Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis

The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.


Introduction
Human Visceral Leishmaniasis (VL), a lethal form of leishmaniasis caused by Leishmania donovani (L. donovani) contributes significantly to the annual burden of infectious diseases in India [1]. Failure in management strategies has led to the rescheduling deadline of kala-azar eradication program from 2012 to 2015 and further upto 2020.Unsatisfactory diagnosis for certain forms of VL is one of the major hurdles in a VL elimination program. The conventional diagnostic test for VL is the microscopic demonstration of L. donovani amastigotes in aspirates from visceral organ [2]. Among the parasitological diagnosis, the sensitivity of microscopic examination of lymph node, spleen and bone-marrow aspirates varies from 52-58%, 93.1-98.7% and 52-85% respectively [3]. The overall sensitivity governs by these diagnostic methods appears highly unsatisfactory, if spleenic aspiration is not considered. Additionally, aspiration of lymphoid tissue is invasive, requires expertise and crowded with complications [3]. PCR is capable of diagnosing relapse or re-infection cases of VL. But, it is complex and not suitable for mass screening in the field [3]. Serological tests like ELISA, rapid immunochromatography, direct agglutination test (DAT) etc. were although less invasive, but its reliability is not assured due to cross-reactivity in other disease conditions, co-infection cases and relapse [4]. The sensitivity and specificity of ELISA are greatly influenced by the antigen used. Whereas, in various studies, DAT has been found to be 91-100 percent sensitive and 72-100 percent specific [5]. In earlier studies, rK-39 showed 100% sensitivity and 98% specificity respectively, but it has no prognostic values as well as it is not reliable for co-infection cases and relapse cases of VL. The previous reports have also evidenced that the sensitivity of rK-39 is as low as 71% in HIV patients co-infected with Leishmania [4]. Further, a report from Sudan has shown the 67% sensitivity of the rK-39 test in immuno-compromised patients [6]. The Ld-rKE-16 antigen by Sa pn diagnostic ltd. Is an another successful example of commercially available rapid antibody detection antigen based on membrane filtration technology [5].
All these tests cannot discriminate between active and past infection as the IgG persists in sera even after two years of cure [3]. Since antigen levels are expected to broadly correlated with the parasite load, it is comparatively more convincing as parasite antigen is eliminated quickly from body system as soon as the disease is cured [5,7]. Therefore, antigen detection systems may be an ideal alternative to antibody detection assay. It may also be ideal for diagnosis of immuno-compromised patients and more particularly with advanced cases of HIV coinfection, where the immune response is impaired [5,7]. However, the efforts are far beyond satisfactory in the development of antigen targeted diagnostic tool. In 2001, a new latex agglutination test (KATEX) developed with 73.5% sensitivity and 99% specificity in detecting Leishmanial antigen in urine samples of VL patients [8]. Moreover, in our recent study B-cell epitopes RFFVQGDGIGQHSLQEALERR (P 1 ) and RRVAVLVLLDRL (P 2 ) from a hypothetical protein [Acc No: XP_003861458.1] of L. donovani were used as potential antigen based diagnostic as well as prognostic candidate in VL detection [9].
This study was designed to identify Leishmania antigen in CICs for the diagnosis of different clinical stages of L. donovani infection like fresh VL cases (VL before anti-Leishmanial therapy), treated cases (patient received a full course of treatment and declared clinically cure), relapse (re-emergence of L. donovani in VL treated cases within six months after anti-leishmanial therapy) and re-infection (Re-infection of L. donovani in cured VL cases after six months from the end of anti-leishmanial therapy). The present data reveals that CICs in VL patients carries some Leishmania-specific antigen fragments, which may have good diagnostic as well as prognostic value.

