dr. r. sathishkumar prof. julian ma bharathiar st. george's

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Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George’s University Medical School

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Page 1: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George’s University Medical School

Page 2: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 3: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar 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

Page 4: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 5: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 6: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 7: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Genetically Modified Tobacco Plants Expressing Chikungunya Antigens

Page 8: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 9: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 10: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Prof. Julian Ma with Indian group

With the all the Research Students of the Dept.

Page 11: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 12: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 13: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 14: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Autumn School in Biotechnology for College Teachers, Sept. 26-30, 2011

Page 15: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

UKIERI Activity Press Coverage

Page 16: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 17: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 18: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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

Page 19: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Partial Purification of E1 and E2 Ectodomain in Tobacco

Page 20: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

Indian PhD Student Mr. Balamurugan in St. George’s

Page 21: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar 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

Page 22: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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.

Page 23: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

• 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.

Page 24: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's

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.

Page 25: Dr. R. Sathishkumar Prof. Julian Ma Bharathiar St. George's