1. Sornkanok Vimolmangkang, Ph.D.
Soybean-based viral subunit vaccine against porcine reproductive and respiratory syndrome (PRRS)
Sornkanok Vimolmangkang, Ph.D.
Dept. Pharmacognosy and Pharmaceutical botany, Faculty of Pharmaceutical sciences, Chulalongkorn University
Dept. Natural Resources and Environmental Sciences,
University of Illinois at Urbana-Champaign

2. Porcine Reproductive and Respiratory Syndrome
Deadly swine disease caused by PRRS virus
Economic losses in swine production worldwide and up to 560 million US dollars (Neumann et al., 2005)

3. Impact of PRRS on farm Increased mortality (1.9 to 10.2 %)
Reduced average daily gain (0.38 to 0.26 kg)
Increased treatment cost per pig (US$ 1.77 to 1.91)
Reduced feed efficiency (1.77 to 1.91 kg feed per kg gain)
Dee S. et al., 1997

4. Clinical signs
No symptoms to death observed in pregnant sows, piglets, and gilts
Respiratory distress observed in all ages
Reproductive distress leads to premature farrowing or abortion in pregnant sows
Stillborn piglet
Mummified PRRS piglet

5. PRRS control strategies
Innovative diagnostic tools
Farm management strategies
Temperature control
Strict sanitation
Effective PRRS vaccine
Modified live vaccine
Viral subunit vaccine

6. Despite current vaccines are available in the market, it remains unsatisfied by producer needs.
The development of an effective PRRS vaccine that is reliable, easy to administer, and safe remains a major challenge.
an alternative vaccination strategy by using plant production systems for antigenic proteins

7. Benefits of plant-based vaccine
Inexpensive
Safe
Scalable
Parenteral or oral administration (orally used without purification)
Systemic and mucosal immune responses

8. Plant-based oral vaccine against swine diseases
porcine epidemic diarrhea virus (PEDV) (Kang et al., 2005; Oszvald et al., 2007)
porcine transmissible gastroenteritis virus (Streatfield et al., 2001; Lamphear et al., 2002 )
PRRSV (Chia et al., 2010; 2011; Chen and Lu, 2011)

9. Objective
To transfer and generate transgenic soybean carrying candidate PRRS viral subunit genes by using Agrobacterium- mediated transformation

10. Development of plant-based vaccine
Transfer
Transformation
Select a target
Identify a coding sequence
Agrobacterium-mediated transformation
Somatic embryogenesis
Regeneration
SEs
Animal feeding studies
Transgenic soybean

11. PRRS viral structure Non structural protein region
1 nt
15428 nt
Non structural protein region
Structural protein region
ORF 1a 1b 2 3 4 5 6 7
Single-stranded RNA
15 kDa N protein (ORF 7 product)
kDa GP4 (ORF 4 product)
kDa M protein (ORF 6 product)
kDa GP5 (ORF 5 product)

12. Immunogenicity
Structural proteins encoded from ORF 2 to 7 were tested in pigs and found their ability to induce immune responses
The product of ORF6 contributed to the highest response, stimulated cellular immunity (Bautista et al., 1999)
Neutralizing antibodies were detected with the highest titres in ORF5 vaccinated pigs, the consistent induction of antibodies were observed in ORF7 vaccinated pigs after three vaccinations (Barfoed et al., 2004).

13. Plasmid construction
Intermediate vector
Expression vector

14. Soybean host system
An ideal system for testing the concept of “value-added crops”
Protein-rich animal feed
Easily transported worldwide in large quantities with no special care
Ability to be transformed

15. Soybean transformation

16. Transgene expression 8 putative transgenic T0 lines
5 from Jack (Ga1, Ga2, Ga3, Ha1, Ha2)
3 from Kunitz (Oa1, Pa1, Pa2)
Jack: 1/5 PCR-positive (Ga1)
Kunitz: 3/3 PCR-positive (Oa1, Pa1, Pa2)

17. Southern blot analysis for transgene integration and T-DNA copy number

18. RT-PCR showed the presence of mRNA with different level of expression among transgenic events
Ga1 ~ Oa1
Pa1 ~ Pa2
Ga1,Oa1 > Pa1, Pa2
a. PRRSV-ORF7 primers; b. soybean tubulin primers (internal control)

19. The amount of the antigenic protein produced in transgenic lines was determined using ELISA
Jack
Kunitz

20. Conclusion
Four transgenic plants were developed: one individual from cv. Jack and three from cv. Kunitz.
All plants regenerated from Kunitz were transformed.
The presence of transgene DNA, mRNA, and protein prove the concept that PRRS viral subunit can be expressed in soybean

21. Future works Expression of other PRRS viral subunits in soybean
Generation of homozygous transgenic lines
Antigenicity assessment of these candidate oral plant-based vaccines in mice and swine

22. Acknowledgement Dr. Schuyler S. Korban Dr. Ksenija Gasic
Dr. Yuepeng Han
Lab-mates
Royal Thai Government