Material and method 2.1. Sample selection
Approval and recommendations were obtained from the Institutional Ethics Committee (IEC) of the Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, India, for the written informed consent form (printed in the language of donor), sample size and procedures as per Helsinki declaration. Left over serum samples of patients (of both sex and age groups, 5 to 45 years) were collected during the diagnosis process at an institute OPD after getting written informed consent from the patient or from next to kin or guardians in case of minors in the presence of a witness signatory. Blood sample of healthy subjects was pooled directly for this purpose after getting the written informed consent. Total sample (n = 115) includes 50 active VL cases (VL-BT that represents fresh VL plus reinfection/ relapse cases), of which, patients were followed up to the end of amphotericin B treatment (15 injections of 1 mg/kg body weight applied with very slow infusion of 5% dextrose on alternate days) (VL-AT). Besides, blood from 25 other diseased cases (comprising 10 tuberculosis cases with positive sputum culture, 5 microfilariae positive cases of lymphatic filariasis, 5 clinically confirmed cases of asthma having chronic airway hyper responsiveness, 2 cases of dengue positive to ELISA, 2 cases of Japanese Encephalitis positive to ELISA and one case of influenza A positive to ELISA) and 40 healthy subjects (25 endemic and 15 non-endemic) were also collected. Samples were collected from sera bank and OPD of RMRIMS after getting informed consent. Each healthy subject had no apparent history of VL or other disease in recent past. These sera samples were used for evaluation of concentration of CICs. VL subjects were diagnosed parasitologically [42] at Rajendra Memorial Research Institute of Medical Sciences, Patna. 2 ml peripheral blood samples were collected from above 40 VL-BT, 10 VL-AT, 10 other diseases and 10 healthy subjects for isolation of antigen from CICs and study through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Six samples of VL-BT and VL-AT were studied through two-dimensional electrophoresis.

L. donovani culture and isolation of SLA
Soluble Leishmania antigen (SLA) was included in this study as a control. For this, a reference strain of L. donovani (MHOM/IN/83/AG83; repository number: RMRI/PB-0078) was used for preparation of SLA. The Promastigote culture was maintained as described elsewhere. Further, late log phage parasites were incubated at 30˚C and 37˚C for two hours in CO 2 incubator [43,44]. The cultures were centrifuged in 50 ml centrifuge tubes (Tarson, India) at 840 × g for 20 minutes at 4˚C in a cooling centrifuge (Hermle, Germany). The pellet was washed twice with PBS (Phosphate buffer saline) by centrifuging at 840 × g for 20 minutes at 4˚C followed by six freezing and thawing cycle. The lysate was centrifuged at 30,000 × g for 30 minutes and the supernatant was collected in aliquots and stored at -80˚C for further use.

Isolation of CICs
The Peripheral blood was collected in serum collection tube and allowed to coagulate in slanting position for 2 hours at room temperature (RT). After coagulation, the blood samples were centrifuged at 500 × g for 15 minutes. Sera supernatant (free from RBCs) was collected in vials. Serum was precipitated in buffer containing 5% Polyethylene glycol (PEG, Merck, India) and 0.1M Sodium borate (Sigma), pH 8.5 for overnight at 4˚C [34]. The precipitated CICs was isolated by centrifugation at 6000 × g for 45 minutes. The pellet was further washed for three times at 6000 × g in 2.5% PEG containing borate buffer (0.1 M) at 4˚C for three times, dissolved in Dulbecco's Phosphate Buffered Saline (Sigma), pH 7.2 and stored at -80˚C in aliquots for further use. The protein content of isolated CICs was estimated with protein estimation kit (Merck Biosciences) based on Lowry's method [45].

Acid dissociation of CICs and removal of antibody using staphylococcal protein A agarose
Acid dissociation of antigen and antibody present in CICs was performed using the glycin-HCl buffer at pH 2.5-2.8 (to obtain final pH 3) as described by Gupta and Tan [46]. Further, it was incubated with equilibrated protein A agarose (Sigma), at RT on shaker incubator for 45 minutes. The mixture was centrifuged at 500 × g for 10 minutes. The antigen free from antibody was obtained from the supernatant due to adsorption of antibody fraction on swollen protein A agarose. Supernatants were dialyzed using Micro Dispo Dialyzer TM apparatus, (Pall Life Sciences, India) against three changes of 300 ml PBS for 24 hours at 4˚C. Immunoglobulin bounded protein A agarose (present in pellet) was washed twice with PBS at 840 × g for 15 minutes. Washed pellet was incubated in 2 ml (or twice with the volume of pellet) of 3.5 M MgCl 2 for 15 minutes, at RT followed by centrifugation at 500 × g for 10 minutes. Supernatant containing antibody was dialyzed and washed as described above. Protein concentration was evaluated by Lowry method [45].

