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

Soybean-based viral subunit vaccine against porcine

reproductive and respiratory syndrome (PRRS)

Porcine Reproductive and Respiratory Syndrome

Deadly swine disease caused by PRRS virusEconomic 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

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

Clinical signsNo 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

PRRS control strategiesInnovative diagnostic toolsFarm management strategies

Temperature control Strict sanitation

Effective PRRS vaccineModified 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 InexpensiveSafeScalableParenteral 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)

ObjectiveTo transfer and generate transgenic

soybean carrying candidate PRRS viral subunit genes by using Agrobacterium-mediated transformation

Development of plant-based vaccine

Select a targetIdentify a coding sequence

Agrobacterium-mediated transformation

Somatic embryogenesis

SEs

Transfer Transformation

Regeneration

Transgenic soybean

Animal feeding studies

PRRS viral structure1 nt 15428 nt

Non structural protein region Structural protein region

ORF1a

1b2

34

56

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 systemAn 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 ~ Pa2Ga1,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

Kunitz

Jack

ConclusionFour 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 worksExpression of other PRRS viral subunits in

soybean Generation of homozygous transgenic linesAntigenicity assessment of these candidate

oral plant-based vaccines in mice and swine

AcknowledgementDr. Schuyler S. Korban Dr. Ksenija GasicDr. Yuepeng HanLab-matesRoyal Thai Government