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. 2026 May 5;16(1):20–28. doi: 10.4103/tp.tp_10_25

Recombinant kinesin protein of Leishmania donovani and Bacille Calmette–Guerin as an immunomodulatory agent: Insights into their role against visceral leishmaniasis

Saumya Srivastava 1, Anil Kumar Gupta 1, Amit Singh 1,2, Sarman Singh 1,3,✉
PMCID: PMC13200747  PMID: 42199681

Abstract

Introduction:

Visceral Leishmaniasis (VL), caused by Leishmania donovani, is a fatal disease, necessitating an effective vaccine. This study aims to develop a vaccine by evaluating the combination of recombinant kinesin protein (rKIN) with Bacille Calmette–Guerin (BCG) as an adjuvant against experimental VL.

Materials and Methods:

The immune response was analyzed against the purified rKIN of L. donovani with and without BCG adjuvant by using BALB/C mice. Mice were divided into 6 groups comprising of 6 animals in each group for the vaccine intramuscularly. All 6 Groups were immunized either with BCG, rKIN, or combination of the two, with different doses. Saline was used as negative control. Each group was further divided into 2 subgroups. One subgroup of animals from each group was challenged with L. donovani promastigotes 1 × 106 cells/100 μl per animal. The extent of protection was evaluated by estimating the reduction in the number of parasites in the spleen, quantity of nitric oxide (NO), reactive oxygen species (ROS) in the peritoneal cells, and production of cytokines in blood serum.

Results:

Significant parasite reduction (70%–90%) was observed in the spleens of groups receiving rKIN (50µg or 100µg) with BCG. NO and ROS production increased by 60%–95% and 70%–90%, respectively. The 100µg rKIN with BCG group demonstrated substantial protection (P < 0.001) with upregulated interferon-gamma (IFN-γ), tumor necrosis factor, interleukin-2 (IL-2), and downregulated IL-4, IL-10, and IL-17. Statistical analysis confirmed significant differences (P < 0.001) between vaccinated and control groups.

Conclusions:

The combination of 100µg rKIN with BCG shows potential as a vaccine candidate against VL.

Keywords: Bacille Calmette–Guerin, immunity, recombinant kinesin protein, Th1/Th2 response, vaccine, visceral leishmaniasis

INTRODUCTION

Visceral leishmaniasis (VL), caused by Leishmania donovani, threatens over 350 million people in endemic regions.[1] Despite elimination efforts, India reported 777 new cases in 2022.[2] Current treatments are costly, have severe side effects, require prolonged use, and face drug resistance.[3] Vaccine development is critical, yet no effective vaccine exists.[4,5,6,7,8,9,10] The kinesin protein of L. donovani has shown potential as a vaccine candidate due to its ability to induce a strong Th1 immune response.[11,12] This study examines the use of recombinant kinesin protein (rKIN) with Bacille Calmette–Guerin (BCG) as an adjuvant for experimental VL.

MATERIALS AND METHODS

Ethics statement

This study was approved by the Institutional Animal Ethics Committee (IAEC), reference no. 596/IAEC/11, and experiments were conducted using 4–6-week-old male BALB/c mice from the Central Animal Facility.

Study design

Expression and purification of recombinant kinesin protein

The rKIN had been cloned in the pRSETC vector and characterized earlier at our laboratory, AIIMS, New Delhi, India.[12] The plasmid was isolated using Qia miniprep (Qiagen, Germany) and transformed in Escherichia coli BL21 (DE3) competent cells, grown overnight at 37°C in Luria Bertani (LB) agar containing ampicillin (100 mg/L). Following this, 10 colonies of E. coli BL21 were picked randomly and grown overnight in 5ml of LB with 100µg/ml ampicillin at 37°C with constant shaking at 225 rpm. Five milliliters (1% inoculum) of this overnight culture was used to inoculate in 500 ml of LB medium containing 100 µg/ml ampicillin and incubated on a shaker incubator at 37°C. When O.D600 reached ~ 0.6, protein expression was induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation at 10,000 rpm for 10 min at 4°C. The bacterial pellets were resuspended in the cell lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole [pH 8.0], containing 1 mM phenylmethylsulfonyl fluoride) and 10µl/ml protease inhibitor cocktail and were disrupted by sonication followed by centrifugation at 10,000 rpm for 30 min at 4°C. 6×His-tagged rKIN was purified under native conditions by immobilized metal affinity chromatography using Ni-NTA (Ni²+–nitrilotriacetic acid) resin, as per the manufacturer’s instructions (Qiagen, Germany).[13] Further, the purified rKIN was subjected to a protein purification system (AKTA PRIME PLUS, GE) and collected the target protein fraction and then concentrated using a protein concentrator (Amicon® Ultra Centrifugal Filter, 10 kDa Cat no. UFC9010). These purified proteins were used for animal study.