Immunoprecipitation, antibody isolation and HRP conjugation
The antibody of confirmed Kala-azar (VL-BT) subject was immunoprecipitated with Protein A agarose (Sigma) (Fig 1). Antigen was eluted from antigen-antibody-protein A agarose tricomplex in a column with the help of glycin-HCl buffer followed by washing with PBS. Antibody-protein A agarose complex was dissociated using 3.5M MgCl 2 solution and eluted from the column. Further, the antibody was passed through Nanosep 1 centrifugal device (Pall Corporation) for concentrating and desalting the sample. Finally, the concentrated and purified antibody was dissolved in PBS. This antibody was labelled with (horse reddish peroxidase) HRP by using HRP labeling Kit (Bangalore Genei, India).

SDS-PAGE and immunoblotting of CICs antigen resolved by SDS-PAGE
50μg CICs antigen of different individuals of different study groups was electrophoresed in discontinuous SDS-PAGE. For this, 10% resolving gels were prepared with 30% acrylamide and bis-acrylamide solution, 1.5 M Tris-HCl (pH-8.8), 10% SDS, 10% APS, and 5 μl TEMED. The electrophoresis were done in mini Protein III cell (BioRad, USA) using electrophoresis buffer (tris 0.025 M, glycin 0.19 M and SDS 0.1%) by following the protocol of Laemmli [47]. Further, the gels were stained with 0.25% Coomassie brilliant blue R250 and analyzed in comparison to standard Marker [Fermentas (broad range), USA]. The Image of the gel and nitrocellulose paper was captured by Gel Dock (Bio-Rad, USA). The molecular weight of polypeptides was calculated by using standard protein molecular weight marker on Quantity One software (Bio-Rad, USA). Western blotting was performed using HRP conjugated anti-leishmanial antibody (antibodies isolated from VL-BT patients, conjugated with HRP using HRP labeling kit, Bangalore Genei, India) following the protocol of Singh and his colleagues [43].

2D gel electrophoresis and mass spectrometry compatible silver staining
The two-dimensional gel electrophoresis was performed with Ready prep TM 2D starter kit (Bio-Rad). Antigen samples were purified by micro-bio-spin chromatography column (Bio-Rad) to remove salts. 40 μg of protein were run on 7 cm IPG strip (pH 3-10, 7 cm, Bio-rad) according to protocol mentioned in ready prep TM 2D starter kit in PROTEIN IEF cell in step wise fashion viz. step1: 250V, linear for 20 minutes; step 2: 4,000V, linear for 150 minutes; step 3: VH-10000, rapid; step 4: Hold Step-500V for 4 hrs. The IPG strip was subjected to SDS-PAGE (2 nd Dimension). 12% resolving gel was prepared and poured in the Bio-Rad gel cast. IPG strip was laid onto the polyacrylamide gel bed. It was electrophoresed in Tris-glycine SDS-PAGE running buffer (1X) at constant 200V for 40 minutes. Silver staining of the electrophoresed gel was performed using Plus One Silver Staining Kit (GE healthcare) following manufacturer's manual. MS compatible staining procedure for gel was adapted from Plus One Staining Kit manual (Amersham). By omitting the glutaraldehyde from the sensitizer and formaldehyde from the silver solution, the method becomes compatible for mass spectrometry analysis, however, at the expense of sensitivity (10 ng). Therefore, only the prominent spots were carefully excised from the gel.

