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

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)

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

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 http://www.thepigsite.com/pighealth/article/142/porcine-reproductive-and-respiratory-syndrome-prrs

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

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

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

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)

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

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

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) 30 - 40 kDa GP4 (ORF 4 product) 18 - 19 kDa M protein (ORF 6 product) 24 -26 kDa GP5 (ORF 5 product)

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).

Plasmid construction Intermediate vector Expression vector

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

Soybean transformation

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)

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

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)

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

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

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

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