END OF PROJECT REPORT
A. Project number: 06-02-05-0008
Project title: Mutation of hemA and M gene of Vibrio development of attenuated V. cholerae vaccine
Project leader: Dr. Manickam Ravichandran
Tel: 09 7664592 Fax: 09 7648673
frnc.-;1:
h'lYtt viB. Summary for the MPKSN Report (For publication in the Annual MPKSN Report, please summarise the project objectives, significant results achieved, research approach and team structure)
Mutation of hemA and M gene of Vibrio cholerae: Towards the development of attenuated \1. cholerae vaccine
Introduction
Cholera remains a major public health problem in developing countries. In some countries, including Malaysia, cholera outbreak often occur and continue to be a major cause of concern. Vaccines represent the most cost effective means to prevent this infection. Live attenuated vaccine has been developed by mutating the ctxA gene that codes for subunitA of cholera toxin and other minor toxin genes (Kaper et at , 1984, Hase et at 1994). These vaccine strains are protective but causes mild diarrhoeagenic side effects. In order to overcome this problem, it is important to include an additional mutation in certain house keeping gene (like hem gene) to limit the in-vivo growth of V. cho/erae to avoid mild diarrhoeagenic side effects and shedding of vaccine strain in the environment.
The current study is aimed to characterize, clone the hemA gene of V choferae Bengal in mobilizable conjugative plasmid vector and mutate the hem gene using kanamycin/GFP gene cassette. Further, the mutated hem gene will be transferred to V.cholerae using a mobilizable plasmid vector system by conjugation and site-specific recombination. The resulting mutation of hem gene in V.cholerae will lead to depletion of ALA which inturn leads to it's limited growth in normal in-vitro and in vivo condition.
Objectives
1. To study the functional role of hemA and hemM gene of Vibrio cholerae in 8- Aminolevulinic acid (ALA) synthesis
2. To clone the hem gene onto mobilizable conjugative plasmid vector (pAR0180) for the purpose of insertional mutagenesis
3. To mutate the hem gene using kanamycin or Green fluorescent protein (GFP) gene cassette or by frame shift mutation.·
4. To incorporate the hem mutation in V. cholera by conjugation and site specific recombination
5. To determine the protective efficacy of V. cholera hem mutant in an animal model.
End Of Project Report Form
-
Results
For the first time in the literature, the hemA gene, which encodes glutamyl t-RNA reductase has been isolated and cloned by our research group from V cholerae Bengal strain. The hemA gene was cloned on to pAR0180 vector at the EcoRI restriction enzyme site. To mutate the hemA gene a kanamycin gene cassette was inserted at the BstXI site, which is present at the middle of hemA gene. The mutated hemA gene construct was then cloned on to a conjugative suicide vector, pWM91. The mutated hemA gene construct in pWM91 was transferred to V.cholerae Bengal by conjugation and site- specific recombination. The successfully developed V.cholerae Bengal hemA mutants grew only in the presence of ALA, due to the loss of function of hemA gene.
The hemA mutant strain of V cholerae (VCUSM1) has been tested in mouse model for colonization and immune efficiency. The VCUSM1 mutant strain could successfully colonize the small intestine of infant mouse as seen in wild type V cholerae. The protective efficacy was tested on rabbits by RIT ARD assay. All the rabbits rabbits vaccinated with the VCUSM1 mutant strains survived when challenged with lethal dose of wild type V cholerae, however all the unvaccinated rabbits died when challenged with wild type V cholerae. Currently there is no vaccine available for cholerae caused by V cholerae Bengal, this prototype vaccine will form a base for the development of vaccine for cholera.
C. Objectives achievement
• Original project objectives (Please state the specific project objectives as described in Section II of the Application Form)
Objectives
1. To study the functional role of hemA and hemM gene of Vibrio cholerae in o-Aminolevulinic acid (ALA) synthesis
2. To clone the hem gene onto mobilizable conjugative plasmid vector (pAR0180) for the purpose of insertional mutagenesis
3. To mutate the hem gene using kanamycin or Green fluorescent protein (GFP) gene cassette or by frame shift mutation.
4. To incorporate the hem mutation in V. cholera by conjugation and site specific recombination 5. To determine the protective efficacy of V. cholera hem mutant in an animal model.
• Objectives Achieved (Please state the extent to which the project objectives were achieved) 1. To study the functional role of hemA and hemM gene of Vibrio cholerae in o-Aminolevulinic acid
(ALA) synthesis : The functional role of hemA was studied using E coli ALA auxotrophs. The results obtained with VCUSM1 mutant was comparable with E. coli hemA mutant
2. To clone the hem gene onto mobilizable conjugative plasmid vector (pAR0180) for the purpose of insertional mutagenesis: The hemA and hemM genes were successfully cloned onto mobilizable conjugative plasmid vector (pAR0180), this was confirmed by restriction enzyme digestion and DNA sequencing
3. To mutate the hem gene using kanamycin or Green fluorescent protein (GFP) gene cassette or by frame shift mutation.: The hemA gene was successfully mutated using kanamycin gene cat;seUe. The $UCcess of the mutation was confirmed by restriction enzyme digestion, PCR and sequencing.
4. To incorporate the hemA mutation in V. cholera by conjugation and site specific recombination: The hemA mutation in V. cholerae was incorporated by conjugation and site specific recombination.