De-staining and tryptic digestion of silver stained spots
Silver stained spots were excised from 2D gel and dried. To enrich protein samples, spots from three gels in triplicate were pooled. Excised spots were destained in solution containing 30 Blood serum was isolated from parasitologically confirmed VL patient sera. Anti-leishmanial antibodies present in VL-BT was immunoprecipitated with protein A agarose. MgCl 2 was used to dissociate VL specific antibody. Eluted antibody was tagged with and HRP using HRP labeling kit to prepare Kala-azar antigen detection antibody. mM potassium ferricyanide and 100 mM sodium thiosulfate solution (1:1), twice by incubating for 10 minutes each. Further, destained gel spots were washed twice for 1 hour each with water, followed by 10 minutes washing with 25 mM ammonium bicarbonate in acetonitrile (ACN) and water in the ratio of 1:1. The excised gel was vacuum dried [48] and subjected for digestion in 10 μl trypsin containing solution (12.5 mg/ml trypsin, 25 mM ammonium bicarbonate) and incubated overnight at 37˚C [47]. The digested polypeptide was extracted using 20 μL ACN containing 1% Trifluoroacetic acid (TFA). The mixture was incubated twice for 20 minutes and pooled extract was dried for further use.

HPLC-MS and identification of peptides
Prior to MS, different peptides present in polypeptide solution were fractionated with HPLC. Peptides were analyzed by electrospray ionization mass spectrometry using the Ultimate 3000 nano-HPLC system [Dionex] coupled to a 4000 QTRAP mass spectrometer [Applied Biosystems]. Tryptic peptides were loaded onto a C18 PepMap100, 3 μm [LC Packings] and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Further, spectra were analyzed to identify proteins of interest using the following parameters: database; Ludwig

Validatory evaluation of the presence of L. donovani antigen in CICs
To validate the presence of L. donovani antigen in circulation 50μg CICs, antigen of different individuals of different study groups was electrophoresed in discontinuous SDS-PAGE. Western blotting was performed with anti-Ld actin antibody (kind gift from Dr. Amogh A. Sahasrabuddhe, CDRI, Lucknow, India), which were used at 1:5000 dilution, whereas alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Santa Cruz) was used at 1:2000 dilution. The blots were visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Santa Cruz) according to the manufacturer's recommendations. Presence of tubulin was displayed with the help of anti-tubulin antibody following protocol of Sardar et al. 2013 [49].
To validate the MS derived peptide sequence, representing B cell epitopes of L. donovaniproteins XP_003861271.1 (RFFVQGDGIGQRSLQEALERRVAVLVLLDR) and XP_0038 61300.1 ARNELYDMLEIDPPAARAANAGESANE were synthesized commercially [9]. Spot ELISA were performed to validate the presence of antibody against these peptides in VL patients. 0.5 μg of SLA and synthetic peptide in PBS were loaded as a dot on ELISA plate coated with Nitro cellulose paper (NCP) for overnight, following protocol of Jamal et al.
(2016) [9]. As positive and negative control, SLA and PBS was used. Blocking was done with 5% BSA in PBS, for 2 hours followed by three washing with TBS-T. TBS-T with 0.2% BSA and 0.1% tween-20 was used as wash buffer. Experiment was performed in two different sets. In first set PBS, SLA and peptides were incubated with VL sera, in another set with healthy sera, for two hours followed by three washing with TBS-T. HRP tagged anti-human IgG was used as secondary antibody (Merck biosciences), incubated for 1 hour followed by three washing with TBS-T. DAB was used as substrate for detection.

Statistical analysis
Statistical analysis was performed using Graph Pad Prism 6. One way ANOVA was performed to evaluate the statistical significance of the obtained data on CICs concentration and densitometry of bands in SDS-PAGE and NCP.

Higher accumulation of CICs in human VL subjects during L. donovani infection
The concentrations of CICs in samples from VL subjects are depicted in Fig 2. The graph Pad Prism 6.0 data revealed 16 μg/μl as a cut-off concentration of CICs. Overall, a 35 fold higher concentration of CICs was observed in VL-BT subjects, which reduced significantly in samples from VL-AT (P = 0.0001). The reduced CICs concentration in VL-AT subjects was even eight folds higher than healthy subjects. Besides, we also identified an elevated CICs in samples from other disease in comparison to control but the overall increase was towards a much higher side in VL-BT subjects (P = 0.0001).

