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Manuscript Title: Identification and characterization of a novel 37 kDa Leishmania donovani 1
Antigen for Diagnosis of Indian Visceral Leishmaniasis 2
Running Title: Serodiagnosis of Indian VL with a novel antigen 3
Article type: Major Article 4
Order of Authors: Subodh Kumar*, Dinesh Kumar*, Jaya Chakravarty, Shyam Sundar 5
*Joint first author 6
Affiliations: Department of Medicine, Institute of Medical Sciences, Banaras Hindu University 7
Varanasi-221005, India 8
Word Count: Abstract 210 words, Text 1,841 words 9
Corresponding Author: 10
Dr. Shyam Sundar* 11
Infectious Disease Research Laboratory 12
Department of Medicine 13
Institute of Medical Sciences 14
Banaras Hindu University 15
Varanasi-221005 16
Phone: +91 542 2369 632 17
Fax: +91 542 2367 568 18
E-mail: [email protected] 19 20
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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00559-10 CVI Accepts, published online ahead of print on 16 March 2011
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Footnotes: 31
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Corresponding Author’s Name: Dr. Shyam Sundar 33
Corresponding Author’s Mailing Address: Professor of Medicine 34
Infectious Disease Research Laboratory 35
Department of Medicine 36
Institute of Medical Sciences 37
Banaras Hindu University 38
Varanasi 221005, Uttar Pradesh 39
India 40
Corresponding Author’s Phone Number, Fax number, and 41
E-mail address: Tel: + 91-542-2367795 42
Fax: + 91-542-2367568 43
E-mail:- [email protected] 44
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Abstract 54
The biggest challenge in serological diagnosis for Visceral Leishmaniasis (VL) is to find out a 55
biomarker with high specificity. This study was undertaken to identify novel Leishmania 56
donovani antigens to solve the existing problem. The soluble L. donovani promastigotes antigen 57
was separated by SDS-PAGE and western blot was probed with pooled sera of five subjects 58
with confirmed VL pre ( n = 9 pool) and post-treated ( n = 9 pool) patients, six month follow up ( 59
n = 9 pool), non endemic healthy controls (NEHC, n = 9 pool) and endemic healthy controls 60
(EHC). Antibody response to the identified partially purified antigen was ascertained by ELISA 61
on 70 parasitologically confirmed VL, 48 endemic healthy, 60 non endemic healthy and 42 62
different disease groups. The eluted protein was subjected to 2D gel electrophoresis, western 63
blotted and probed with sera of confirmed VL and NEHC. The antigenic protein was further 64
characterized by MALDI-TOF mass spectrometry. The identified protein (BHUP2) corresponds to 65
a Cytochrome - C like synthesis protein of 37 kDa. ELISA results were 94% sensitive whereas 66
specificity with EHC, NEHC and disease control were 98%, 100% and 97% respectively. The 67
antigen identified via a proteomics based approach has a strong potential for further development 68
as a diagnostic tool for VL. 69
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Key Words: Diagnosis, ELISA, L. donovani, MALDI-TOF, Visceral Leishmaniasis, 71
western blotting, 2D gel electrophoresis. 72
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Introduction 77
Visceral Leishmaniasis (VL) commonly known as kala-azar is the life threatening clinical form of 78
leishmaniasis characterized by fever, loss of weight, splenomegaly, hepatomegaly and anaemia. It 79
is a major public health problem in several countries, particularly India, Bangladesh, Nepal, Sudan 80
and Brazil. (2). Timely and accurate diagnosis is necessary for cure and also because the 81
antileishmanial drugs for treatment are expensive and associated with adverse events which can 82
occasionally be serious. The gold standard for the diagnosis of VL is through direct microscopic 83
detection of amastigotes in giemsa stained splenic or bone marrow smears with reported 84
sensitivity ranging from 93.1% -98.7% (4, 8, 17) for splenic smears and 52% to 85% (1, 17) for 85
bone marrow aspirates. These methods are invasive, painful and risky. High levels of parasite 86
specific antibodies are observed prior to detection of antigen specific T cell responses (3). One 87
approach adopted to improve diagnostic methods to detect infection with L. donovani is to 88
identify dominant antigens that elicit specific antibody detectable by serological tests. In Indian 89
subcontinent, rK39 antigen, available in the ELISA and ICT (Immunochromatographic) format, is 90
widely used for the diagnosis of VL with excellent sensitivity (10, 12, 14, 15) The specificity in 91