5. To determine the protective efficacy of V. cholera hem mutant in an animal model.: The hemA mutant strain of V cholerae (VCUSM1) has been tested in mouse model for colonization and immune efficiency. The VCUSM1 mutant strain could successfully colonize the small intestine of infant mouse as seen in wild type V cholerae. The protective efficacy was tested on rabbits by RITARD assay. All the rabbits rabbits vaccinated with the VCUSM1 mutant strains survived when challenged with lethal dose of wild type Vcholerae, however all the unvaccinated rabbits died when challenged with wild type V cholerae.
• Objectives not achieved (Please identify the objectives that were not achieved and give reasons)
End Of Project Report Form
NIL
3
D. Technology Transfer/Commercialisation Approach (Please describe the approach planned to transfer/commercialise the results of the project)
1. VCUSM1 mutant V cholerae strain has been filed for Malaysian patent on
8.10.2003
with the numberP120033828
E. Benefits of the Project (Please identify the actual benefits arising from the project as defined in Section Ill of the Application Form. For examples of outputs, organisational outcomes and sectoral/national impacts, please refer to Section Ill of the Guidelines for the Application of R&D Funding under IRPA)
• Outputs of the project and potential beneficiaries (Please describe as specifically as possible the outputs achieved and provide an assessment of their significance to users)
Ministry of Health : Based on the efficacy of V cholera hem mutant strain and further trials, it will be possible to use this indigenously produced cholera vaccine.
• Organisational Outcomes (Please describe as specifically as possible the organisational benefits arising from the project and provide an assessment of their significance)
One Ph.D and one MSc student has been trained during this project. Other outcomes of this project include establishing USM as the Malaysian centre of excellence in indigenous vaccine development.
• National Impacts (If known at this point in time, please describe as specifically as possible the potential sectoral/national benefits arising from the project and provide an assessment of their significance)
1. Malaysian patent has been filed on 8.1 0.2003 with the number Pl20033828
2. BEST PRESENTATION AWARD Atif Ali, S, Nur Haslindawaty, A.R , Lai, C.T, Afifi, S.A.B., Lalitha, P, Zainuddin, Z.F and Ravichandran M. Evaluation of a live oral attenuated cholerae vaccine for Vibrio cholerae 0139 Bengal. 25th Malaysian Microbiology Society Symposium and 5th UNESCO National Workshop for the Promotion of Microbiology in Malaysia, 8-11 September 2002. Kota Bharu, Kelantan, Malaysia
3. ANUGERAH MERIT: Ravichandran, M., Atif Ali, S., Nur Haslindawaty A.R., Lai C.T., Chan Y Y, Lalitha, P., Zainuddin, Z.F. 200~. A Genetically Engineered, Non-Toxic and Environmentally Safe Vaccine Strain (Vcusm1) for Cholera. Pameran Penyelidikan, Kategori Genetik Mlkrob, Pameran Penyelidikan dan Pembangunan IPTA 2003.
4. Best poster award, Atif Ali S, Kurunathan S, Nur Haslindawaty A R, Chan Y Y, Lalitha P, Zainuddin Z F and Ravichandran M 'Safety And lmmunogenicity of Oral Auxotrophic Cholera Vaccine Candidates'. 14th National Biotechnology Seminar Universiti Sains Malaysia Penang Dec 1 0-131h 2003
5. OUTSTANDING PROJECT AWARD, Second prize , Development of a Genetically Engineered, Orally Administrable, Live Cholera Vaccine against Vibrio cho/erae 0139 Bengal, Given by Universiti Sains Malaysia, 18 December 2003.
F. Assessment of project structure
• Project T earn (Please provide an assessment of how the project team performed and highlight any significant departures from plan in either structure or actual man-days utilised).
End Of Project Report Form
NIL
5
• Collaborations (Please describe the nature of collaborations with other research organisations and/or industry)
G. Assessment of Research Approach (Please highlight the main steps actually performed and indicate any major departure from the planned approach or any major difficulty encountered)
No difficulty encountered during this project
H. Assessment of the Project Schedule (Please make any relevant comment regarding the actual duration of the project and highlight any significant variation from plan)
NIL
L Assessment of Project Costs (Please comment on the appropriateness of the original budget and highlight any major departure from the planned budget)
The budget approved for this project was insufficient (RM144.000). Since we work as research clusters, we got good support to carry out the project in terms of infrastructure, equipments and man power from our Medical Biotechnology cluster at, Health Campus, USM.
J. Additional Project Funding Obtained (In case of involvement of other funding sources, please indicate the source and total funding provided)
NIL
K. Other Remarks (Please include any other comment which you feel is relevant for the evaluation of this project)
We tried to recruit postdoctrol candidate in this project, since the renumeration was too low (RM2,500) when compared to other countries and there was a long delay in the processing (now it is better) we could not recruit any postdoctoral candidates in this project under MOSTE postdoctoral scheme.
Date:
?-) / :) l.oo 1
Signature:fv/.~'~ ~,..
()no copy of
the~End of ProJect Hopon is to bP rnalJPd to:
DR. MANICKAM F~AVICHANDRAN
I r<PA Sucretariat
f\·ilnistry of Science. Technolog;' and the Bled< c:s. Parccd C
Putrajaya
End Of Project Report Form 7
lecturer
Depor!menl ol Medical Mboblolog:~ And i'arcsiklogy . School of Ml)dico! Sci~;;·~c~s . Envlro.nr::ntJ:nMQrsii~ Sains tv1ol~r~ia .;X:f:rP'.;/
'"''~L.l6l50 ~,1.1bong Ksn~•n 7~v
~ Kelankm: __ ..Q>,...-'