Up-regulated expression of CICs antigen in samples from VL subjects
The antigens isolated from CICs of different study groups were electrophoresed in 10% SDS-PAGE to evaluate the molecular weight of polypeptide fractions (Fig 3A.i, Table 1). Multiple bands of molecular weight 100kDa, 90kDa, 70 kDa, 65kDa, 63kDa, 62kDa, 55 kDa, 45 kDa, 42 kDa, 41kDa, 37 kDa, 31.6 kDa, 30kDa, 28 kDa, 23 kDa, 21kDa and 19 kDa were observed in SLA (Lane 7, Fig 3A.ii & 3C). The fourteen peptide fractions of CICs antigen was common between VL-BT and VL-AT subjects (182 kDa, 100kDA, 90kDa, 82kDa, 65 kDa, 62 kDa, 55 kDa, 42 kDa, 37 kDa, 31.6kDa, 30 kDa, 28kDa, 23kDa, 21 kDa). Except 182 kDa and 82 kDa bands, all bands identified in CICs were also present in SLA. One additional fragment of 36  For this, one ml of peripheral blood serum was taken from the different study groups. It was incubated with precipitation buffer (5% polyethylene glycol and 0.1 M sodium borate) for overnight. The precipitated CICs was washed with wash buffer containing 2.5% PEG 6000 and re-dissolved in 100 μl PBS. Protein content (in μg/μl) was evaluated using Lowry method. 16 μg/μl was evaluated as cut-off concentration analyzed using graph pad prism 6.0. kDa was observed in sample of tuberculosis patient (Fig 3C). Similarly, there was an additional band of 92 kDa in CICs antigen of Asthma. In addition to the above, there were some sporadic bands in some of the samples; all bands explained above were reproducible within the study group. Notably, a dominant expression of 55 kDa and 23 kDa in CICs antigen of VL-BT was not observed in VL-AT (Fig 3A and 3C). Densitometry analysis (Quantity One software, Bio-Rad) revealed differences in relative quantity of bands ( Fig 3E). Relative quantities of 55 kDa band were 27.4, 5.5 and 10.5 in VL-BT, VL-AT and SLA respectively. Likewise, relative quantities of 23 kDa band were 19.3, 9.6 and 7.9 in VL-BT, VL-AT and SLA respectively. However,

Many of the antigenic fractions isolated from CICs were immunoreactive in Western immunoblotting
Out of the total antigenic fragments, obtained of SLA in SDS-PAGE (90 kDa, 70 kDa, 63 kDa, 62 kDa, 55 kDa, 45 kDa, 41 kDa, 31.6 kDa, 30kDa, and 23 kDa) Eight polypeptides showed immunoreactivity to anti-L. donovani primary antibody (Fig 3B). Amongst them, three polypeptides (55 kDa, 37kDa and 23 kDa) were immunodominant. Especially, the 55 kDa and 23 kDa polypeptide displayed reactivity in all VL-BT samples (Fig 3B). The specificity in them was confirmed through a negative reaction observed in the sample of asthma, tuberculosis, filarial, viral flu etc. (Fig 3D, lane 6-9). Comparatively, a less immunoreactive band of 62 kDa was also observed, which showed its immunoreactivity to anti-L. donovani primary antibody in samples of another disease. A sporadic band of 37 kDa was also observed which was marked as reactive in few samples of VL-BT and healthy subjects, but its sensitivity was less. Except in two samples, 23 kDa band was not recognizable but 55 kDa band was observed with 4.9 fold less band intensity in VL-AT samples.
The Relative quantity of 55 kDa and 23 kDa bands in Western blotting in the given figure were 36.9 and 12.7 in VL-BT subjects in comparison to SLA (Fig 3E), which was 5.4 and 11 respectively. Thus, the immunoreactivity established L. donovani as a source of these proteins. The Relative density of 55 kDa band in VL-AT sample was 9.2 (down-regulated). Therefore, we identified a strong discriminative ability of both the polypeptide between active and treated cases of VL.

