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-reproductiveand-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 Agrobacteriummediated transformation
Development of plant-based vaccine
Transfer
Select a target
Identify a coding sequence
Transformation
Agrobacterium-mediated
transformation
Somatic embryogenesis
Regeneration
Animal feeding studies
Transgenic soybean
SEs
PRRS viral structure
1 nt
15428 nt
Non structural protein region
Structural protein region
ORF
1a
1b
2
3
4
5
6
7
Single-stranded RNA
30 - 40 kDa GP4 (ORF 4 product)
24 -26 kDa GP5 (ORF 5 product)
18 - 19 kDa M protein (ORF 6
product)
15 kDa N protein (ORF 7 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