Preparation of soluble Leishmania antigen

The soluble Leishmania antigen (SLA) was prepared from washed L. donovani (Dd8 strain) promastigotes (109/ml) through 8–10 freeze-thaw cycles between −196°C (liquid nitrogen) and 37°C, followed by 5 min on ice.[14] The promastigotes were ultrasonicated, centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant containing SLA was collected.[15] Protein concentration was determined using the Bradford assay, and the antigen was aliquoted and stored at −80°C. Recent ex vivo studies show SLA effectively stimulates nitric oxide (NO) and reactive oxygen species (ROS) production,[16] with optimal levels achieved at 5 μg/ml SLA. Peritoneal cells were stimulated with SLA,[14] and NO and ROS levels were measured.

Immunization of animals (mice)

For the immunization study, a total of 36 male BALB/c mice (4–6 weeks old) were used. These animals were randomly divided into six groups (six mice in each group). Group 1 was immunized with saline (control), Group 2 with only BCG, Group 3 with rKIN 50µg/0.1ml, Group 4 with rKIN 100µg/0.1ml, Group 5 with rKIN 50µg/0.1ml with BCG, and Group 6 with rKIN 100 µg/0.1 ml with BCG. 2 × 103 unit/0.1 mL BCG was used for each injection [Figure 1]. The vaccine construct was diluted in phosphate buffer saline (PBS) and injected via intramuscular (I.M) route in the mid muscle of the left thigh of BALB/c mice.[17,18] All mice were immunized with total of three doses, each administered at 2-week intervals for cell proliferation and cytokine production, and 14 days after the last immunization, animals (n = 3 per subgroup) were euthanized, and blood samples were collected for serum preparation and cytokine assay. Peritoneal cells were also aspirated from these mice and were used for the detection of NO and ROS. The remaining mice from each group (n = 3 per subgroup) were challenged with 1 × 106/0.1 ml of stationary phase promastigotes of the L. donovani Dd8 strain. Promastigotes were injected via the tail vein with 100 µl of PBS/mouse. As demonstrated in previous studies, the rate of L. donovani infection in BALB/c mice is maximal at ~4–5 weeks.[19] Four weeks after the infection, all mice were euthanized, and their serum samples were used for the detection of cytokine levels. The peritoneal cells were aspirated for NO and ROS detection, spleen touch smear was microscopically examined to quantitate the level of infection. All slides were fixed with methanol and stained with Giemsa stain.

Figure 1.

Figure 1

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Experimental groups of the study

Quantification of nitric oxide

NO production was assessed by measuring nitrite accumulation in the culture medium of peritoneal cells from mice. For this, 1 × 105 cells/well from various experimental groups were incubated with 5 μg/ml SLA in RPMI-1640 medium with 10% fetal bovine serum (FBS) in a 96-well plate at 37°C in a CO2 incubator for 48 h. The culture supernatant was then collected, and nitrite content was analyzed using Griess reagent (Cat no. MAK367, Nitrite Assay Kit, Griess Reagent, Sigma). The reagent was added to the supernatant (1:1 ratio) and incubated at 37°C for 20 min in the dark. Absorbance was measured at 540 nm,[20] and nitrite concentration was determined using a sodium nitrite standard curve.

Measurement of reactive oxygen species

ROS are by-products of enzymatic reactions and oxygen reduction during aerobic respiration, playing crucial roles in cell signaling and host defense. ROS levels are typically proportional to cellular hydrogen peroxide concentration, measurable at 530 nm. In our experiment, mice peritoneal cells (1 × 106 cells/ml) were cultured in 24-well plates with RPMI-1640 medium and 10% FBS, incubated for 24 h at 37°C with 5% CO2 in the presence of 5 μg/ml SLA. After incubation, 25 μM dichloro-dihydro-fluorescein-diacetate (H2DCFDA, Thermo Fisher Cat no. D399) was added, followed by a 30-min incubation. Cells were washed with PBS, and ROS levels were measured via flow cytometry, expressed as fluorescence intensity units.[21]

Th1, Th2, and Th17 cytokine assay

Serum levels of interleukin-4 (IL-4), IL-10, IL-2, IL-6, IL-17A, tumor necrosis factor (TNF), and interferon-gamma (IFN-γ) in all mouse groups, both before and after L. donovani challenge, were measured using flow cytometry (LSR, BD Biosciences) with the BD cytometric bead array. The Mouse Th1/Th2/Th17 Cytokine Kit (Cat No. 560485) was used following the manufacturer’s instructions.

