Centre for Health & Population Research
RESEARCH PROTOCOL
NUMBER:
RRC APPLICATION FORM
FOR OFFICE USE ONLY
RRC Approval:

ERC Approval:
AEEC Approval:

Yes /
No
Date: 23/12/2002
Yes / No Date: 2/01/2003
Yes / No Date:
Project Title: Towards an immunodiagnostic kit for leprosy
Short protocol title (in 50 characters including space): Towards an immunodiagnostic kit for leprosy
Theme: (Check all that apply)
Nutrition

Emerging and Re-emerging Infectious Diseases
Population Dynamics
Reproductive Health
Vaccine evaluation
HIV/AIDS
Environmental Health
Health Services
Child Health
Clinical Case Management
Social and Behavioural Sciences
Key words: Leprosy, antigens, diagnostics
Despite global coverage of multidrug therapy (MDT) and a major decrease in
prevalance, the incidence of leprosy remains obstinately high in many countries including Bangladesh.
Unfortunately, there is no potential diagnostic method to diagnose leprosy in the early stage of the disease
process. Most of the cases the disease is diagnosed depending on clinical symptoms. It has long been
desired that a efficient diagnostic method is available so that early infection and the presence of multiplying
Mycobacterium leprae can be detected as soon as possible. Now that the complete genome sequences for
M. leprae and the principal members of the M. tuberculsosis complex, three environmental mycobacteria
and M. ulcerans are available, it is the time to discover M. leprae specific antigens which could be used as
the diagnostic tools for leprosy.
Relevance of the protocol:
Centre’s priority: The Centre has taken initiative to work in the field of mycobacterial diseases including
tuberculosis and leprosy. A state-of-the-art laboratory has been set up at the centre to continue research
work on mycobacteriology.
Programmes
Child Health Programme
Nutrition Programme

Programme on Infectious Diseases & Vaccine Science
Poverty and Health Programme
Principal Investigator: Dr. Sayera Banu (ICDDR,B)
Health and Family Planning Systems Programme
Population Programme
Reproductive Health Programme
HIV/AIDS Programme
Division: LSD
1
Phone: 8802-8811751-60
(should be a Centre’s staff)
Address: Tuberculosis Unit, LSD, ICDDR,B
Mahakhali 1212
Email: [email protected]
Co-Principal Investigator(s):
Internal
Co-Principal Investigator(s): Prof. Stewart Cole (Institut Pasteur, 28, rue du Docteur Roux 75724 Paris Cedex 15)
External
(Please provide full official address and Gender)
Co-Investigator(s): Dr. Firdausi Qadri, Dr. Rubhana Raqib, (ICDDR,B),
Internal
Co-Investigator(s): Dr. Nadine Honore (Institut Pasteur, 25-28, Rue du Docteur Roux, 75724 Paris Cedex 15), Dr. S. K. Abdul
Hadi (Leprosy Control Institute & Hospital, Mahakhali, Dhaka)
External
(Please provide full official address and Gender)
Student Investigator/Intern:
External
(Please provide full address of educational institution and Gender)
Student Investigator/Intern:
Internal (Centre’s staff)
Collaborating Institute(s): 1. Institut Pasteur, Paris, 2. Leprosy Control Institute & Hospital, Dhaka
Please provide full address
Country:
1. France
2. Bangladesh
Contact person: 1. Prof Stewart Cole 2. Dr. S. K. Abdul Hadi
Department:
1. Genetic Molecular Bacteriology 2. Leprosy Control Institute & Hospital
(including
Division, Centre,
Unit)
Institution:
Directorate:
(in case of GoB
e.g., DGHS)
Ministry
(in case of GOB)
2
Population: Inclusion of special groups (Check all that apply):
Gender

Male

Females
Age
0 – 5 years
5 – 9 years

10 – 19 years

20 – 64 years
65 +
Pregnant Women
Fetuses
Prisoners
Destitutes
Service providers
Cognitively Impaired
CSW
Others (specify ____________________________)
Animal
NOTE: It is the policy of the Centre to include men, women and children in all biomedical and behavioural research
projects involving human subjects unless a clear and compelling rationale and justification (e.g. gender
specific or inappropriate with respect to the purpose of the research) is there. Justification be provided in case
inclusiveness of study participants is not proposed in the study.
Project / study Site (Check all the apply):
Dhaka Hospital
Matlab Hospital
Matlab DSS area
Matlab non-DSS area
Mirzapur

Dhaka Community
Chakaria
Abhoynagar
Mirsarai
Patyia

Other areas in Bangladesh ____________________
Outside Bangladesh
name of country: ________________________
Multi centre trial
(Name other countries involved)
______________________________________
Type of Study (Check all that apply):

Case Control study
Community based trial / intervention
Program Project (Umbrella)
Secondary Data Analysis
Clinical Trial (Hospital/Clinic)
Family follow-up study
NOTE:
Cross sectional survey
Longitudinal Study (cohort or follow-up)
Record Review
Prophylactic trial
Surveillance / monitoring
Others
All clinical studies/trials should be registered in appropriate websites, preferably at
www.clinicaltrials.gov. When the study is registered in website(s), the PI should provide website
address, registration number, and date of registration to the Committee Coordination Secretariat for
entering these information into the Centre’s database of your research protocol.
Targeted Population (Check all that apply):

No ethnic selection (Bangladeshi)
Bangalee
Tribal groups
Expatriates
Immigrants
Refugee
Consent Process (Check all that apply):

Written
Oral
None


Bengali language
English language
Proposed Sample size:
Total sample size: 204 (case-68, control-136
Sub-group ____________________________________
___________________________________________
_____________________________________________
___________________________________________
Determination of Risk: Does the Research Involve (Check all that apply):
3
Human exposure to radioactive agents?
Fetal tissue or abortus?
Investigational new device?
(specify________________________)
Existing data available from Co-investigator
Human exposure to infectious agents?
Investigational new drug
Existing data available via public archives/source

Pathological or diagnostic clinical specimen only
Observation of public behaviour
New treatment regime
Yes/No

Is the information recorded in such a manner that study participants can be identified from information provided
directly or through identifiers linked to the subjects?

Does the research deal with sensitive aspects of the study participants behaviour; sexual behaviour, alcohol use or
illegal conduct such as drug use?
Could the information recorded about the individual if it became known outside of the research:

a. place the study participants at risk of criminal or civil liability?

b. damage the study participants financial standing, reputation or employability; social rejection, lead to stigma,
divorce etc.
Do you consider this research (Check one):

greater than minimal risk
no more than minimal risk
only part of the diagnostic test
Minimal Risk is "a risk where the probability and magnitude of harm or discomfort anticipated in the proposed research are not
greater in and of themselves than those ordinarily encountered in daily life or during the performance of routine physical,
psychological examinations or tests. For example, the risk of drawing a small amount of blood from a healthy individual for
research purposes is no greater than the risk of doing so as a part of routine physical examination".
Yes/No