Antigenic peptides of Leishmanial proteins were identified in 2D spots by LC-ESI-MS/MS
2DE spots of 93.3 kDa, 87 kDa, 63 kDa, 55 kDa, 45 kDa, 37 kDa, 24.5 kDa all at pI 8.5, 25.7 kDa (pI 7.7) kDa and 23 kDa (pI 6.2 and 6.8) (spot A-J), which were prominent immune-reactive, were further selected for LC-MS for in-depth characterization. In total, 46 well-resolved spots of varying intensity were detected in the silver-stained gel (Fig 4A). Among them 13 spots were immunoreactive. Ten spots were exclusively immunoreactive in VL-BT. 39 proteins have been identified from VL-BT antigens, which are very noteworthy proteins. The mass spectra of peptides obtained from each spot provided sufficient signals to search Databases  Table 2. Significant protein hits were obtained in each spot. Proteins identified during LC-ESI-MS/MS were associated either with characterized proteins or hypothetical conserve protein or hypothetical proteins of unknown function (S1 Dataset). The Peptides of cytosolic dynein heavy chain were predominant in the spot I and spot J. On the basis of their biological function, identified proteins were classified into 11 groups (Fig 5). Fifty percent of identified protein was the hypothetical protein of unknown function. Most of the known protein was concerned with the physio-pathological function of L. donovani. Motor protein was the second largest group (18.42%) of the identified protein, whereas protein involved in phosphorylation and synthesis (5.26%) was the third largest group. Protein involved in cytoskeleton organization, carbohydrate metabolism, transcription, DNA replication, cell adhesion, signal transduction and nuclear assembly were remaining functional category of identified protein reported in this work.

Validation of the presence of L. donovani antigen in CICs and MS derived peptides
The Presence of anti-Ld actin antibody reactive against 42 kDa band was identified in VL-BT (Fig 6A). Similar band with five-fold reduction was also revealed in VL-AT subjects. However, it was absent in healthy sample. Interestingly, presence of alpha-tubulin (49 kDa) was found in 55 kDa region of CICs antigen (Fig 6B), which was in agreement with earlier report [50]. MS data was validated with spot ELISA (Fig 6C). It confirmed the reactivity of synthetic peptides with VL sera. SLA validates its L. donovani origin. Reactivity of healthy sera was negative in both cases.