Determination of parasitic load in the spleen after the challenge

To evaluate protection, 3 mice per group were challenged with 1 × 106 stationary-phase L. donovani Dd8 promastigotes via intravenous injection (100 µl PBS/mouse) 2 weeks after the final booster dose. Four weeks postinfection, mice were euthanized, and spleen touch smears were prepared, fixed with methanol, and stained with Giemsa for microscopic examination. The Leishman Donovan units (LDU) were calculated as the number of amastigotes per 100 tissue nuclei to quantify infection levels.[22]

Statistical analysis

Two sets of experimental groups were formed, one immunized but uninfected and the other as immunized and infected. Each group had 6 subgroups (with different vaccine formulations and saline), each having 3 animals per subgroup. Data were expressed as mean ± standard deviation (SD). A two-way ANOVA and one-way ANOVA tests were performed using GraphPad Prism version 8 for Windows.

RESULTS

Recombinant kinesin protein expression and purification

Induction of rKIN expression in the pRSETc vector with 1 mM IPTG for 4 h resulted in high-level expression, yielding a molecular mass of approximately 35kDa [Figure 2a]. Ni²⁺-NTA agarose purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed an expression level of 0.4 mg of purified protein per liter of culture, with >90% purity [Figure 2a]. The presence of kinesin-specific antibodies in VL-positive serum was confirmed by western blot, indicated by a brown spot on the nitrocellulose membrane when reacted with LD-positive patient serum [Figure 2b].

Figure 2.

Figure 2

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot of purified recombinant kinesin protein (rKIN): (a) rKIN stained with Coomassie Brilliant Blue R-250, (b) Western blot of purified rKIN with kala Azar positive patient serum. M: Protein marker (PG-PMT0782), WCL: Whole cell lysate, FT: Flow through, P: Purified protein

Quantification of nitric oxide

NO production in peritoneal cells differed significantly between control (saline) and immunized groups in both immunized and immunized + infected mice. All vaccinated groups showed increased NO production compared to the control. The rKIN 50 µg + BCG group produced significantly higher nitrite levels (44.19 ± 1.99 µM) than the saline group (5.48 ± 0.577 µM), and the rKIN 100 µg + BCG group showed even higher levels (57.14 ± 2.51 µM). After 4 weeks of infection, these levels increased further to 65.54 ± 4.03 µM and 106.53 ± 1.52 µM, respectively, significantly higher than the saline group (7.215 ± 0.003 µM). Groups immunized with BCG alone, rKIN 50 µg, and rKIN 100 µg also showed elevated nitrite levels, but to a lesser extent [Figure 3].

Figure 3.

Figure 3

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Quantification of nitric oxide (NO) (mM): Peritoneal cells of mice were stimulated with the 5 μg/ml soluble Leishmania antigen. The estimation of NO production was done using Griess reagent in supernatants collected from peritoneal cell culture, 48 h after incubation, and the absorbance of the reaction product was measured at 540 nm. Data represent mean ± standard deviation of three animals per group. The significance between different experimental groups was calculated by a two-way ANOVA test using graph pad Prism (version 8.0.1), (a) Significance values indicate the difference between the vaccinated and control (saline) mice (*P = 0.2, ***P < 0.002, ****P = 0.0001), (b) Significance values indicate the difference in all vaccinated group including control, before and after infection (ns = non-significant, **P < 0.009, ****P < 0.0001)

Measurement of reactive oxygen species

Similar to NO, ROS production in peritoneal cells showed significant differences between control (saline) and immunized groups in both immunized and immunized + infected mice. All vaccinated groups exhibited higher ROS levels compared to the saline group. Mice immunized with rKIN 50 µg + BCG produced significant (**P < 0.001) ROS levels (721.33 ± 4.04), and rKIN 100 µg + BCG induced even higher ROS (883.66 ± 0.57), representing >6- and >8-fold increases over the saline group (107.33 ± 8.50). Postinfection, ROS levels rose further to 1107 ± 7.2 in the rKIN 50 µg + BCG group and 1593.45 ± 1.27 in the rKIN 100 µg + BCG group, >5- and >8-fold higher than saline (187 ± 5). Groups immunized with BCG alone, rKIN 50 µg, and rKIN 100 µg also showed increased ROS, with postinfection levels reaching 277 ± 7.9, 469.66 ± 9.01, and 775 ± 5, respectively (***P < 0.001) [Figure 4a and b].