Is the proposal funded?
If yes, sponsor Name: WHO-TDR
____________________________________________________________________
Yes/No
Is the proposal being submitted for funding ?
If yes, name of funding agency: (1) ___________________________________________________________
(2) ___________________________________________________________
Do any of the participating investigators and/or their immediate families have an equity relationship (e.g. stockholder)
with the sponsor of the project or manufacturer and/or owner of the test product or device to be studied or serve as a
consultant to any of the above?
IF YES, submit a written statement of disclosure to the Executive Director.
4
Dates of Proposed Period of Support
Cost Required for the Budget Period ($)
(Day, Month, Year - DD/MM/YY)
Beginning date: January 1, 2004
Direct Cost
Indirect Cost
US$ 52,500
----------------
Year-1
Year-2
US$ 55,500
Total Cost
----------------
----------------
End date: December 31, 2006
Year-3
Year-4
---------------
-----------------
---------------
----------------
----------------
---------------
----------------
----------------
---------------Year-5
TOTAL: US$ 108,000
Approval of the Project by the Division Director of the Applicant
The above-mentioned project has been discussed and reviewed at the Division level as well by the external reviewers.
The protocol has been revised according to the reviewer’s comments and is approved.
_________________________
Name of the Division Director
_____________________
Signature
_______________________
Date of Approval
Certification by the Principal Investigator
Signature of PI
I certify that the statements herein are true, complete
and accurate to the best of my knowledge. I am aware
that any false, fictitious, or fraudulent statements or
claims may subject me to criminal, civil, or administrative penalties. I agree to accept responsibility for the
scientific conduct of the project and to provide the required progress reports if a grant is awarded as a result
of this application.
Date:
Name of Contact Person (if applicable)
5
Table of Contents
Page Numbers
Face Page………………………………………………………………………………………
Project Summary………………………………………………………………………. 7
Description of the Research Project…………………………………………………. 8
Hypothesis to be tested…………………………………………………………….. 8
Specific Aims ……………….……………………………………………………... 8
Background of the Project Including Preliminary Observations…………………... 9
Research Design and Methods……………………………………………………...12
Facilities Available………………………………………………………………….16
Data Analysis………………………………………………………………………. 17
Ethical Assurance for Protection of Human Rights…………………………………17
Use of Animals………………………………………………………………………17
Literature Cited………………………………………………………………………18
Dissemination and Use of Findings………………………………………………….19
Collaborative Arrangements………………………………………………………....19
Biography of the Investigators…………………………………………………………20
Detalied Budget…………………………………………….……………………………24
Budget Justifications………………………………………………………………….. 26
Other Support……………………………………………….…………………………..26
Ethical Assurance : Protection of Human Rights ………….………………………………
Appendix…..…………………………………………………………………………………..
Consent Forms in English
Consent Forms in Bangla
Check here if appendix is included
6
ROJECT SUMMARY: Describe in concise terms, the hypothesis, objectives, and the relevant background of the project.
Describe concisely the experimental design and research methods for achieving the objectives. This description will serve as a
succinct and precise and accurate description of the proposed research is required. This summary must be understandable and
interpretable when removed from the main application. ( TYPE TEXT WITHIN THE SPACE PROVIDED).
Principal Investigator: Dr. Sayera Banu (ICDDR,B) and Prof. Stewart Cole (Institut Pasteur)
Project Name: Towards an immunodiagnostic kit for leprosy
Total Budget (Direct): US$ 108,002
Beginning Date: 1/1/2003
Ending Date: 31/12/2004
Leprosy, caused by Mycobacterium leprae, a close relative of tubercle bacillus, remains a major public
health problem worldwide. According to WHO report, more than 690,000 new cases are notified annually.
Leprosy is a public health problem in Bangladesh with an estimated case load of 136,000 (prevalence rate
of more than 10 per 10,000) as of 1999. Armauer Hansen discovered the leprosy bacillus in skin biopsies
but failed to culture M. leprae. Subsequent efforts enabled large quantities of bacillus to be isolated from
armadillo which was used as a surrogate host but falied to grow in synthetic media causing diagnosis of the
disease difficult. One of the reasons of this failure might the exceptionally slow growth of the bacillus. So,
the diagnosis of leprosy now mainly based on clinical symptoms and sometimes the presence of acid-fast
bacilli in skin smear by light microscopy. However, increasingly fewer centres have trained microscopists
and, in any case, microscopy lacks specificity being unable to distinguish between the various mycobacteria
that are pathogenic for humans.
The aim of this project is to apply a post-genomic approach to the discovery of potential protein antigens of
Mycobacterium leprae that could be used for the development of a test to identify paucibacillary leprosy
patients and/or individuals who have been exposed to the bacillus. Using comparative genomics and bioinformatics a panel of <50 proteins has been defined; these are restricted to M. leprae or show limited
distribution. Some are predicted to contain CD8 T-cell epitopes and others to be secreted or surface
exposed. Immunological characterisation is planned for forty of these proteins which will be overexpressed
and purified in recombinant form. Initial results are encouraging. Proteins that are recognised by the
immune system and induce interferon gamma production and lymphocyte proliferation in tuberculoid
patients will be characterised further. Overlapping synthetic peptides will then be used to define T-cell
epitopes and immune responses. This strategy will identify a combination of M. leprae-specific
proteins/peptides suitable for inclusion in a skin test. Earlier identification of leprosy will result in patients
receiving treatment sooner and, in turn, this should help to reduce further the prevalence and incidence of
the disease.
Sera and clinical data of 68 adult patients with paucibacillary leprosy will be collected from the National
Leprosy Hospital in Mohakhali, Dhaka. An equal number (68) healthy adult controls will be enrolled from
the relatives of the leprosy patients who are not close contacts of the patients. Age and sex matched 68 TB
patients as controls will be taken from the Institute of Diseases of Chest and Hospital, Dhaka.
7
KEY PERSONNEL (List names of all investigators including PI and their respective specialties)
Name
Professional Discipline/ Specialty
1. Dr. Sayera Banu
2. Prof. Stewart Cole
3. Dr. Nadine Honore
4. R. Araoz
5. Dr. Firdausi Qadri
6. Dr. Rubhana Raqib
8. Dr. S. K. Abdul Hadi
Molecular Microbiologist
Molecular Biologist
Molecular Biologist
Researcher
Immunologist
Immunologist
Physician
Role in the Project
PI, ICDDR,B
Co-PI
Co-investigator
Co-investigator
Co-investigator
Co-investigator
Co-investigator
DESCRIPTION OF THE RESEARCH PROJECT
Hypothesis to be tested:
Concisely list in order, in the space provided, the hypothesis to be tested and the Specific Aims of the proposed study. Provide
the scientific basis of the hypothesis, critically examining the observations leading to the formulation of the hypothesis.
Based on some preliminary data concerning expression and purification of the M. leprae specific
recombinant proteins, we hypothesize that sets of potential antigens can be defined that are restricted to M.
leprae and these new antigenic targets could serve as the basis of a new diagnostic test for paucibacillary
leprosy patients.
Specific Aims:
Describe the specific aims of the proposed study. State the specific parameters, biological functions/ rates/ processes that will be
assessed by specific methods (TYPE WITHIN LIMITS).
1. To apply a post-genomic approach to the discovery of potential protein antigens specific to
Mycobacterium leprae.
2. To develop a diagnostic test for identifying paucibacillary leprosy patients and/or individuals who
have been exposed to the bacillus.
8
Background of the Project including Preliminary Observations
Describe the relevant background of the proposed study. Discuss the previous related works on the subject by citing specific
references. Describe logically how the present hypothesis is supported by the relevant background observations including any
preliminary results that may be available. Critically analyze available knowledge in the field of the proposed study and discuss
the questions and gaps in the knowledge that need to be fulfilled to achieve the proposed goals. Provide scientific validity of the
hypothesis on the basis of background information. If there is no sufficient information on the subject, indicate the need to
develop new knowledge. Also include the significance and rationale of the proposed work by specifically discussing how these
accomplishments will bring benefit to human health in relation to biomedical, social, and environmental perspectives. (DO NOT
EXCEED 5 PAGES, USE CONTINUATION SHEETS).
Leprosy is a unique infectious disease with a prolonged incubation period and a predilection for skin and
nerves. The introduction of WHO recommended multi drug therapy (MDT) over the last two decades has
