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

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) 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.

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

1. Evaluation of PRRS field samples 11 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

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

MJPRRS® Grouping for PRRS Viruses 2. PRRSV Evaluation MJPRRS® Grouping for PRRS Viruses PRRS Isolates N. American strain European Group D Group 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

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

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

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

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.

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

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

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 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

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.

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.

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

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

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

6. Evaluate the Trial Batch of Vaccine ELISA

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

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

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.

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”

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