Figure 4.

Figure 4

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Measurement of reactive oxygen species (ROS) (a.u.). Peritoneal cells of mice were stimulated with the 5 μg/ml soluble Leishmania antigen. The estimation of ROS production was measured by flow cytometry. Data represent mean ± standard deviation of three animals per group. The significance between different experimental groups was calculated by a two-way ANOVA test using graph pad Prism (version 8.0.1), (a) Significance values indicate the difference between the vaccinated and control (saline) mice (***P < 0.001), (b) Significance values indicate the difference in all vaccinated group including control, before and after infection (***P < 0.001)

Th1 and Th2 cytokine assays

In both experimental sets (immunized but uninfected and immunized + infected), we compared the Th1, Th2, and Th17 immune responses induced by different vaccine formulations of rKIN and BCG with the control (saline).

Interferon-gamma and interleukin-2 expression

The two-way ANOVA showed that the mean square of IFN-γ and IL-2 between all vaccinated groups was highly significant compared to other Th1 and Th2 cytokines (***P < 0.001). In the immunized but uninfected mice, rKIN 50 µg + BCG produced 475.1 ± 3.95 pg/mL IFN-γ, and rKIN 100 µg + BCG produced 675.1 ± 3.95 pg/mL IFN-γ, both >4- and ~6-fold higher than the saline group (115.58 ± 4.27 pg/mL) (***P < 0.001). Similarly, rKIN 50 µg + BCG produced 223 ± 1 pg/mL IL-2, and rKIN 100 µg + BCG produced 451.5 ± 2.29 pg/mL IL-2, both >6- and >10-fold higher than the saline group (33.66 ± 1.52 pg/mL) (***P < 0.001). After 4 weeks of infection, rKIN 50 µg + BCG produced 822.33 ± 1.46 pg/mL IFN-γ, and rKIN 100 µg + BCG produced 1295.66 ± 21.95 pg/mL IFN-γ, both >10-fold higher than the saline group (77.33 ± 2.0 pg/mL). Similarly, rKIN 50 µg + BCG produced 499.33 ± 1.52 pg/mL IL-2, and rKIN 100 µg + BCG produced 800.33 ± 3.21 pg/mL IL-2, both >15- and >20-fold higher than the saline group (28.66 ± 0.57 pg/mL) (***P < 0.001). The BCG alone, rKIN 50 µg, and rKIN 100 µg groups also showed increased production of IFN-γ (201.33 ± 0.5 pg/mL, 252.33 ± 2.51 pg/mL, and 381.33 ± 7.09 pg/mL) and IL-2 (56.66 ± 2.08 pg/mL, 76.33 ± 1.52 pg/mL, and 121.66 ± 1.52 pg/mL) in the uninfected mice group (***P < 0.001). After infection, these groups showed increased IFN-γ (405.33 ± 4.72 pg/mL, 460.66 ± 1.52 pg/mL, and 557.33 ± 2.08 pg/mL) and IL-2 (156 ± 1 pg/mL, 184 ± 4.5 pg/mL, and 243.33 ± 1.52 pg/mL) [Figures 5a, b and 6a, b].

Figure 5.

Figure 5

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Production of cytokine; interferon-gamma (IFN-γ), interleukin-2 (IL-2), IL-4, IL-10, tumor necrosis factor (TNF), and IL-17, in blood serum of all Immunized v/s control (saline) mice. The results were expressed as picograms of cytokine/ml, based on the standard curves of the respective cytokine provided in the kit. Data represent the mean ± standard deviation of three animals per group. The significance between different experimental groups was calculated by two-way ANOVA test using graph pad Prism (version 8.0.1) Significance values indicate the difference between the control (saline) mice and vaccinated for IFN- γ (a), IL-2 (b) and TNF (e) ***P < 0.001 (significant), for IL-4 (c) and IL-10 (d) ***P < 0.001, **P < 0.01 (significant) between control versus all vaccinated except Bacille Calmette–Guerin (BCG) in (c) which was nonsignificant. Difference between the control and vaccinated mice for IL-17 (f) ***P < 0.001, *P < 0.02 (significant) except BCG and K 50 µg

Figure 6.