terms of non endemic healthy control is excellent in Indian subcontinent whereas it shows �20-92
32% positivity with healthy subjects living in endemic areas (13, 15) and and this has kept alive 93
the search for a better test. Another antigen rK28, is in its evaluation phase in different countries, 94
it claims to replace the rK39, as having excellent sensitivity (96.8%) and specificity (96.2%) 95
tested on Sudanese population (7). The objective of this study was to identify L. donovani specific 96
antigen having high sensitivity and specificity and can have the potential to overcome the 97
problems associated with existing serological biomarkers in Indian subcontinent. 98
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Patients and Methods: 101
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Study Population: 102
The study was conducted at Infectious Disease Research Laboratory (IDRL) of Banaras Hindu 103
University (BHU), Varanasi, Uttar Pradesh and at its field site at Kala-Azar Medical Research 104
Center (KAMRC), Muzaffarpur which is highly endemic for VL. The study was approved by the 105
Ethical Committee of Institute of Medical Sciences, BHU, Varanasi and a written informed 106
consent was taken. 107
Patients: 108
Sera from 70 parasitologically confirmed VL patients and 48 healthy individuals living in 109
endemic regions (with no past history of kala-azar) were collected from endemic region. Healthy 110
individuals (n=60) from non-endemic areas constituted control cohort and 42 samples were 111
collected from those who were suffering from diseases like amoebic liver abscess, tuberculosis, 112
malaria etc. from Sir Sunderlal Hospital, BHU. The samples were stored at -20°C. 113
Crude soluble antigen (CSA) preparation 114
1 X 108
parasites were harvested from stationary phase promastigote culture in cold 1 X phosphate 115
buffer saline (PBS) at pH 7.2 for CSA preparation. After washing and centrifugation, pellet was 116
resuspended in 1X PBS and equal volume of complete protease inhibitor cocktail (Sigma, USA) 117
was added. Lysis of parasite cells was done by 6 alternate cycle of freezing (at -70 0C), and 118
thawing (at room temperature) followed by sonication. Supernatant was collected by 119
centrifugation at 4000 rpm for 10 min, and the protein was quantified by BCA (Bicinchoninic 120
acid) kit (Thermo scientific) (9). 121
SDS-PAGE 122
CSA extract was electrophoresed on 12% polyacrylamide gel following the method of Laemmli et 123
al (6). 124
Western Blotting 125
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CSA (45µg/well) of L. donovani was subjected to 12% sodium dodecylsulfate-polyacrylamide gel 126
electrophoresis (SDS-PAGE). CSA was then immunoblotted (16), by western blotting (Bio-Rad 127
Mini-Protean II, Multi Screen), to a PVDF membrane (0.45 µm pore size, Millipore, USA) at 20 128
V for 30 minutes. The membrane was further treated with sera (1:100 in PBS) of different study 129
groups, for 1 hour at room temperature. Alkaline phosphatase conjugated with goat anti human 130
IgG (1:1000) was used as a secondary antibody. At the end, color was developed using BCIP-131
NBT (5-Bromo-4-Chloro-3-indolylphosphate + Nitro Blue Thiazole) as a substrate (Promega, 132
USA). The obtained bands were analyzed by Alpha Imager (Alpha Inno. Tech, USA). 133
Partial purification of protein from SDS-PAGE gel 134
37 kDa protein band corresponding to protein marker was excised with sterile scalpel directly 135
from the SDS-PAGE gel, crushed and incubated overnight in an elution buffer (50 mM Tris-HCl, 136
150 mM NaCl and 0.1 mM EDTA, pH 7.5) at 370C. The solution was centrifuged at 10,000 rpm, 137
10oC for 20 minutes, and the obtained supernatant was quantified for protein by BCA method. 138
Enzyme Linked Immunosorbent Assay (ELISA) 139
Assay was done as described elsewhere with some modifications (5). Microtitre plates (Nunc, 140
USA) were coated with eluted 37 kDa BHUP2 protein (100ng/well) as a target antigen in 141
carbonate buffer (pH 9.6) for overnight at 4oC and then blocked the plate with 1% Bovine serum 142
albumin (BSA) in 1 X PBS for 2 hour at room temperature to prevent nonspecific binding. Sera 143
(1:100 dilution) of different sets were added and incubated at 25 0C for 1 hour. Serum antibody 144
titers were measured with HRP conjugated goat anti human IgG (1:16000) secondary antibody, 145
using tri-methylene benzidine (TMB, Promega) as a substrate. The reaction was stopped by 146
addition of 1N H2SO4, and OD was measured at 450 nm by an ELISA plate reader (Molecular 147
Device, Spectromax 190). The cutoff value is determined as mean ± 2 Standard deviations (SDs) 148