Discussion
Formation of immune complexes occurs during the various scavenging process as a phenomenon of humoral immunity to remove antigen or pathogen [51]. Higher expression of the immune complex is associated with L. donovani induced inversion of the normal albumin to globulin ratio [52] and hypergamma-globulinemia in human VL subject [6,17,53,54]. The Gamma-globulins trap released Leishmania amastigotes or its antigen and forms immune complex during various complexes forming action of humoral immune response. Usually, immune complexes are removed by scavenging macrophages. But, excessive production and defective clearance of these immune complexes lead to accumulation in circulation [55]. Previously, Evan and Pearson [17] had observed 140 fold higher accumulations of CICs in American VL subject in comparison to healthy subjects. However, it was observed 36 higher fold in the present study. This may be due to a difference in Leishmania strain of American and Indian VL as well as due to a difference in dynamics of immunity in patients. Expectedly, the higher concentration of CICs in VL subject due to hypergamma-globulinemia as reported  a.(A) and (B). The sample of VL-BT subject showed 100 kDa, 93.3 kDa, 87 kDa, 67.6 kDa, 63 kDa, 55 kDa, 45.7 kDa, 41.6 kDa, 37 kDa all having pI 8.5 as well as 24.5 kDa (pI 8.5), 23 kDa (pI 6.8, 6.2) immunoreactive spots. Besides, 55 kDa (pI7.7) 26.3 kDa (pI 8) and 25.7 kDa (pI 7.7) dots were upregulated in the immunoblot of the sample from VL-BT subjects in comparison to the sample from healthy subjects. (d). Figure showing comparative densitometry analysis of immunoreactive spots. Densitometry data of immunoreactive 2D spots of VL-BT and healthy subject after western immune-blotting with anti-Leishmania antibody isolated from patients are given in term of volume (INT*mm2), and area (mm 2 ). Immuno-reactive 2D spots present in both VL-BT and healthy are indicated in " 1, " 2 indicates 2D spots only present in the healthy subject and 2D spots only present in VL-BT subject indicated by " 3. https://doi.org/10.1371/journal.pone.0182474.g004 Targeting antigen as diagnostic marker in visceral leishmaniasis   Targeting antigen as diagnostic marker in visceral leishmaniasis [56]. However, infectious diseases like tuberculosis, malaria, filarial, leprosy, influenza are known for comparatively lower expression of CICs [24,31,57,58]. Asthma was also observed with higher expression of CICs. Anti-leishmanial therapy particularly Amphotericin B treatments improves the ratio of albuminuria to hypergammaglobulinemia, which is being used as a nonspecific biochemical parameter of prognosis in VL [59]. Anti-leishmanial therapy controls the parasite load in the patient, as well as the release of antigen in circulation. In the present study, lower expression of CICs in treated subjects is associated with lowering of parasite load as well as lowering of hypergamma-globulinemia. Though, there was statistically significant difference in concentration of CICs in study groups; this cannot be recommended for diagnosis as it is not specific only to leishmaniasis. The thrust area to explore in this study was the identification of Leishmania antigen present in CICs. But the presence of higher concentration of immunoglobulins in CICs may induce shielding effect during epitope detection. Therefore, the effort was needed to minimize the concentration of immunoglobulins in samples of CICs. Previously, Gupta and Tan [46] and Hoffken et al [60] had successfully segregated antigens and antibodies from CICs. Methodologies applied in those studies were adopted here to minimize the concentration of immunoglobulin fragments in CICs samples. Through this, we could minimize the concentration of antibody fragments from the CICs samples.
The start-up phase of the study revealed 55 kDa and 23 kDa antigen, which showed an overexpression with consistency and immunodominance in VL-BT subjects and also showed a discriminating ability to differentiate active from a treated VL cases. Comparatively, 23kDa antigen appeared more promising diagnostic marker in comparison to 55 kDa band, which was recognizable in samples of few treated VL cases. In immunoblotting studies, the 55 kDa and 23 kDa antigen fractions were not only immunoreactive against anti-Leishmania polyclonal sera in samples from VL-BT; it was also present and reactive in SLA. Previously, Sanyal et al. [50] had also reported the presence of 55 kDa fraction in their study but not 23 kDa. Additionally, they had also confirmed the presence of the fragments of gp63, tubulin-like protein and IgG heavy chain in 55 kDa region through western blotting [50,61]. 55kDa and 23kDa fractions was absolute in VL-BT subjects. Moreover, immuno-reactivity revealed low relative intensity band in VL-AT, which was significant in some samples from treated VL subjects. Therefore, it was impossible to ascertain the prognostic value of these bands unless considering band intensity. Since one of the aims of this study was to identify an antigen of prognostic value, further segregation of antigen was needed through 2D for unambiguous characterization of circulating antigens. Additionally, above referenced studies are evident that the 55 kDa bands contain multiple peptides; it was essential to go for 2D segregation of CICs antigen samples. Since some other immunodominant 2D spots were identified in the whole antigen in addition to 2D spots segregated from 55 kDa and 23kDa; all the immunodominant spots present in whole antigens were included for further analysis. Some of the 2D spots recognized in the immunoblotting were those previously reported [50,61,62]. The analysis revealed of twelve immunodominant 2D spots, which were absent in healthy samples and treated samples, hence be of diagnostic value.