produced dramatic changes in the management and control of leprosy. But leprosy still remains the most
common cause of peripheral neuropathy in developing countries. The MDT regimens for both
paucibacillary (PB) and multibacillary (MB) leprosy include rifampicin (RMP), dapsone (DDS) and
clofazimine (CLO). Rifampicin is the key component and and has more bactericidal effect than the other
components in the chemotherapeutic regimens.
Leprosy is not evenly distributed in the 64 districts of the country. Over 120 sub-districts out of 460 were
known to be endemic for leprosy. Leprosy control programme in Bangladesh started in 1965 with dapsone
monotherapy in a few sub-districts and extended to about 120 sub-districts till 1984. Dapsone monotherapy
has been replaced with MDT since 1985 in a phased manner covering the 120 endemic thanas.
Consequent to the adoption May 1991 resolution by the World Health Assembly recommending leprosy
elimination as a public health problem by the year 2000, the Government of Bangladesh has committed to
achieve the goal and a national plan for leprosy elimination has been developed. Leprosy elimination as a
public health problem implies reduction of leprosy prevalence to less than 1 per 10,000 population in the
country.
Leprosy is diagnosed on the basis of its clinical signs and symptoms and in some cases with the help of
laboratory investigation. Persons with the presence of one or more of the following cardinal signs can be
diagnosed as leprosy.
i)
ii)
iii)
Hypo-pigmented or erythematous patch or patches on the skin with diminished or loss of
sensation.
Enlargement of peripheral nerves.
Demonstration of M. leprae in skin smear.
Leprosy shows wide variation in clinical manifestations, associated with differences in immunology,
evolution and epidemiology. For the purposes of treatment, leprosy is primarily classified into two major
forms depending on the bacillary load:
(1) Multibacillary (MB) type: bacillary load is high, the skin smear is positive and/or the
number of skin lesions is 6 or more.
(2) Paucibacillary (PB) type: bacillary load is low, the skin smear is negative and/or the number
of skin lesions is 1 to 5.
If a patient is clinically diagnosed as PB, shows skin-smear positivity, he/she is classified as MB and given
MB treatment.
9
Multi-drug treatment (MDT): All newly detected cases of leprosy are provided with MDT (rifampicin,
clofazimine and dapsone) free of cost. The duration of treatment is 6 months for paucibacillary cases and
12 months for multibacillary cases. In both cases, treatments are supervised. This means that the patient
swallows the drugs under the direct supervision of the leprosy program staff member, when the patient
makes the monthly visit i.e. once in 4 weeks at the assigned place. The patient is thereafter given the
required drugs for daily consumption at home (self administered) for the following 27 days.
The availability of a diagnostic method has long been desired that would detect infection and the presence
of multiplying Mycobacterium leprae as soon as possible and preferably before clinical signs became
apparent. The discovery and characterization of phenolic glycolipid 1 (PGL-1), an M. leprae-specific
antigen, provided hope of developing such a test and much effort was devoted to detecting the presence of
antibodies to this molecule as a test for infection (7, 8). Anti-PGL-1 antibodies, which are largely IgM, are
abundant in lepromatous (LL) or multibacillary (MB) leprosy patients but scarce or completely absent in
tuberculoid (TT) or pauciibacillary (PB) patients. PGL-1 itself is an unusually stable biomolecule often
persisting in patients who have completed MDT. Consequently, both the detection of PGL-1 and
circulating anti-PGL1 antibodies have become less attractive as early diagnostic indicators.
Diagnostic tests based on cellular immunity have also been the subject of some attention and these are
analogous to the tuberculin test and often involve intradermal inoculation of a heat-killed extract of M.
leprae, or lepromin. A DTH-type response could sometimes be detected prior to the onset of clinical
disease. However, M. leprae induces anergy in LL patients and these individuals are lepromin-negative,
therefore reducing the utility of this diagnostic test. Nevertheless, tests based on protein or peptide antigens
still offer great promise for identifying recently-infected individuals or TT and other PB patients as their
cell-mediated immunity remains vigorous. A confounding factor, however, is prior immunisation with
related cross-reacting antigens such as those present in Mycobacterium bovis BCG or environmental
mycobacteria.
Two new approaches have been used to develop improved skin test antigens. Firstly, armadillo-derived M.
leprae cells have been fractionated and proteins associated with the cell wall and the cytoplasm identified
then immunologically characterized (5). For technical reasons, true secreted proteins will largely escape
this analysis which yields proteins in their native conformation. Secondly, peptide antigens representing
potentially M. leprae-specific epitopes were synthesized using a post-genomic approach and used to
evaluate responses in leprosy patients and healthy individuals from non-endemic areas (14). While the
initial findings were promising, poor specificity and sensitivity were observed possibly as a result of crossreactivity or to some of the epitopes being MHC-restricted, respectively.
With the availability of complete genome sequences for M. leprae (11) (http://genolist.pasteur.fr/Leproma/)
and the principal members of the M. tuberculosis complex (6, 10) (http://genolist.pasteur.fr/TubercuList/),
and near complete sequences for three environmental mycobacteria (M. avium, M. marinum
(http://www.sanger.ac.uk/Projects/M_marinum/)
and
M.
smegmatis
(http://www.tigr.org/tdb/mdb/mdbinprogress.html)
and
M.
ulcerans
(http://genopole.pasteur.fr/Mulc/BuruList.html), we are now ideally positioned to adopt a more rational
route to antigen discovery. Using genomics and bio-informatics, sets of potential antigens can be defined
that are restricted to M. leprae, these include secreted or surface-exposed proteins and screened in silico
for potential T-cell epitopes (9). After validation, these new antigenic targets could serve as the basis of a
new diagnostic test capable of revealing infection before clinical symptoms are recognizable or of detecting
disease in PB patients. Earlier disease detection will result in prompt initiation of MDT thereby increasing
the likelihood that both the prevalence and incidence of leprosy can be further reduced. Clearly, patients
stand to gain considerable benefit from earlier diagnosis and risks of nerve damage will be greatly reduced
from earlier treatment.
10
If we are to develop a specific immunological test for the early diagnosis of leprosy, in particular the
tuberculoid form of the disease, it is essential to define the protein repertoire accurately through a
combination of genomic and proteomic studies, and to identify those proteins that are confined to M. leprae
or show extensive diversity from their counterparts in other mycobacteria. Using various bio-informatic
procedures, including database similarity searches and classification into families, we have conducted a
search for genes that fulfill the following criteria: I) present in M. leprae but absent from all other genomes
(class 8 in Table 1); II) present in M. leprae but absent from all other mycobacterial genomes; III) show a
strong likelihood of encoding secreted or surface-exposed proteins of restricted distribution.
There are 136 genes that belong to class I but many of these are likely to correspond to gene fragments or
pseudogenes given the high level of gene decay found in M. leprae (11). Consequently, all 136 were reinspected using more stringent criteria (codon usage, GC content, positional base preference and avoiding
location in gene-poor areas of the chromosome) to identify those most likely to express proteins. This
reduced the sample to 18 candidates of the highest priority (Table 1). Of the 29 genes belonging to class II,
15 were retained for further analysis. Class III contains 20 proteins and these will be described briefly
hereafter. Roughly 10% of the M. tuberculosis genome encodes ~170 proteins belonging to the novel,
glycine-rich PE (proline-glutamate) and PPE (proline-proline-glutamate) families and these proteins appear
to be confined to mycobacteria. Several of them have been shown recently to contain T-cell epitopes
and/or to localize to the cell envelope of M. tuberculosis (3, 4, 12). The more distantly-related M. leprae
proteins may therefore represent good candidates for antigen discovery programmes. Of considerable
interest is the finding that one of the PPE proteins, the serine-rich antigen, is well recognised by sera from
leprosy patients (19) and strikingly different from the equivalent M. tuberculosis protein. Furthermore, an
M. tuberculosis PPE protein, Rv1196, has been found to be immunodominant and is currently being
developed as part of a TB sub-unit vaccine (13). The secreted or surface-exposed proteins in class III
belong to the lipoprotein, twin-arginine or ESAT-6 (early secreted antigenic target 6 kDa protein) families.
The latter are of considerable interest owing to their remarkable ability to induce T-cell responses (1, 17),