above the mean absorbance of NEHC. The diagnostic accuracy of BHUP2 protein was evaluated 149
by calculating the ROC (receiver operating characteristic) value, which was 0.98. 150
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151
Two Dimensional gel Electrophoresis (2D-PAGE) 152
Isoelectric focusing (IEF) was done in immobilized pH gradient gel strips (IPG strips, Bio-Rad) 153
with pH range of 3-10. Five microgram eluted protein was applied in 125 µl of rehydration buffer 154
per IPG strip. The sample containing rehydration buffer was loaded overnight at room 155
temperature by in gel re-swelling under mineral oil to prevent oxidation of protein and drying of 156
the gel strip. The loaded IPG strip was connected with the electrode of PROTIEN IEF cell 157
(BioRad, India), followed by electric parameters 20 minute, 100 V & 50 µA; 30 minute, 250 V & 158
50 µA; 2 hours, 4000 V and 10,000 V for 3 hours. The IPG strip was then equilibrated in 159
equilibrium buffer then after run for second dimension on resolving gel of SDS-PAGE. The gel 160
was stained with highly sensitive silver staining kit (PierceR Silver stain kit, Thermo Scientific) 161
according to the manufacturer’s instructions. 162
Mass Spectrometry 163
The protein spot after silver staining was excised and subjected to protein sequencing analysis by 164
MALDI-TOF at Molecular Biophysics Unit (MBU) of Indian Institute of Science, Banglore, 165
India. 166
Statistical Analysis 167
Data analysis was done through SPSS 16.0 software. The comparative evaluation was done by 168
non-parametric t-test. The peak lists of the mass spectra were used for peptide mass fingerprint 169
analysis with the Mascot software (Matrix Science; 170
http:www.matrixscience.com/search_form_select.html) together with NCBI sequence database. 171
Protein was identified using the following parameter: database: eukaryote (eukaryotes); enzyme: 172
trypsin; variable modification: oxidation (M), fixed modification: Carbamidomethyl (C); mass 173
value: monoisotopic; protein mass: unrestricted; peptide mass tolerance: ± 100 ppm, peptide 174
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charge state: +1, maximum missed cleavages: 1. The analyses of the PSD datasets were done by 175
peptide mass fingerprint with Mascot. 176
Results 177
Western Blot: Protein profile of CSA of L. donovani promastigotes showed a number of protein 178
bands of different molecular weight when separated on 12% SDS-PAGE gel (Fig 1A). Soluble 179
antigen trapped in PVDF membrane were reacted with pooled sera of each group. A number of 180
antigenic bands were recognized by the serum sample of different groups with different frequency 181
and intensity. 37 kDa (BHUP2) soluble antigen of L. donovani promastigote showed strong 182
reactivity with all serum samples (9/9) of VL patient’s sera (Fig. 2). Out of nine only three 183
(33.3%) serum samples of six month follow up hybridized with this protein. No reaction was 184
observed sera of endemic and non endemic healthy subjects. The identified protein was eluted 185
from SDS-PAGE gel through a specific gel elution method as shown in Fig 1B. 186
ELISA: ELISA with 37 kDa (BHUP2) protein was 94% (66/70; 95% CI: 86.2-97.7) sensitive 187
whereas the specificity in endemic healthy, non-endemic healthy and disease controls was 98% 188
(47/48; 95% CI: 92.6-100), 100% (60/60; 95% CI: 92.6-100) and 97% (41/42; 95% CI: 92.6-100), 189
respectively ( Fig 3). 190
2D gel electrophoresis and MALDI-TOF characterization 191
2D gel electrophoresis profile of 37 kDa fraction revealed a mixture of 3-4 proteins (Fig. 4A). On 192
performing western blotting of the resolved 2D gel and probing with the sera of confirmed VL 193
and NEHC separately, only two protein spots reacted with VL patients sera (Fig. 4B) whereas 194
none reacted with NEHC sera. We excised first of the two recognized protein spot from 2D gel for 195
MALDI-TOF characterization. The peak lists of the MALDI-TOF were used for peptide mass 196
fingerprint analysis with the MASCOT software of the Matrix Science together with NCBI 197
sequence data-base . The search of the protein sequence obtained from the mass spectrometry 198
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analysis recognized it as a Cytochrome-C like synthesis protein of 37 kDa . Attempts to 199
characterize the second protein failed due to low yield of protein. 200
Discussion: 201
Through a series of experiments we identified a novel 37 kDa L.donovani antigen (BHUP2) 202
having excellent sensitivity and specificity as confirmed by ELISA. This protein was recognized 203
as Cytochrome-C like synthesis protein on blasting peptide mass fingerprints (PMFs) with NCBI 204
