Background: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. Methods: Citrate-reduced GNPs (∼39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ∼46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ∼48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet–visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. Results: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ∼78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ∼5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory T_h1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ∼5.5 and ∼3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ∼2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). Conclusion: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

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.

Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P₁) and RRVAVLVLLDRL (P₂) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P₁P₂) is 97–100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P₁P₂) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient’s sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16–96.27)% and specificity 100%; Sp Cl95% (84.56–100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P₁P₂ antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P₁P₂ antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

Towards Identifying Immunogenic Targets in Visceral Leishmaniasis: Role of 17kDa and 63kDa Phosphoproteins