highly desirable properties for the development of immunodiagnostic tests such as a skin test based on
DTH. Initial work by Geluk et al. has confirmed the immunodominance of the M. leprae ESAT-6
ortholog, ML0049, (15) and shown that problems of potential cross-reactivity arise in individuals likely to
have been infected or exposed to M. tuberculosis. For this reason, we will concentrate on other more
divergent members of the ESAT-6 family of M. leprae (e.g. ML2531, ML2532). Bioinformatic analysis
predicts the presence of at least one epitope likely to induce CD8 T-cell responses in most of the candidates
(http://syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm) but, owing to the lack of a
suitable algorithm, we have been unable to apply this approach to the longer CD4 epitopes.
So far, the genes for 26 potential antigenic targets of high specificity have been cloned into Gateway
vectors and genetically tagged at either the N- or C-terminal end with Histidine tails to facilitate their
purification at the Unit of Genetic and Molecular Bacteriology, Institut Pasteur, Paris. A further 14
constructions are in progress. Thirteen of the 16 constructs tested in E. coli were found to overexpress the
corresponding proteins although in many cases problems of insolubility were encountered. There is a
strong chance that these can be overcome by reducing the temperature of the cultures to <20° C or by
changing the host to a fast-growing mycobacterium. Based on past experience, it is anticipated that around
30 of the 40 constructions will give suitable material for further analysis. The His-tagged proteins will be
affinity-purified by chromatography on Ni-columns then used in subsequent studies. Antibodies to the
recombinant proteins will be raised in rabbits and used to confirm the presence of the corresponding native
proteins in extracts of M. leprae.
11
Research Design and Methods
Describe in detail the methods and procedures that will be used to accomplish the objectives and specific aims of the project.
Discuss the alternative methods that are available and justify the use of the method proposed in the study. Justify the scientific
validity of the methodological approach (biomedical, social, or environmental) as an investigation tool to achieve the specific
aims. Discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them. Discuss the
ethical issues related to biomedical and social research for employing special procedures, such as invasive procedures in sick
children, use of isotopes or any other hazardous materials, or social questionnaires relating to individual privacy. Point out safety
procedures to be observed for protection of individuals during any situations or materials that may be injurious to human health.
The methodology section should be sufficiently descriptive to allow the reviewers to make valid and unambiguous assessment of
the project. (DO NOT EXCEED TEN PAGES, USE CONTINUATION SHEETS).
The overexpreesed and purified M. leprae specific recombinant proteins will be screened in dot-blot
assays for the presence of B-cell epitopes using well-characterized sera from patients to further appraise
their antigenicity. Controls will be performed using sera from tuberculosis patients or healthy contacts to
assess specificity. Proteins harbouring B-cell epitopes are generally antigenic targets for T-cells as well.
Those proteins that react with patients' sera will then rescreened in Western blots and ELISA and, after
confirmation, tested for their ability to stimulate the proliferation of lymphocytes in peripheral blood from
TT patients and matched controls, and their capacity to induce IFN- measured by capture enzyme-linked
immunosorbent assay (ELISA) using commercial monoclonal antibodies. Where possible, we will
implement whole blood assays for IFN- (interferon-). We intend to examine 68 PB patients and an equal
number of healthy individuals from the same setting.
In the second phase of the project, those proteins that emerge from the screen with the best immunological
criteria will be subjected to further analysis. Overlapping peptides of 20 amino acids, spanning the proteins,
will be designed and chemically synthesized in order to delimit the T-cell epitopes. The peptides will then
be used in lymphocyte proliferation and IFN- production assays to evaluate their potential as diagnostic
reagents. Peptides yielding positive results from different proteins will be pooled and re-tested to
determine whether a combinatorial approach can improve sensitivity. Matched controls corresponding to
specimens from naive individuals or healthy contacts will be included and, if available, lepromin will also
be used for comparative purposes.
Sample collection: Sera and related clinical data of patients will be collected from the National Leprosy
Hospital in Dhaka from paucibacillary (PB) leprosy cases. A total of 68 adult (age between 18-50 yrs) PB
leprosy cases will be recruited in the study. Diagnosis of leprosy will be bases on clinical signs and
symptoms. In doubtful cases, skin smear examination for acid-fast bacilli will be performed to confirm the
diagnosis. Patients aged between 18 to 50 yrs without having any other illness and willing to be enrolled in
the study will be selected for recruitment in the study. Skin smear is generally negative in PB leprosy. If a
patient clinically diagnosed as PB, shows skin-smear positivity, he/she will be considered as MB case and
will be excluded from the study. Patients having any other major illness will also be excluded from the
study. The sample will be stained using Ziehl-Neelson method and will be seen under light microscopy at
the collection site.
Blood (6 ml) will be collected aseptically from an antecubital vein from all enrolled PB leprosy patients at
the time of enrollment. Blood will be utilized for (1) dot-blot assay for 1st screening using recombinant
proteins (2) Western blot and ELISA for 2nd screening (3) lymphocyte proliferation assay and (4) capture
enzyme-linked immunosorbant assay (ELISA) using commercial monoclonal antibodies to test capacity to
induce IFN-.
12
Table. Potential antigenic targets selected by bioinformatics
CDS
ML0008
ML0126
ML0308
ML0333
ML0336
ML0376
ML0394
ML0397
ML0398
ML0410
ML0447
ML0458
ML0466
ML0568
ML0578
ML0678
ML0757
ML0841
ML0842
ML0957
ML1053
ML1055
ML1056
ML1057
ML1182
ML1183
ML1419
ML1420
ML1553
ML1603
ML1795
ML1828
ML1829
ML1915
ML1979
ML2088
ML2177
ML2242
ML2244
ML2249
ML2252
ML2264
ML2283
ML2341
ML2346
ML2498
ML2531
ML2532
ML2534
ML2567
ML2649
ML2667
Class
8
8
10
10
3
3
8
3
3
6
10
7
3
10
7
8
8
3
10
8
6
3
3
8
6
6
5
8
2
8
0
6
8
8
8
7
7
10
8
8
8
8
8
9
8
1
3
6
6
8
10
3
Function
unknown
unknown
conserved hypo
conserved hypo
ABC-tranporter ATP-binding protein
membrane protein
unknown
unknown, possibly pseudo
D-ribose-binding (lipo)protein
PE family
P450, pseudo
oxidoreductase
possible secreted protein
unknown, pseudo
phosphoenolpyruvate carboxylase
unknown
unknown
major membrane protein I
nifS-like protein
unknown
PE family
ESAT-6 family
ESAT-6 family
unknown
PPE family
PE family
regulatory protein, PFAM matches
unknown
prolyl tRNA synthetase
unknown
HSP 16.7
PPE family
unknown, pseudo
unknown
unknown, secreted?
cytochrome p-450
uridine phosphorylase
unknown
unknown
unknown
unknown
unknown
unknown
adenylate cyclase
unknown
enoyl-CoA hydratase
ESAT-6 family
PE/ESAT-6 family
PE family
unknown
TWIN-ARG
membrane transport protein, MntH-like
Cloned Expressed
Y, yes; N, No; nt, not tested yet
13
Y
Y
Y
Y
Y
nt
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
nt
nt
nt
Y
Y
nt
Y
Y
Y
Y
Y
nt
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
Y
Y
Y
Y
Y
Y
nt
nt
Y
N
Controls: Healthy age and sex matched adult (18 to 50 yrs) controls will be enrolled from the relatives of
the leprosy patients who are not close contacts of the patients. Healthy relatives (as control) willing to
participate in the study will be asked to come to the Leprosy Hospital for physical examination and blood
collection. Clinical data to be collected from controls will include age, sex, history of previous illnesses
and physical examination. Blood (6 ml) will be collected from healthy controls. TB patients as controls will
be recruited from the IDCH. A study on tuberculosis is ongoing with the collaboration of IDCH. Age and
sex matched 68 adult (age ranges 18 to 50 yrs) TB patients without having any other diseases from the
ongoing study on TB will be enrolled in the study. Clinical data will be collected and recorded. Same
volume (6 ml) of blood will be collected from the TB patients. Serum will be used to perform the tests
mentioned above.
Laboratory methods
Dot-blot assay
As the first screening the recombinant proteins will be screened by dot-blot assay for the presence of Bcell epitopes using well-characterized sera from patients. The recombinant proteins will be dot blotted on
to the nylon membrane. The membrane will be kept on the bench for 30 min for drying and then will be
stained with Ponceau S solution for one min. The filter will be destained completely in water for 10 min.
Nonspecific antibody sites on the nitrocellulose will be blocked with blocking solution [1 g of instant
nonfat dry milk in 100 ml phosphate biffered saline (PBS)] for 1 hr at room temperature on a rocking
platform. To test the immune responses of antibodies from pateints’ sera to recombinant proteins (primary
