Dr. R. Sathishkumar
Bharathiar
University
Prof. Julian Ma
St. George’s
Medical School
Name:
Position:
Institute:
•
•
•
•
•
Dr. R. Sathishkumar
Assistant Professor
Bharathiar University
Group Leader- Plant Genetic Engineering Lab
UKIERI and DAAD Fellow
Deputy Coordinator- UGC-SAP
IBSC in-charge
Academic Visitor, St. George’s
Name:
Position:
Institute:
Professor Julian K-C Ma
Hotung chair of molecular immunology,
Joint
Head of the Infection and
Immunity Research
Centre
St. George’s University of London
•
Scientific co-ordinator of Pharma-Planta, a European
Union Framework Programme 6 project to develop
pharmaceuticals from plant-derived proteins.
•
Vice Chair for the European COST Action on Molecular
Farming that co-ordinates research and policy
activities in 28 European countries.
UKIERI’s Support
2007 –
2008
• RESEARCH FELLOWSHIP Awarded to work with Prof.
Julian Ma, St. George’s University of London, London, UK
2009 2011
• Awarded the project “DEVELOPING
RECOMBINANT VACCINE AGAINST
under Thematic Partnerships
PLANT BASED
CHIKUNGUNYA”
2011
• Financial support to organize
FARMING WORKSHOP”
“PLANT
2011
• Financial support to organize “AUTUMN SCHOOL IN
BIOTECHNOLOGY FOR COLLEGE TEACHERS”
2012 –
2014
• Awarded the project “PURIFICATION OF PLANT BASED
CHIKUNGUNYA ANTIGEN AND EVALUATION OF ITS
VACCINE POTENTIAL” under Thematic Partnerships
MOLECULAR
UKIERI Supported Man Power Exchanges Between
Bharathiar University and
St. George’s University of London
Year
India to UK
UK to India
2007
1
-
2008
-
-
2009
-
2
2010
2
-
2011
-
2
2012
1
-
2013
2
-
2014
-
3
Events Conducted with the Support of
UKIERI in India
Date
Name of the
event
April 7-8, 2011
Indo-UK Joint
Plant Molecular
Farming
Workshop
September
26 – 30, 2011
Autumn School
in Biotechnology
for College
Teachers
No. of
Participants
30
30
Mode of
Publicity
• Internet
• Website
• News Papers
• Scientific
JournalCurrent
Science
Dr. R. Sathishkumar was awarded one year UKIERI
Research Fellowship to work with Prof. Julian K. C. Ma
Activities
First exchange between the Bharathiar University and
St. George’s University of London
Developed stable lines of transgenic tobacco plant
expressing Chikungunya antigen E1 and E2
Confirmed the presence of E1 and E2 in transgenic
tobacco plants by Western blots
Genetically Modified Tobacco Plants Expressing Chikungunya Antigens
Developing plant based recombinant vaccine against
Chikungunya
Activities
Indian PhD Student Mr. Varghese trained for one year in
Prof. Julian Ma’s lab
Transiently Expressed Chikungunya antigen E1 and E2,
but could not purify
One visit by Indian PI to UK and UK PI to India for
discussions.
Activities
Prof. Julian Ma made his first visit to Bharathiar University
during October 17 – 22, 2009.
Had a detailed discussion about the progress of the
project with Indian lead scientist Dr. R. Sathishkumar.
Visited all the labs in the Department of Biotechnology
and interacted with research scholars in the Department.
Delivered a lecture to the Research scholars as well as for
the Masters students.
Prof. Julian Ma with Indian group
With the all the Research Students of the Dept.
Indian PhD student Mr. Varghese worked one year in Prof Julian
Ma’s lab from March, 2010 – February, 2011
Activities
Developed a methodology for expressing E1 and E2
transiently in plants
Presented a poster for the St. Georges research day and was
awarded the prize for best poster
Work was selected for a poster presentation in “Plant Based
Vaccine and Antibodies (PBVA 2011)” held at Porto, Portugal
Presentations
Indian Student in UK
Capsid
E3
6K
E2
E1
E2
E1
1.3 kb
Structural
polyprotein
precursor
Proposed Chik virus
antigens for expression
1.4 kb
BP recombination to Gateway entry vector
pDON R/Zeo
LR recombination to Gateway
destination plant expression vectors
pEAQ-HT-DEST3
pEarley Gate-HA
pEarley Gate-His
Agroinfiltration and Protein Extraction
50 kDa
37 kDa
Western blot of plant-expressed E1 using
ChikV-specific antiserum in denaturing condition
1
Department of Biotechnology, Bharathiar University, India
2 Division
Memorable Moments during Mr. Varghese stay in St. Georges
Transient Expression of E1 and E2 in tobacco
Developing Vaccines Against
Chikungunya Virus
Inchakalody VP1,van Dolleweerd CJ2, Reljic R2, Paul M2, Ramalingam S1, Ma JKC2
Introduction:
of Clinical Sciences, St. George's, University of London
Materials and Methods
 Chikungunya virus is a mosquito-born alpha virus in the family of Togoviridae.