Though our main goal was to decode Leishmania antigen in 2D spots, we encrypted antigenic peptide through MS. LC-ESI-MS/MS data of the spots evaluated in the present study after analysis through search database (NCBI, UniProt, Ludwig NR database) revealed more than one protein in a single spot, for example, cytosolic dynein heavy chain, kinesin, 60 S ribosomal proteins in spot number B. There were also presences of many hypothetical conserved proteins in different spots. These proteins may be used in the diagnosis and other purposes. Peptide fractions reported from different spots were identified as hypothetical/uncharacterized Leishmania proteins along with putative kinesin of L. donovani, major surface protease gp63, putative tubulin, DNA polymerase epsilon catalytic subunit, GTP-binding protein, and tyrosyl-methionyl t-RNA synthetase-like protein. Obtained data on gp63 confirmed the reports of Chakraborty et al. [62] in which, they had reported fractioned gp63 at dislocated site using monoclonal antibody. Besides, tubulin was also reported earlier with the help of monoclonal antibodies [50,61,62]. In this study, we also recognized it through LC-ESI-MS/MS data analysis. Protein like t-RNA synthetase, ribosomal protein, DNA-directed RNA polymerase subunit, and protein kinase was also reported by Kumar et al. [63]. Some of the peptides reported in this study are associated with GTP-binding protein and increase in expression of 60S ribosomal subunit as well as nucleosome assembly protein. These are important parasite antigen being associated with the possible loss of infectivity of L. amzonensis [64]. Madalhaes et al, 2014 had reported the decrease in expression level of these proteins in SLA [65]. Whereas, the expression of all the three proteins was increased. The presence of highly conserved ribosomal protein, proteins of the DNA replication and transcription identified in the samples of VL-BT is confirmed by the earlier finding of Requena et al. [66].
In fact, uncharacterized hypothetical peptides of unknown function were observed in high concentration. Kumar et al. [63] had reported the presence of 22% hypothetical proteins in SLA from L. donovani. Likewise in the present study, 50% hypothetical protein is being reported in CICs of VL-BT subjects. These proteins could be of prime concern understanding functional aspect, their role in pathogenicity, drug resistance, disease control and intracellular survival. Future, these novel proteins can be proved crucial not only for diagnosis but also for potential drug target, vaccine development, and therapeutics. Most of the reported proteins of known function are associated with physio-pathology. Other identified peptides were from motor proteins, proteins involved in phosphorylation and synthesis, cytoskeleton organization, carbohydrate metabolism, transcription, DNA replication, cell adhesion, signal transduction and nuclear assembly.
This study is reporting the presence of L. donovani specific antigens in CICs of VL-BT subjects. Previously, several diagnostic kits viz. rapid Dengue NS-1 antigen test [30,67,68], P24 HIV test [69], Carsinoembryonic antigen test (CEA test) [70], HRP2 antigen test [71,72] etc. have been successfully developed targeting immune complex antigens. Due to highly perishable nature of protein antigen and due to scavenging and salvage process, antigens disappear or decrease in the subject receiving anti-leishmanial therapy. The residual amount of antigen also disappears quickly in comparison to antibodies from treated subjects. The presence of the Leishmania antigen may be detected in peripheral blood using antibodies which will be reverse to the existing RDT.
In this respect, we further validated the MS derived peptide for its reactivity with patience in section 3.6. These synthetic peptides revealed reactivity with VL sera, but not with healthy sera. However, the presence of L. donovani antigens in CICs (Fig 6A & 6B) further strengthen the L. donovani origin of circulating antigen. Earlier, presence of anti-actin and anti-tubulin antibody was also reported [50,73,74]. Hypothetical protein (XP_003861271.1, spot C) successfully shown the efficiency of targeted MS derived peptide through Immuno chromatographic test (ICT) [9]. Above protein is under process of translational research for application. In another attempt, peptide from known protein of L. donovani i.e. tubulin folding cofactor D from spot E (AccNo.XP_003861300.1, spot D, under review) was characterized employing antigen capture ELISA. Therefore, the peptides reported in the present study can be used for development of diagnostic as well prognostic kit.  Table. Table showing molecular weight (kDa) and isoelectric point (pI) of silver-stained 2D spots in VL-BT and healthy subjects. Calculation was done using Quantity One software. (DOCX) S2 Table. Table showing relative intensity (mm 2 ) of silver-stained 2D protein spots in VL-BT and healthy subjects. Calculation was done using Quantity One software. (DOCX) S3 Table. Table showing