protein sequence data base. The recognized 37 kDa (BHUP2) protein, revealed a great potential as 205
a serological marker for the diagnosis of Indian Visceral leishmaniasis. ELISA results with 206
BHUP2 protein was 94% (66/70) sensitive and 98.6% (148/150) specific. Thus less than 2% 207
healthy individuals from endemic area were positive with BHUP2 antigen as opposed up to 32% 208
positivity reported with rk39 antigen (12, 13). There are several other diseases like malaria, 209
typhoid fever, tuberculosis etc. whose signs and symptoms overlap with VL, also show positivity 210
with rK39(11) . Thus, there is an urgent need of a diagnostic assay which could specifically 211
diagnose active VL. This study was done to discover a specific immunoreactive marker for the 212
early diagnosis of visceral leishmaniasis, So in our experimental finding we chose this 37 kDa 213
immunoreactive fraction for evaluation in this disease which was compared with endemic healthy, 214
Non-endemic healthy control and different diseases to give a comparable specificity and 215
sensitivity data. 216
Our results suggest that BHUP2 protein can be used as a diagnostic marker for VL. Though the 217
limitation of BHUP2 antigen is its sensitivity being slightly lower than benchmark of 95%. 218
However, these experiments were done with antigen eluted from gel; a recombinant antigen 219
devoid of impurities might provide better results. Since the antibody response against the antigen 220
persists in significant proportion of patients, it does not have a prognostic value and can not be 221
used to detect relapses; however, high specificity is its highlight. 222
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To summarize, the result of this study showed that crude antigen of 37 kDa fraction gave 223
encouraging results with ELISA in terms of antibodies reactivity. Our protein shows high 224
specificity, which is one of the highest of all the antigen used in diagnosis of leishmaniasis. If a 225
result on recombinant form is similar or better, this will be another candidate to be used in the 226
diagnosis of VL. 227
228
Acknowledgements 229
The author would like to thanks all the staff of Kala Azar Medical Research Centre (KAMRC), a 230
unit of Sitaram Memorial Trust for their assistance in collection of samples used in the 231
experiments. 232
233
Financial support: The study was partially funded by National Institute of Allergy and Infectious 234
disease (NIAID), DMID funding mechanism: Tropical Medicine Research Center Grant number: 235
P50AI074321. SK and DK thanks to Council of Scientific and Industrial Research (CSIR), New 236
Delhi, India for providing fellowship. 237
238
Potential conflicts of interest: None declared 239
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Figure legends: 301
302
Fig. 1A: Protein profile of crude soluble antigen (6) of L. donovani promastigotes separated on 303
12% SDS-PAGE. Lane 1 = Protein marker, Lane 2 = Crude soluble antigen 304
305
Fig. 1B: Showing the eluted protein as a single protein band stained with sensitive silver stain. 306
Lane 1 = Protein marker, Lane 2 = Eluted protein 307
308
Fig. 2: Western blotting profile of crude soluble antigen of L. donovani promastigotes hybridized 309
with the serum of different groups. In each panel we have taken parasitologically confirmed VL 310
pre and post treated, six month follow up, Endemic healthy control and Non endemic healthy 311
control sera. Arrow indicates BHUP2 protein which was recognized by the sera of pre and post 312
treated VL patients but not with our control groups (EHC, NEHC). 313
314
Fig. 3: ELISA Reactivity of sera with 37 kDa (BHUP2) protein. The study group contains 315
visceral leishmaniasis (VL, n = 70), non endemic healthy control (NEHC, n = 60), endemic 316
healthy control (EHC, n = 48) and different disease (DD, n = 42). MALDI-TOF result showed 317
that the identified protein belongs to Cytochrome-C like synthesis protein. The Full lines indicate 318
the average ELISA Index (EI) for each assayed group. Dashed line represent cut-off value. Each 319
dot represent individual subject. 320
321
Fig. 4A: 2D gel electrophoresis of BHUP2 Eluted protein. Circle around the spot is our 322
eluted protein 323
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Fig 4B: Western blotting result of eluted protein separated on 2D gel, hybridized with 325
parasitologically confirmed VL patient’s sera. 2D gel electrophoresis of eluted protein revealed 3-326
4 protein spot and Out of these proteins spot only two of them were showed antibody reactivity 327
against eluted protein. 328
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