The virions are spherical in shape, 60 to 70 nm in size, with an icosahedral nucleocapsid
enclosed in a lipid-protein envelope.
Chikungunya virus infection can cause a debilitating illness, most often characterized by the
following, fever, headache, fatigue, muscle pain, joint pain etc.,
The genetic material is a single stranded positive sense RNA (11.7 kb long) which is
responsible for the synthesis of four non structural proteins (nsP1, nsP2, nsP3, nsP4) and five
structural proteins synthesised from a polyprotein precursor (Capsid, E3, E2, 6K, E1).
Capsid
E3
E2
6K
E1
6K
E1
E2
Structural polyprotein
precursor
E1
1.3 kb
E3
Proposed Chik virus
antigen for expression
1.4 kb
E2
2.9 kb
BP recombination to Gateway entry vector
Chikungunya Genome Organization and Replication
pDON R/Zeo
LR recombination to Gateway destination vector
Mammalian cell expression
(pcDNA-DEST40 - C terminal
His tag)
Plant expression
(pEarleyGate His, pEAQ-HT-DEST3His)
Lyovec-mediated transfection
of CHO cells
Expression in CHO cells
Agro-infiltration
Expression in Nicotiana benthamiana
Protein extraction and western blotting.
Results:
Worldwide Epidemiology of Chikungunya Virus
Confirmation of Chikungunya Antigen E1 and E2 by Western Blotting
M E2 +ve WT pEARLY pEAQ
Control
His
His
Tanzania (1952)
M E2 +ve pEARLY pEAQ
Control
His
His
M WT pEAQ pEAQ
E1
E2
50 kDa
50 kDa
Philippines (1954)
50 kDa
?
37 kDa
37 kDa
India (1963)
37 kDa
Fig.1.
India, Madagascar
(2005-2006)
Sumatra, Java,
Moluccas Islands
(1982-1985)
French island of
Réunion,
Mauritius (2005)
Fig.2.
Fig.3.
Fig.1&2 – Shows the western blot analysis of plant expressed E1 and E2 against Chik
polyclonal antibodies. Mammalian cell expressed E2 protein was used as the positive
control. The recombinant proteins were expressed with a N terminal His tag .
Fig.3.shows the western blot of plant expressed E1 and E2 against anti His antibodies.
Malaysia in 1999
M
Indonesia(1999 - 2003)
E1
E2
50 kDa
37 kDa
The Vectors for Chikungunya Virus
Fig.4.
The CHO cells expressed E1 & E2 contains a V5 tag at N terminal end. Fig.4. shows
the western blot of E1 and E2 against anti V5 antibodies.
Future Work:
Aedes aegypti
Aedes albaptycus
Aims:
 To express different combinations of Chikungunya virus structural proteins in mammalian and
plant systems and assess these systems for yield.
 To assess whether these expression systems are able to produce virus-like particles (VLPs)
The expression of the E3-E2-6K-E1 protein still remains to be assessed.
E1& E2 proteins will be purified and used to determine the ability of these
recombinant antigens to induce protective immune responses in a mouse challenge
model.
Reference:
Wataru, A., Zhi-Yong, Y., Hanne, A., Siyang, S., Heather, AH., Wing-Pui, K., Mark, GL.,
Stephen, H., Michael, GR., Srinivas, R & Gary, JN. 2010, ‘A virus-like particle vaccine for
epidemic Chikungunya virus protects nonhuman primates against infection’, Nature Medicine,
vol. 16, pp. 334 – 338.
 Purification of Chikungunya virus antigens.
Acknowledgement:
 To assess the ability of the purified recombinant proteins to induce protective immune responses
in a mouse model system.
Thanks to the UK-India Education and Research Initiative (UKIERI) for awarding this
collaborative research project.