antibodies), blots will be hybridized with serum samples diluted 1:100. The blot with diluted primary
antibody will be incubated at room temperature for 1 hr on a rocking platform. Nonspecifically bound
primary antibody will be removed by washing the filter 4 times by agitating with 200 ml of PBS each time.
Bound antibody will be revealed by incubation with peroxidase-conjugated anti-human immunoglobulin.
Western blotting
Western blotting technique will be used as described previously (2). Proteins will be separated by SDSPAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in PBS on slab gels of polyacrylamide.
Ten g of protein will be loaded in each lane of the gel. A mixture of standard protein markers will be used
for the determination of molecular mass. Proteins will be transferred from polyacrylamide gels onto
nitrocellulose membrane (18). Electrophoretic transfer of the proteins from the gel to the nitrocellulose will
be done at 14 V constant voltage for 2 hrs. Nonspecific antibody sites on the nitrocellulose will be blocked
with blocking solution. Blots will be hybridized with serum samples diluted 1:100.
ELISA assay
The ELISA assays for detection of IgM, IgA and IgG antibodies to purified proteins will be used in the
laboratory at the ICDDR,B (16). Briefly, wells of the ELISA plates will be coated with 100 l of 2 g/ml of
purified recombinant proteins overnight at room temperature, will be washed with PBS and blocked with
0.1% BSA (bovine serum albumin) in PBS. The initial dilution of serum will be 1/30 followed by threefold dilutions of the serum samples. Serum dilutions will be incubated with the antigen-coated ELISA
plates for 1.5 hours at 370C. The plates will be then washed with phosphate-buffered saline containing
Tween-20 (0.05%) and anti-human antibody of different isotypes conjugated to HRP (horse radish
peroxidase) will be added and the incubation will be continued for 1.5 hours at 370C. After extensive
washing, substrate (3,3’, 5,5’ tetramethyl benzidine) will be added and, after 10 minutes, the color
development will be stopped by the addition of 1.0 M H2SO4 (50 l/well). Color development is measured
14
in a spectrophotometer at 450 nm. The end-point antibody titer will be measured using a computer-assisted
program MULTI (Data Tree Inc., Watthams, MA) (19). All study samples will be evaluated in duplicate.
Lymphocyte proliferation response.
Mononuclear cells will be cultured in RPMI 1640 (Gibco BRL, Life Technologies, Grand Island, NY)
supplemented with 20% heat inactivated human AB sera, 2 mM glutamine, 1 mM Na-pyruvate, 100 U/ml
penicillin and 1
2. Phytohemagglutinin A
(PHA, 100 ng/ml; Sigma, St. Loius, Mo) will be used as the mitogen. Cells will be pulsed with tritiated
(3H-) thymidine (Amersham, Pharmacia Biotech, UK) specific activity 5
hours before cell harvest. Labeled cells will be collected on glass fiber filter paper (Whatman International
Ltd., Maidstone, England) by using a cell harvestor (Automesh 2000, Dynatech, Denkendorf, germany) and
the 3H-thymidine incorporation into lymphocytes will be counted as counts per minute (cpm) with a beta
counter (LS 6500, Beckman, Calif.). The result per individual will be expressed as mean stimulation index
of triplicates (cpm of stimulated cells/cpm of unstimulated cells).
Sample size estimation
Calculation of the sample size is based on several assumptions. First, we assume that serum samples will
be obtained from patients attending the National Leprosy Hospital, IDCH (Institute of Diseases of Chest
and Hospital) and from healthy controls for this study. Secondly, we assume that clinical diagnosis based
on clinical signs and symptoms of patients is 100% sensitive for diagnosis of paucibacillary leprosy.
Thirdly, we assume that the serological assay to be tested would be 80% sensitive and 85% specific.
Therefore, for validation of this serological assay for leprosy, approximately each of 68 leprosy patients,
TB patients and healthy controls would have to be tested in order to detect at least a sensitivity of 80% and
specificity of 85% with 95% confidence and 10% precision allowing for a drop out of 10%.
Flow-chart for different tests to be performed
Steps of experimental procedure to be taken
Definition of optimum result
1. Identification of M. leprae specific proteins by
Comparative genomics and bioinformatics
Must be retricted to M. leprae
2. Cloning and overexpression of proteins
Must be cloned and overexpressed
3. Dot-blot assay for presence of B-cells in sera of
patients and controls (1st screening)
Must be identified by sera from leprosy
patients but must be absent in controls
4. 2nd screening using ELISA and Western blotting
Must be identified by leprosy patients’
sera with specific size of each
ecombinant protein
15
5. Lymphocyte proliferation assay in peripheral blood
Must have ability to stimulate the
proliferation of lymphocytes
6. IFN-production by capture ELISA
Must have the capacity to induce IFN-
7. Designing and synthesizing overlapping peptides of
20 amino acids, spanning the proteins in order to
delimit the T-cell epitopes
Should be designed and synthesized
properly
8. Peptides will be tested for capacity to proliferate
lymphocytes and produce IFN- to evaluate their
potential as diagnostic reagents
Must be capable of proliferating
lymphocyte and produce inducing
IFN- production in patients’ sera
Experimental procedures mentioned in steps 1, 2 and 7 will be performed at Institut Pasteur, Paris and all other
experiments (steps 3, 4, 5, 6 and 8) will be performed at ICDDR,B laboratory. All purified proteins and peptides
will be produced at Institut Pasteur and will be sent to ICDDR,B for further analysis. No specimen will be sent to
Pasteur from ICDDR,B.
Facilities Available
Describe the availability of physical facilities at the place where the study will be carried out. For clinical and laboratory-based
studies, indicate the provision of hospital and other types of patient’s care facilities and adequate laboratory support. Point out
the laboratory facilities and major equipment that will be required for the study. For field studies, describe the field area
including its size, population, and means of communications. (TYPE WITHIN THE PROVIDED SPACE).
Dr. Sayera Banu has been trained on mycobacteriology at the Institut Pasteur, Paris. Prof. Stewart Cole,
Head of Genetics and Molecular Bacteriology Unit of Institut Pasteur, Paris, is the foreign principal
investigator and will provide technical support for the study.
Sample collection will be done in collaboration with the staff of National Leprosy Hospital in Dhaka and
Damien Foundation Bangladesh. The leprosy clinic at Mohakhali, Dhaka, is part of the National Control
Programme and receives 30 – 40 new cases per month and these fall into roughly equal groups of PB and
MB. They routinely take punch biopsies for diagnostic purposes. The Mohakhali clinic is very close to the
International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B) where a laboratory for
mycobacterial research has been established recently. Samples could be quickly transferred to ICDDR,B
which is suitably equipped, then processed for immunological analyses, these would be mainly from new
cases.
Part of the study work will be performed at the TB laboratory of ICDDR,B and part of it will be done at
Pasteur Institute.
16
Data Analysis
Describe plans for data analysis. Indicate whether data will be analyzed by the investigators themselves or by other professionals.
Specify what statistical software packages will be used and if the study is blinded, when the code will be opened. For clinical
trials, indicate if interim data analysis will be required to monitor further progress of the study. (TYPE WITHIN THE PROVIDED
SPACE).
Data will be entered, cleaned and analyzed by the investigators themselves. SPSS Windows software will
be used to analyze data. Sensitivity, specificity and predictive values of the tests will be determined
following standard statistical methods. These indices will be compared among the leprosy patients and
healthy controls. The indices are defined as follows:
Sensitivity is defined as the ability of a test to identify a disease when it is really present, that is, the
proportion positive of those who have the disease. Specificity is the ability to identify the absence of a
disease when it is really not present. It is the proportion negative of those who do not have the disease. The
positive predictive value is the ratio of true-positive test results to all positive test results. The negative
predictive value is the ratio of true-negative test results to all negative test results.
Ethical Assurance for Protection of Human Rights
Describe in the space provided the justifications for conducting this research in human subjects. If the study needs observations
on sick individuals, provide sufficient reasons for using them. Indicate how subject’s rights are protected and if there is any
benefit or risk to each subject of the study.
The study will only be initiated after it has been approved by the Research Review Committee (RRC) and
the Ethical Review Committee (ERC) of ICDDR,B. Informed signed consent is obtained from the patients.
The consent form is written in Bangla in a language and format that is easily understood by the study
subject of even little or no educational background. The consent form is read out to the subject if he/she is
