Trial Batch of Vaccine Virus #1 Virus #2 Virus #3 Virus #4 Virus #5 Virus #6 S 2x 3x 4x v 2x 3x 4x v2x 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

  17. 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.

  18. 6. Evaluate the Trial Batch of Vaccine 6.2. 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.

  19. 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

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

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

  22. 6. Evaluate the Trial Batch of Vaccine ELISA

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

  24. 6. Evaluate the Trial Batch of Vaccine Western Blots S Pig #1 Pig #10 Pig #2 Pig #3 Pig #4MJ S Pig #5 Pig #9 Pig #6 Pig #7 Pig #8MJ 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 S Pig #1 Pig #2 Pig #7 Pig #3 Pig #4MJ S Pig #5 Pig #6 Pig #8 Pig #9 Pig #10MJ 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

  25. 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. • 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 autogenousMJPRRS ®vaccine in nursery pigs”

  27. Thank you for your attention Central Vietnam Veterinary Institute

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