dr. r. sathishkumar prof. julian ma bharathiar st. george's
TRANSCRIPT
Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George’s University Medical School
Name: Professor Julian K-C Ma Position: Hotung chair of molecular immunology, Joint Head of the Infection and Immunity Research Centre Institute: 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.
Name: Dr. R. Sathishkumar Position: Assistant Professor Institute: Bharathiar University • Group Leader- Plant Genetic Engineering Lab • UKIERI and DAAD Fellow • Deputy Coordinator- UGC-SAP • IBSC in-charge • Academic Visitor, St. George’s
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 PLANT BASED RECOMBINANT VACCINE AGAINST CHIKUNGUNYA” under Thematic Partnerships
2011
• Financial support to organize “PLANT MOLECULAR FARMING WORKSHOP”
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
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 No. of
Participants Mode of Publicity
April 7-8, 2011
Indo-UK Joint Plant Molecular Farming Workshop
30 • Internet
• Website
• News Papers
• Scientific Journal- Current Science
September 26 – 30, 2011
Autumn School in Biotechnology for College Teachers
30
Dr. R. Sathishkumar was awarded one year UKIERI Research Fellowship to work with Prof. Julian K. C. Ma
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
Activities
Genetically Modified Tobacco Plants Expressing Chikungunya Antigens
Developing plant based recombinant vaccine against Chikungunya
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.
Activities
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
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
Activities
Developing Vaccines Against
Chikungunya Virus
Inchakalody VP1,van Dolleweerd CJ2, Reljic R2, Paul M2, Ramalingam S1, Ma JKC2
1 Department of Biotechnology, Bharathiar University, India
2 Division of Clinical Sciences, St. George's, University of London
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)
Purification of Chikungunya virus antigens.
To assess the ability of the purified recombinant proteins to induce protective immune responses
in a mouse model system.
Results:
M E2 +ve WT pEARLY pEAQ
Control His His
37 kDa
50 kDa
37 kDa
50 kDa
Confirmation of Chikungunya Antigen E1 and E2 by Western Blotting
Materials and Methods
1.3 kb 1.4 kb
2.9 kbBP recombination to Gateway entry vector
LR recombination to Gateway destination vector
Mammalian cell expression
(pcDNA-DEST40 - C terminal His tag)
Protein extraction and western blotting.
Capsid E3 E2 6K E1
pDON R/Zeo
Plant expression
(pEarleyGate His, pEAQ-HT-DEST3-His)
Expression in CHO cells Expression in Nicotiana benthamiana
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.
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.
Acknowledgement: Thanks to the UK-India Education and Research Initiative (UKIERI) for awarding this
collaborative research project.
Fig.1.
Worldwide Epidemiology of Chikungunya Virus
Tanzania (1952)
Philippines (1954)
Sumatra, Java,
Moluccas Islands
(1982-1985)
Malaysia in 1999
Indonesia(1999 - 2003)
French island of
Réunion,
Mauritius (2005)
India, Madagascar
(2005-2006)
?India (1963)
Lyovec-mediated transfection
of CHO cells Agro-infiltration
The Vectors for Chikungunya Virus
Aedes aegypti Aedes albaptycus
Structural polyprotein
precursor
E2 E1
E3 E2 6K E1
Proposed Chik virus
antigen for expression
M E2 +ve pEARLY pEAQ
Control His His
Fig.2. Fig.3.
M WT pEAQ pEAQ E1 E2
50 kDa
37 kDa
Fig.4.
50 kDa
37 kDa
M E1 E2
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: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.
Introduction: 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).
Chikungunya Genome Organization and Replication
Presentations
Poster Presentation at St. Georges University of London
Poster Presentation at PBVA 2011, Porto , Portugal
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Transient Expression of E1 and E2 in tobacco
Capsid E3 E2 6K E1 Structural
polyprotein
precursor
1.3 kb 1.4 kb
BP recombination to Gateway entry vector
pDON R/Zeo
E2 E1 Proposed Chik virus
antigens for expression
LR recombination to Gateway
destination plant expression vectors
pEAQ-HT-DEST3 pEarley Gate-His pEarley Gate-HA
Agroinfiltration and Protein Extraction
37 kDa
50 kDa
Western blot of plant-expressed E1 using ChikV-specific antiserum in denaturing condition
Indian Student in UK
Dinathanthi:
Chief Guests during Inauguration Function
Prof. Julian Ma interacting with participants
Dr. Mathew Paul interacting with participants during
practical time
Participants
Indo-UK Joint workshop on Plant Molecular Farming April 7 – 8, 2011
Autumn School in Biotechnology for College Teachers, Sept. 26-30, 2011
UKIERI Activity Press Coverage
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.
Activities
Transient Expression of E1 and E2 Ectodomain in Tobacco
E2 E1 C E3
E1
C-Terminal
HIS Myc E2
C-Terminal
HIS Myc
N-Terminal N-Terminal
pDONR Zeo
pEAQ & pTRAK
Agroinfiltration
Protein Purification
Animal Studies
Entry
vector Entry
vector
Destination
vector
Destination
vector
Chik Structural protein E3
9/12/2013 18
Cloning of Chikungunya E1 and E2
Ectodomain in pEAQ and confirmation by
PCR and Restriction Digestion
Cloning of Chikungunya E1 and E2
Ectodomain in pTRAK and confirmation by
PCR and Restriction Digestion
(a) (b)
Cloning of CHIK E1 and E2 Ectodomain
Partial Purification of E1 and E2 Ectodomain in Tobacco
Indian PhD Student Mr. Balamurugan in St. George’s
Outlook
Production and Purification of Chikungunya antigens from plants as a production platform
Immunogenic potential of plant expressed chikungunya antigens
Clinical trials for the commercial applications
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.