unable to read. Signed consent or the left thumb impression will be taken on the form from the study
subjects prior to enrollment onto the study. Consent will be taken both for participation in the study and for
collecting blood. Confidentiality about subjects and their health status will be strictly maintained. Only the
investigators and the subjects will be aware of the health status.
Use of Animals
Describe in the space provided the type and species of animal that will be used in the study. Justify with reasons the use of
particular animal species in the experiment and the compliance of the animal ethical guidelines for conducting the proposed
procedures.
Not applicable for ICDDR,B but needed for Institut Pasteur.
17
Literature Cited
Identify all cited references to published literature in the text by number in parentheses. List all cited references sequentially as
they appear in the text. For unpublished references, provide complete information in the text and do not include them in the list
of Literature Cited. There is no page limit for this section, however exercise judgment in assessing the “standard” length.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Alderson, M. R., T. Bement, C. H. Day, L. Zhu, D. Molesh, Y. A. Skeiky, R. Coler, D. M.
Lewinsohn, S. G. Reed, and D. C. Dillon. 2000. Expression cloning of an immunodominant family
of Mycobacterium tuberculosis antigens using human CD4(+) T cells. J Exp Med 191:551-60.
Banu, S., N. Honore, B. Saint-Joanis, D. Philpott, M. C. Prevost, and S. T. Cole. 2002. Are the PEPGRS proteins of Mycobacterium tuberculosis variable surface antigens? Mol Microbiol 44:9-19.
Brennan, M. J., and G. Delogu. 2002. The PE multigene family: a 'molecular mantra' for
mycobacteria. Trends Microbiol 10:246-9.
Brennan, M. J., G. Delogu, Y. Chen, S. Bardarov, J. Kriakov, M. Alavi, and W. R. Jacobs, Jr. 2001.
Evidence that mycobacterial PE_PGRS proteins are cell surface constituents that influence
interactions with other cells. Infect Immun 69:7326-33.
Brennan, P. J. 2000. Skin test development in leprosy: progress with first-generation skin test
antigens, and an approach to the second generation. Lepr Rev 71 Suppl:S50-4.
Brosch, R., A. S. Pym, S. V. Gordon, and S. T. Cole. 2001. The evolution of mycobacterial
pathogenicity: clues from comparative genomics. Trends Microbiol 9:452-8.
Chin-a-Lien, R. A., W. R. Faber, M. M. van Rens, D. L. Leiker, B. Naafs, and P. R. Klatser. 1992.
Follow-up of multibacillary leprosy patients using a phenolic glycolipid-I-based ELISA. Do
increasing ELISA-values after discontinuation of treatment indicate relapse? Lepr Rev 63:21-7.
Cho, S. N., S. H. Kim, R. V. Cellona, G. P. Chan, T. T. Fajardo, G. P. Walsh, and J. D. Kim. 1992.
Prevalence of IgM antibodies to phenolic glycolipid I among household contacts and controls in
Korea and the Philippines. Lepr Rev 63:12-20.
Cole, S. T. 2002. Comparative mycobacterial genomics as a tool for drug target and antigen
discovery. Eur Respir J Suppl 36:78s-86s.
Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier,
S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R.
Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K.
Jagels, B. G. Barrell, and et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from
the complete genome sequence. Nature 393:537-44.
Cole, S. T., K. Eiglmeier, J. Parkhill, K. D. James, N. R. Thomson, P. R. Wheeler, N. Honore, T.
Garnier, C. Churcher, D. Harris, K. Mungall, D. Basham, D. Brown, T. Chillingworth, R. Connor,
R. M. Davies, K. Devlin, S. Duthoy, T. Feltwell, A. Fraser, N. Hamlin, S. Holroyd, T. Hornsby, K.
Jagels, C. Lacroix, J. Maclean, S. Moule, L. Murphy, K. Oliver, M. A. Quail, M. A. Rajandream, K.
M. Rutherford, S. Rutter, K. Seeger, S. Simon, M. Simmonds, J. Skelton, R. Squares, S. Squares,
K. Stevens, K. Taylor, S. Whitehead, J. R. Woodward, and B. G. Barrell. 2001. Massive gene decay
in the leprosy bacillus. Nature 409:1007-11.
Delogu, G., and M. J. Brennan. 2001. Comparative immune response to PE and PE_PGRS antigens
of Mycobacterium tuberculosis. Infect Immun 69:5606-11.
Dillon, D. C., M. R. Alderson, C. H. Day, D. M. Lewinsohn, R. Coler, T. Bement, A. CamposNeto, Y. A. Skeiky, I. M. Orme, A. Roberts, S. Steen, W. Dalemans, R. Badaro, and S. G. Reed.
1999. Molecular characterization and human T-cell responses to a member of a novel
Mycobacterium tuberculosis mtb39 gene family. Infect Immun 67:2941-50.
Dockrell, H. M., S. Brahmbhatt, B. D. Robertson, S. Britton, U. Fruth, N. Gebre, M. Hunegnaw, R.
Hussain, R. Manandhar, L. Murillo, M. C. Pessolani, P. Roche, J. L. Salgado, E. Sampaio, F.
Shahid, J. E. Thole, and D. B. Young. 2000. A postgenomic approach to identification of
Mycobacterium leprae-specific peptides as T-cell reagents. Infect Immun 68:5846-55.
Geluk, A., K. E. van Meijgaarden, K. L. Franken, Y. W. Subronto, B. Wieles, S. M. Arend, E. P.
Sampaio, T. de Boer, W. R. Faber, B. Naafs, and T. H. Ottenhoff. 2002. Identification and
18
16.
17.
18.
19.
characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity
with Mycobacterium tuberculosis. Infect Immun 70:2544-8.
Qadri, F., C. Wenneras, M. J. Albert, J. Hossain, K. Mannoor, Y. A. Begum, G. Mohi, M. A.
Salam, R. B. Sack, and A. M. Svennerholm. 1997. Comparison of immune responses in patients
infected with Vibrio cholerae O139 and O1. Infect Immun 65:3571-6.
Sorensen, A. L., S. Nagai, G. Houen, P. Andersen, and A. B. Andersen. 1995. Purification and
characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.
Infect Immun 63:1710-7.
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci
U S A 76:4350-4.
Vega-Lopez, F., L. A. Brooks, H. M. Dockrell, K. A. De Smet, J. K. Thompson, R. Hussain, and N.
G. Stoker. 1993. Sequence and immunological characterization of a serine-rich antigen from
Mycobacterium leprae. Infect Immun 61:2145-53.
Dissemination and Use of Findings
Describe explicitly the plans for disseminating the accomplished results. Describe what type of publication
is anticipated: working papers, internal (institutional) publication, international publications, international
conferences and agencies, workshops etc. Mention if the project is linked to the Government of Bangladesh
through a training programme.
It is expected that this post-genomic diagnostic approach will give rise to a test with the desired sensitivity
and specificity for the detection of leprosy in suspected PB patients and that the reagents will be compatible
with a non-invasive intervention such as in a trans-dermal formulation. In addition, there is also a
possibility that some of the proteins/peptides identified here may be of use for identifying individuals with
sub-clinical infection and thus provide more reliable epidemiological information about the extent of
transmission of M. leprae.
Collaborative Arrangements
Describe briefly if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other national or
international organizations or individuals. Indicate the nature and extent of collaboration and include a letter of agreement
between the applicant or his/her organization and the collaborating organization. (DO NOT EXCEED ONE PAGE)
This is a collaborative study between the International Centre for Diarrhoeal Disease Research, Bangladesh
(ICDDR,B), National Leprosy Control Program, Bangladesh, and, the Genetic and Molecular Microbiology
Unit, Pasteur Institute, Paris.
19
Biography of the Investigators
Give biographical data in the following table for key personnel including the Principal Investigator. Use a photocopy of this page
for each investigator.
1
Name
: Dr. Sayera Banu
2
Present position
: National Research Fellow, LSD (Tuberculosis section)
Educational background
:
3
(last degree and diploma & training
relevant to the present research proposal)
Mymensingh Medical College
University of Dhaka
M.B.B.S
1989
Medical Science
University of Tsukuba, Japan
MS
1997
Molecular Microbiology
Institut Pasteur, Paris
Post graduate training
2000
Mycobacteriology
University of Dhaka
PhD (thesis submitted)
2002
Microbiology
List of ongoing research protocols
(start and end dates; and percentage of time)
4.1. As Principal Investigator
Protocol Number
2000--029
Starting date
1.4.02
End date
31.3.04
Percentage of time
80
Starting date
End date
Percentage of time
Starting date
1.6.00
Ending date
31.5.03
Percentage of time
20
4.2. As Co-Principal Investigator
Protocol Number
4.3. As Co-Investigator
Protocol Number
2000-13
20
5 Publications
a)
b)
c)
c)
Types of publications
Original scientific papers in peer-review journals
Peer reviewed articles and book chapters
Papers in conference proceedings
Letters, editorials, annotations, and abstracts in peer-reviewed
journals
Numbers
5
1
2
d) Working papers
b) Monographs
6
Five recent publications including publications relevant to the present research protocol
1. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Banu S, Honoré N, SaintJoanis B, Philpott D, Prévost MC and Cole St. Mol Microbiol 2002 Apr; 44(1): 9-19.
2. Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Shimizu T, Yaguchi
H, Ohtani K, Banu S, and Hayashi H. Mol Microbiol. 2002 Jan; 43(1): 257-65.
3. Identification of novel VirR/VirS-regulated genes in Clostridium perfringens. Banu S, Ohtani K, Yaguchi H, Swe
T, Cole ST, Hayashi H, Shimizu T. Mol Microbiol. 2000 Feb; 35(4): 854-64.
4. Collagenase gene (colA) is located in the 3'-flanking region of the perfringolysin O (pfoA) locus in Clostridium