Figure 6

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Production of cytokine; interferon-gamma (IFN- γ), interleukin-2 (IL-2), IL-4, IL-10, tumor necrosis factor (TNF), and IL-17, in blood serum of all immunized/infected and immunized/uninfected mice. The results were expressed as picograms of cytokine/ml, based on the standard curves of the respective cytokine provided in the kit. Data represent the mean ± standard deviation of three animals per group. The significance between different experimental groups was calculated by two-way ANOVA test using graph pad Prism (version 8.0.1) Significance values indicate the difference between the infected and uninfected group of all vaccinated animals: IFN- γ (a), IL-2 (b), IL-4 (c) IL-10 (d) and TNF (e) *P < 0.1, **P < 0.01 and ***P < 0.001 (significant). IL-17 (f) showed ***P < 0.001 in all groups except control, which was nonsignificant

Interleukin-4 and interleukin-10 expressions

Highest production of IL4 (48 ± 1 pg/mL) and IL10 (28.33 ± 0.5 pg/ml) was found in the saline (control) group in the immunized-uninfected mice group, whereas after 4 weeks of infection, level was increased significantly (***P < 0.001) to 64.33 ± 4.04 pg/mL and 48 ± 1 pg/ml for IL4 and IL10 respectively. IL10 was decreased significantly (***P < 0.001) in all the vaccinated groups; however, IL-4 was decreased significantly (***P < 0.001) in BCG and rKIN 50 µg. Whereas rKIN 100 µg and rKIN 100 µg + BCG showed decreased production of IL4 (**P = 0.003) [Figures 5c, d and 6c, d].

Tumor necrosis factor expression

TNF levels increased significantly over time (***P < 0.001). In the immunized but uninfected mice, rKIN 50 µg + BCG produced 201.33 ± 2.51 pg/mL TNF, and rKIN 100 µg + BCG produced 307.33 ± 1.52 pg/mL TNF, both >9- and >10-fold higher than the saline group (21.66 ± 1.52 pg/mL). After 4 weeks of infection, TNF levels in the immunized mice were also elevated, with rKIN 50 µg + BCG at 307 ± 1.52 pg/mL and rKIN 100 µg + BCG at 660.33 ± 5.50 pg/mL, both >10-fold higher than the saline group (9.66 ± 1.52 pg/mL). In addition, the BCG alone, rKIN 50 µg, and rKIN 100 µg groups showed increased TNF production in uninfected mice (46.33 ± 1.52 pg/mL, 79 ± 1 pg/mL, 139 ± 4.58 pg/mL). After infection, these groups showed higher TNF production (79 ± 1 pg/mL, 139 ± 4.5 pg/mL, 201.33 ± 2.51 pg/mL) [Figures 5e and 6e].

Interlukin-17 expression

IL-17 levels were progressively higher in all immunized groups. Notably, 4 weeks after infection, the immunized-uninfected mice with rKIN 50 µg + BCG had 35 ± 1 pg/mL IL-17, and rKIN 100 µg + BCG had 46 ± 1 pg/mL, both >2-fold higher than the saline group (18.36 ± 0.47 pg/mL) (***P < 0.001). In the infected mice, rKIN 50 µg + BCG produced 254 ± 2.64 pg/mL and rKIN 100 µg + BCG produced 362 ± 2 pg/mL, >10-fold higher than the saline group (18 ± 1 pg/mL) (***P < 0.001). The saline group showed no change after infection. Groups immunized with BCG alone, rKIN 50 µg, and rKIN 100 µg had less IL-17 production in the uninfected state, with levels of 21.73 ± 1.41 pg/mL, 19.41 ± 0.35 pg/mL, and 24 ± 1 pg/mL (non-significant: P =0.3, 0.9, and 0.2). After infection, IL-17 levels in these groups increased (93.66 ± 2.51 pg/mL, 103 ± 1 pg/mL, 175 ± 5 pg/mL) (***P < 0.001) [Figures 5f and 6f].

