1. The researchers aimed to develop a plant-based vaccine for lumpy skin disease (LSD) in cattle by attaching an LSD epitope to the coat protein of tobacco mosaic virus (TMV) and infecting plants. 2. They were unsuccessful in extracting a fully assembled TMV virus with the LSD epitope from infected plants. However, young infected plants did show signs of infection. 3. As next steps, they will try infecting different plant species, using different virus extraction methods, and testing an in vitro infection method using a fluorescent reporter protein instead of the LSD epitope. Developing an effective plant-based LSD vaccine could help protect Botswana's cattle industry.

1. Attachment of Large Lumpy Skin Disease Epitopes to
Tobacco Mosaic Virus Coat Protein
Travis Yiwey Tu, Tianna Sheih, and Dr. Larry Grill
Claremont McKenna College, Scripps College, Ferré/Marquet Vaccine Research Center
INTRODUCTION
METHODS & MATERIALS
1.Werner S, Marillonnet S, House G, Klimyuk V, Gleba Y (2006) Immunoabsorbant
nanoparticles based on a tobamovirus displaying protein A. Proc Natl Acad Sci USA. Applied
Biological Scienes:103 (47) : 17581 – 18026
2. Chapman S, Faulkner C, Kaiserli E, Gracia-Mata C, Savenkov E, Roberts A, Oparka K,
Christie JM (2008) The photoreversible fluorescent protein iLOV outperforms GFP as a reporter
of plant virus infection. Proc Natl Acad Sci USA. Plant Biology:105 (50) : 19563 - 20045, E107 -
E114
RESULTS CONTINUED
RESULTS
Lumpy Skin Disease
(LSD) is a viral
infection that affects
cattle, making it a
serious threat to
Botswana’s economy.
Agroinfection:
1. Design LSD 20 epitope (Figure 3.)
2. Attach to TMV coat protein using glycine-rich linker
(GL) or helical linker (HL)1 via TOPO plasmid
3. Transform into A. tumefaciens
4. Infiltrate N. benthamiana plants
5. Extract and purify virus. Test with Western Blot.
In vitro:
1. Attach iLOV epitope to TMV CP using GL or HL via
TOPO plasmid
2. Use transcription kit to make TMV RNA
3. Infiltrate N. benthamiana plants with TMV RNA
4. TMV self-assembles
5. Extract and purify virus. Test with Western Blot.
ACKNOWLEDGMENTS
We would like to thank Dr. Larry Grill for all of his guidance and
expertise here at the Vaccine Research Center as well as in the lab at
Botswana. We are also indebted to Kelvin Phiri for his unending patience
and support. A major thank you to Caitie Tanaka, Andrea Gochi, and
Ryan McComb at KGI for all of their time and advice. We also want to
thank W. M. Keck Science Department for funding this project.
• TOPO plasmid is re-ligating without iLOV
• Test efficiency of restriction enzymes
• Use alkaline phosphatase on digested vector
plasmids to increase ligation efficiency
• Use agar on the plate as a negative control to
prevent false positive colony PCRs
• Absence of the 35 kDa band suggests that LSD 20’s size
may be hindering the recombinant TMV’s assembly
• Use Western Blot to find unassembled virus in
virus extraction byproducts.
• Infect Glurk plants as a control
• Try other virus extraction protocols
• Avoid plant cell nucleus (In Vitro method)
Figure 5. Left: Agroinfected N. benthamiana with pTRBO
TMV CP + HL + LSD 20. Right: Expected results for in
vitro method with TMV CP + GL/HL + iLOV2.
METHODS AND MATERIALS CONTINUED
Figure 3. Kyte-Doolittle Plot of the amino acid sequence for LSD
coat protein. Hydrophilic portions are below 0 and LSD 20 is taken
from positions 145-285, between the yellow lines. The epitope should
be as large and hydrophilic as possible.
• Young plants showed aggressive signs of infection with
TMV CP + HL + LSD 20 but Coomassie-stained SDS gel
of the virus extractions did not yield the expected band
• Older, flowering plants showed no signs of infection with
TMV CP + GL + LSD 20 or TMV CP+GL+LSD 20
• Extraction of iLOV construct from TOPO plasmid
unsuccessful.
Plant-based vaccines can neutralize this threat. They are
made by attaching an epitope to a plant specific virus such
as tobacco mosaic virus (TMV) making them cheaper and
easier to produce and store than traditional vaccines.
Outline:
1. Attach LSD 20 epitope: 140 a.a long to TMV coat
protein (CP) and infect N. benthamiana by agroinfection.
2. Use iLOV to test new In Vitro infection method.
REFERENCES
Figure 4. SDS gel analysis of TMV CP+HL+LSD 20 extracted
from young plants. No expected band at 35 kDa in “TMV” column
for fully assembled TMV CP+HL+ LSD20
Figure 1. Calf with LSD
HL or GL
(45 bp)
LSD 20 or iLOV (420 or
330 bp, respectively)
Figure 2. TMV with construct attached to its CP
37 kDa
35 kDa
Ladder
PJL24
GPCtrl
GP
SNCtrl
SN
WPCtrl
WP
TMVCtrl
TMV
PJ24= TMV positive control
GP= Green Pellet (initial extract)
SN=Supernatant
WP= White Pellet
TMV= Final extract (fully
assembled virus)
KEY
DISCUSSION AND FUTURE STUDIES