perfringens. Ohtani K, Bando M, Swe T, Banu S, Oe M, Hayashi H, Shimizu T. FEMS Microbiol. Lett.
1997;146:155-159.
5. Genomic diversity in the pfoA region of theta toxin deficient strains of Clostridium perfringens. Ba-Thein W,
Inui S, Shimizu T, Swe T, Banu S, Ohtani K, Oe M, Sakurai N, Nakamura S, Hayashi H. Microbiol. Immunol.
1997;41(8):629-631.
------------------------------------------------------------------------------------------------------------
21
Biography of the Investigators
Give biographical data in the following table for key personnel including the Principal Investigator. Use a photocopy of this page
for each investigator.
1
Name
: Professor Stewart Cole
2
Present position
: Head, Genetics and Molecular Bacteriology Unit, Pasteur Institute
Educational background
:
3
(last degree and diploma & training
relevant to the present research proposal)
1976
Bachelor of Science, University of Wales (Microbiology)
1979
Doctor of Philosophy, University of Sheffield (Molecular Genetics)
List of ongoing research protocols
(start and end dates; and percentage of time)
4.4. As Principal Investigator
Protocol Number
Starting date
End date
Percentage of time
Starting date
End date
Percentage of time
Starting date
Ending date
Percentage of time
4.5. As Co-Principal Investigator
Protocol Number
4.6. As Co-Investigator
Protocol Number
22
5 Publications
a)
b)
c)
c)
Types of publications
Original scientific papers in peer-review journals
Peer reviewed articles and book chapters
Papers in conference proceedings
Letters, editorials, annotations, and abstracts in peer-reviewed
journals
Numbers
c) Working papers
d) Monographs
6
Five recent publications including publications relevant to the present research protocol
1) Cole, S.T., et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature 393, 537-544 (1998).
2) Cole, S.T., et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007-1011 (2001).
3) Cole, S.T. Comparative mycobacterial genomics as a tool for drug target and antigen discovery. Eur. Resp. J. 20,
78-86S (2002).
4) Brosch, R., et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci
U S A 99, 3684-9. (2002).
5) Brosch, R., Pym, A.S., Gordon, S.V. & Cole, S.T. The evolution of mycobacterial pathogenicity: clues from
comparative genomics. Trends Microbiol 9, 452-458 (2001).
------------------------------------------------------------------------------------------------------------
23
Continuation Sheet (Number each sheet consecutively)
Detailed Budget for New Proposal
Project Title: Post-genomic leprosy diagnostics
Name of PI: Dr. Sayera Banu (ICDDR,B), Prof. Steawart Cole (Paris)
Protocol Number:
Name of Division: LSD
Funding Source : WHO-TDR
Overhead (%)
Starting Date:
Amount Funded (direct):
Total:
Closing Date:
Strategic Plan Priority Code(s):
Sl. No
Account Description
Personnel
Dr. Sayera Banu
To be named
Salary Support
Position
Principal investigator
Research Assistant
Sub Total
US $ Amount Requested
Effort
%
40
100
Salary
1st Yr
2nd Yr
NO-B
GS5/1
CSA
3,752
3,078
3,940
3,232
6830
7172
Consultants
Local Travel
400
300
2,500 2,500
2,900 2,800
International Travel
Sub Total
Supplies and Materials (Description of Items)
Supplies
39,000
44,000
Sub Totals
39,000
44,000
Other Contractual Services
Repair and Maintenance
Rent, Communications, Utilities
Training Workshop, Seminars
Printing and Publication
Staff Development
Sub Total
24
100
100
300
200
250
200
550
500
3rd Yr
1st Yr
Interdepartmental Services
Computer Charges
Pathological Tests
Microbiological tests
Biochemistry Tests
X-Rays
Patients Study
Research Animals
Biochemistry and Nutrition
Transport
Xerox, Mimeographs etc.
150
Sub Total
Capital Expenditure
Total Operating Cost
TOTAL DIRECT COST
US$108,002
25
2nd Yr
3rd Yr
150
900
2000
700
100
100
3,150
950
52,530
55,472
Budget Justifications
Please provide one page statement justifying the budgeted amount for each major item. Justify use of man power, major
equipment, and laboratory services.
Funding will be shared equally between the Institut Pasteur and ICDDR,B, except for patient costs and salaries. We
request $3752 towards Dr. Banu's salary and $3,078 to recruit a person (research assistant level, 100%) at ICDDR,B
who will assist in collecting samples in hospitals and at the field level, bring samples to ICDDR,B and help in the
laboratory.
Supplies:
Molecular biology & biochemical reagents: $19,000
Immunological reagents (IFN-gamma detection kits, etc.): $20,000
In year 2, extensive peptide synthesis will be required at $300/20mer and >10 peptides/target protein. This
component may exceed $35,000.
Travel funds are requested to facilitate discussion and execution of the project.
Other Support
Describe sources, amount, duration, and grant number of all other research funding currently granted to PI or under
consideration. (DO NOT EXCEED ONE PAGE FOR EACH INVESTIGATOR)
Part of the costs for producing r-proteins are offset by grant no. 1 RO1 AI47197-01A1 from the National Institutes
of Health, National Institute of Allergy and Infectious Diseases (PI - P.J. Brennan)
Check List
26
After completing the protocol, please check that the following selected items have been included.
1. Face Sheet Included
2. Approval of the Division Director on Face Sheet
3. Certification and Signature of PI on Face Sheet, #9 and #10
4. Table on Contents
5. Project Summary
6. Literature Cited
7. Biography of Investigators
8. Ethical Assurance
9. Consent Forms
10. Detailed Budget
APPENDIX
International Centre for Diarrhoeal Disease Research, Bangladesh
27
Voluntary Consent Form for Leprosy Patient
Title of the Research Project: Towards
an immunodiagnostic kit for leprosy
Principal Investigator: Dr. Sayera Banu
Before recruiting into the study, the study subject must be informed about the objectives, procedures, and potential benefits and risks involved
in the study. Details of all procedures must be provided including their risks, utility, duration, frequencies, and severity. All questions of the
subject must be answered to his/ her satisfaction, indicating that the participation is purely voluntary. For children, consents must be obtained
from their parents or legal guardians. The subject must indicate his/ her acceptance of participation by signing or thumb printing on this form.
(This form will be read and explained clearly in the vernacular to the patient before consent is obtained)
Leprosy is a public health problem in developing countries including Bangladesh. The current estimated number of
cases in Bangladesh is 136,000. To eradicate leprosy completely it is very important that the disease can be
diagnosed at the very early stage of the infection. But unfortunately, it remains difficult diagnosing the early stage
of leprosy due to lack of efficient diagnostic methods. Leprosy is a completely curable disease if it can be detected
early and preferably before clinical signs become apparent.
The International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) with collaboration with the
Pasteur Institute, Paris is conducting a research to develop a rapid and efficient technique for the diagnosis of
leprosy. We request for your permission to include you in our study and help us in our efforts.
Other than a closer observation and the usual good care and treatment provided by this hospital, you would not
receive any other benefit from participating in this study. If you allow us to enroll you in our study, the followings
will be done:
We will ask you questions related to your illness and perform thorough physical examination at enrollment to the
study, and record and use the information for this study. You do not have to answer the questions but by answering
you will improve the quality of the results of the study.
As the routine diagnostic test, a tiny pieces of tissue will be taken from your infected skin. This would be done by a
trained and very experienced investigator of this study who has performed this procedure on hundreds of patients.
For special tests of this study we would collect 6 ml (one teaspoonful) of blood from a vein on your forearm at the
beginning of the study. Other than momentary pain and a very small chance of bruising at the site of insertion of the
needles, drawing of such amount of blood will not cause any harm to you. To minimize the chance of infection, we
will take aseptic precausions and use disposable, sterile syringe and needles for drawing blood. Your blood will be
mixed with special chemicals and an immune reaction measured that will help us to identify leprosy. Afterwards
your sample will be frozen in case we have to repeat the measurement or perform another test related to the study.
If you do not agree with this second test you should say so now and it will not be performed. If you do agree now we
will not ask for your permission again to perform the second test on your sample at a later date. Within one year of
completion of the study any remaining samples will be destroyed.
All information obtained from you will be stored in a secure place, and none other than the investigators of this
study and Ethical Review Committee of this Centre would have access to such information. Your name and identity
will not be used at the time of analysis of data or in publishing the results of this study. We would be happy to
answer your questions related to illness and our study, and to provide you with the results of your laboratory tests as
and when they are available. You are free to accept or reject our proposal to enroll in this study, and you would also