Poster Presentation at St. Georges
University of London
Poster Presentation at PBVA 2011,
Porto , Portugal
Indo-UK Joint workshop on Plant Molecular Farming
April 7 – 8, 2011
Dinathanthi:
Chief Guests during
Inauguration Function
Prof. Julian Ma interacting with
participants
Dr. Mathew Paul interacting
with participants during
practical time
Participants
Autumn School in Biotechnology for College Teachers,
Sept. 26-30, 2011
UKIERI Activity Press Coverage
Activities
Indian PhD Student Mr. Balamurugan in 6th month out of
one year stay in Julian Ma’s lab
Transiently Expressed Chikungunya antigen E1 and E2 and he has
purified. Animal model studies to be started
Indian PI visited UK for a month to discuss with UK counter part
and planned a Indo-UK joint workshop in India in early 2014.
Transient Expression of E1 and E2 Ectodomain in Tobacco
C
C-Terminal
N-Terminal
Myc
E2
E3
E1
Entry
vector
Destination
vector
E3
E1
Chik Structural protein
N-Terminal
Myc
HIS
C-Terminal
E2
pDONR Zeo
Entry
vector
pEAQ & pTRAK
Destination
vector
Agroinfiltration
Protein Purification
Animal Studies
HIS
Cloning of CHIK E1 and E2 Ectodomain
Cloning of Chikungunya E1 and E2
Ectodomain in pEAQ and confirmation by
PCR and Restriction Digestion
(a)
Cloning of Chikungunya E1 and E2
Ectodomain in pTRAK and confirmation by
PCR and Restriction Digestion
(b)
9/12/2013
18
Partial Purification of E1 and E2 Ectodomain in Tobacco
Indian PhD Student Mr. Balamurugan in St. George’s
Outlook
Clinical trials for the commercial applications
Immunogenic potential of plant expressed chikungunya
antigens
Production and Purification of Chikungunya antigens from plants as a production
platform
Journal
1. Mathew Paul, Craig van Dolleweerd, Pascal MW Drake, Rajko Reljic, Harry Thangaraj,
Tommaso Barbi, Elena Stylianou, Ilaria Pepponi, Leonard Both, Verena Hehle, Luisa
Madeira, Varghese Inchakalody, Sammy Ho, Thais Guerra, Julian K-C Ma (2011)
Molecular pharming: Future targets and aspirations. Human Vaccines.
doi: 10.4161/HV.7.3.14456
2. Julian K-C. Ma, Paul Christou, Rachel Chikwamba, Hugh Haydon, Mathew Paul, Merardo
Pujol Ferrer, Sathishkumar Ramalingam, Elibio Rech, Edward Rybicki, Andres
Wigdorowitz , Dai-Chang Yang and Harry Thangaraj (2013) Realising the value of plant
molecular pharming to benefit the poor in developing countries and emerging
economies. Accepted for publication in Plant Biotechnology.
Presentation at Conferences
1. UKIERI sponsored research has been presented as a poster in a Research Day
conference conducted at St. Georges University of London, UK and awarded the prize
for best poster by the Indian PhD student, Mr. Varghese.
1. The research work was also presented at International conference on “Plant Based
Vaccine and Antibodies (PBVA 2011)” at Portugal, by the Indian PhD student, Mr.
Varghese.
• Over these funding period, UKIERI support has created a significant impact on
both research groups, who have jointly addressed the problem of solving an
important developing country infectious disease using cutting edge new
biotechnological tools.
• Our results offer great promise in the control of Chikungunya infection in
resource-poor settings.
• Once we have generated the data regarding immunogenicity of the plant
recombinant proteins, we shall apply for further funding to develop the project
further. We will apply to the Wellcome Trust and the European Union for
funding to allow us to take the project to the next stage, namely pre-clinical
development of a vaccine. Julian Ma has already established manufacturing
capacity through an EU funded consortium (www.pharma-planta.net), and this
will be invaluable in helping us to reach pre-clinical and clinical testing.
• Support from UKIERI has been magnificent and we are extremely grateful to
UKIERI for enabling this important piece of research.
In kind support
PI’s time and University Infrastructure.
Issues
• The cost related to consumables should be increased.
• Split site PhD procedure should be made simple.
Future
•
As production levels of antigen is still less in plants,
we would like to express Chikungunya antibodies.
•
We would like to request UKIERI to consider
this proposal.