be able to withdraw your consent at any time during the study.
If you agree to our proposal of enrollment in this study, please indicate that by putting your signature or the
impression of your left thumb at the specified space below.
I have read the above information/it has
been read to me and understood clearly. I
28
consent voluntarily to participate as a
subject in this study.
Signature of Investigator/ or agents
Date:
Signature or thumb impression of
Subject
Date:
Signature of witness
Date:
International Centre for Diarrhoeal Disease Research, Bangladesh
29
Voluntary Consent Form for Healthy Control
Title of the Research Project: Towards
an immunodiagnostic kit for leprosy
Principal Investigator: Dr. Sayera Banu
Before recruiting into the study, the study subject must be informed about the objectives, procedures, and potential benefits and risks involved
in the study. Details of all procedures must be provided including their risks, utility, duration, frequencies, and severity. All questions of the
subject must be answered to his/ her satisfaction, indicating that the participation is purely voluntary. For children, consents must be obtained
from their parents or legal guardians. The subject must indicate his/ her acceptance of participation by signing or thumb printing on this form.
(This form will be read and explained clearly in the vernacular to the patient before consent is obtained)
Leprosy is a public health problem in developing countries including Bangladesh. The current estimated number of
cases in Bangladesh is 136,000. To eradicate leprosy completely it is very important that the disease can be
diagnosed at the very early stage of the infection. But unfortunately, it remains difficult diagnosing the early stage
of leprosy due to lack of efficient diagnostic methods. Leprosy is a completely curable disease if it can be detected
early and preferably before clinical signs become apparent.
The International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) with collaboration with the
Pasteur Institute, Paris is conducting a research to develop a rapid and efficient technique for the diagnosis of
leprosy. For the purpose of this study we are studying adult leprosy patients. However, we also need to study
healthy adults who do not have leprosy, and we request for your permission to include you in our study and help us
in our efforts. You would not benefit from participation in this study, however, results of this study will improve our
knowledge that could benefit the society in future.
If you allow us to enroll you in our study, the followings will be done:
We will ask you questions related to your illness and perform thorough physical examination at enrollment to the
study, and record and use the information for this study. You do not have to answer the questions but by answering
you will improve the quality of the results of the study.
For special tests of this study we would collect 6 ml (one teaspoonful) of blood from a vein on your forearm at the
beginning of the study. Other than momentary pain and a very small chance of bruising at the site of insertion of the
needles, drawing of such amount of blood will not cause any harm to you. To minimize the chance of infection, we
will take aseptic precausions and use disposable, sterile syringe and needles for drawing blood. Your blood will be
mixed with special chemicals and an immune reaction measured that will help us to identify leprosy. Afterwards
your sample will be frozen in case we have to repeat the measurement or perform another test related to the study.
If you do not agree with this second test you should say so now and it will not be performed. If you do agree now we
will not ask for your permission again to perform the second test on your sample at a later date. Within one year of
completion of the study any remaining samples will be destroyed.
All information obtained from you will be stored in a secure place, and none other than the investigators of this
study and Ethical Review Committee of this Centre would have access to such information. Your name and identity
will not be used at the time of analysis of data or in publishing the results of this study. We would be happy to
answer your questions related to our study, and to provide you with the results of your laboratory tests as and when
they are available. You are free to accept or reject our proposal to enroll in this study, and you would also be able to
withdraw your consent at any time during the study.
If you agree to our proposal of enrollment in this study, please indicate that by putting your signature or the
impression of your left thumb at the specified space below.
30
I have read the above information/it has
been read to me and understood clearly. I
consent voluntarily to participate as a
subject in this study.
Signature of Investigator/ or agents
Date:
Signature or thumb impression of
Subject
Date:
Signature of witness
Date:
International Centre for Diarrhoeal Disease Research, Bangladesh
Voluntary Consent Form for TB Control
Title of the Research Project: Towards an immunodiagnostic kit for leprosy
31
Principal Investigator: Dr. Sayera Banu
Before recruiting into the study, the study subject must be informed about the objectives, procedures, and potential benefits and risks involved
in the study. Details of all procedures must be provided including their risks, utility, duration, frequencies, and severity. All questions of the
subject must be answered to his/ her satisfaction, indicating that the participation is purely voluntary. For children, consents must be obtained
from their parents or legal guardians. The subject must indicate his/ her acceptance of participation by signing or thumb printing on this form.
(This form will be read and explained clearly in the vernacular to the patient before consent is obtained)
Leprosy is a public health problem in developing countries including Bangladesh. The current estimated number of
cases in Bangladesh is 136,000. To eradicate leprosy completely it is very important that the disease can be
diagnosed at the very early stage of the infection. But unfortunately, it remains difficult diagnosing the early stage
of leprosy due to lack of efficient diagnostic methods. Leprosy is a completely curable disease if it can be detected
early and preferably before clinical signs become apparent.
The International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) with collaboration with the
Pasteur Institute, Paris is conducting a research to develop a rapid and efficient technique for the diagnosis of
leprosy. For the purpose of this study we are studying adult leprosy patients. However, we also need to study adults
TB patients, and we request for your permission to include you in our study and help us in our efforts. You would
not benefit from participation in this study, however, results of this study will improve our knowledge that could
benefit the society in future.
If you allow us to enroll you in our study, the followings will be done:
We will ask you questions related to your illness and perform thorough physical examination at enrollment to the
study, and record and use the information for this study. You do not have to answer the questions but by answering
you will improve the quality of the results of the study.
For special tests of this study we would collect 6 ml (one teaspoonful) of blood from a vein on your forearm at the
beginning of the study. Other than momentary pain and a very small chance of bruising at the site of insertion of the
needles, drawing of such amount of blood will not cause any harm to you. To minimize the chance of infection, we
will take aseptic precausions and use disposable, sterile syringe and needles for drawing blood. Your blood will be
mixed with special chemicals and an immune reaction measured that will help us to identify leprosy. Afterwards
your sample will be frozen in case we have to repeat the measurement or perform another test related to the study.
If you do not agree with this second test you should say so now and it will not be performed. If you do agree now we
will not ask for your permission again to perform the second test on your sample at a later date. Within one year of
completion of the study any remaining samples will be destroyed.
All information obtained from you will be stored in a secure place, and none other than the investigators of this
study and Ethical Review Committee of this Centre would have access to such information. Your name and identity
will not be used at the time of analysis of data or in publishing the results of this study. We would be happy to
answer your questions related to our study, and to provide you with the results of your laboratory tests as and when
they are available. You are free to accept or reject our proposal to enroll in this study, and you would also be able to
withdraw your consent at any time during the study.
If you agree to our proposal of enrollment in this study, please indicate that by putting your signature or the
impression of your left thumb at the specified space below.
I have read the above information/it has
been read to me and understood clearly. I
32
consent voluntarily to participate as a
subject in this study.
Signature of Investigator/ or agents
Date:
Signature or thumb impression of
Subject
Date:
Signature of witness
Date:
33
34