Assessment of parasitic load in the spleen after challenge

Parasitic load of the mice spleen was expressed as LDU. The spleen of the mice of group rKIN 50 µg + BCG and group rKIN 100 µg + BCG showed 80 and 90% reduction in LDU in comparison to the infected control animals (***P < 0.001) whereas mice from group BCG, rKIN 50 µg and rKIN 100 µg showed significant (***P < 0.001) but <50% reduction in LDU [Figure 7].

Figure 7.

Figure 7

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Assessment of parasite load after challenge: Schematic representation of the parasite load in the spleen of all experimental mice, expressed in Leishman Donovan units. Data represent the mean ± standard deviation of three animals per group. The significance between different experimental groups was calculated by one-way ANNOVA using GraphPad Prism (version 8.0.1). Significance values indicate the difference between the vaccinated and control (saline) mice (***P < 0.001)

DISCUSSION

This study aimed to evaluate the potential of a rKIN vaccine combined with BCG as an adjuvant against experimental VL. We expressed and purified rKIN, using concentrations of 50 µg/100 µL and 100 µg/100 µL as vaccine formulations. BALB/c mice, the standard experimental model for studying L. donovani immunology, were chosen due to the availability of various immunological reagents. Since many antigens have limited immunogenicity in their pure form, an adjuvant is crucial for enhancing immune responses.[23] BCG is known for its ability to stimulate antigen-presenting cells, induce pro-inflammatory cytokines, and promote Th1 responses. It also enhances macrophage production of NO and upregulates costimulatory molecules essential for priming naïve T-cells. Given its strong immunostimulatory properties, BCG is an ideal candidate to boost specific immune responses, as demonstrated in our study.[20,24] Previous research has shown that BCG can generate robust humoral and cellular immune responses when combined with Leishmania antigens, supporting its use in our rKIN vaccine formulation to enhance immunogenicity and protective efficacy.[24]

Production of NO and ROS is essential for macrophages to kill Leishmania parasites within cells.[25] It is well-established that a Th1 immune response, characterized by increased production of IL-2, IL-12, IFN-γ, and NO synthase, is associated with immunity in cured VL patients, preventing reinfection.[26,27] Both Th1 and Th2 immune responses are observed in VL in mice and humans, with protective immunity linked to elevated Th1 cytokines.[28,29] Therefore, antigens that promote Th1 cell induction and memory Th1 cell formation are potential candidates for VL vaccines.[23] The treatment of VL typically stimulates Th1 cytokines, primarily IFN-γ.[30,31] In our study, the significant increase in IFN-γ and IL-2 levels in the BCG groups (with rKIN 50 µg and 100 µg) correlated with enhanced release of ROS and NO, both crucial for macrophage-mediated intracellular killing of Leishmania parasites.

In Leishmania-induced skin lesions, IL-17 produced by Th17 cells is shown to aggravate the disease, suggesting a role in pathogenesis. It is a fact that individuals who have recovered from a VL infection are capable of inducing long-lasting protection against reinfection and that this protection correlates with elevated IL-17 production,[32,33] suggesting that our vaccine candidate is working in the expected way.

CONCLUSIONS

In our study, we observed significant upregulation of TNF, IFN-γ, and IL-2 levels, along with a downregulation of IL-10 and IL-4 in the immunized groups compared to the control group. These results highlight the crucial interplay between IL-4 and IL-10 cytokines, suggesting their roles in determining the effect of vaccination, with lower production levels of these cytokines in the immunized groups.[34] Beyond Th1 and Th2 immune responses, we also observed a significant increase in IL-17 production in the immunized group, which supports the efficacy of the vaccine. IL-17 plays an important role in clearing intracellular pathogens,[32,33] and its elevated production in recovered individuals is correlated with long-lasting protection against reinfection, further suggesting the efficacy of our vaccine candidate. Although we did not perform quantitative real-time PCR assays, we assessed parasitic burden as a measure of active replication, which is a highly specific tool. Spleens from mice in the rKIN 50 µg + BCG and rKIN 100 µg + BCG groups showed an 80–90% reduction in LDU compared to infected control animals. This reduction in LDU was strongly associated with increased production of NO and ROS, as well as a pronounced Th1 immune response. A summary of all immune parameters, including nitrite, ROS, cytokines (IL-2, IL-4, IL-10, IL-17, IFN-γ, TNF), and LDU values (mean ± SD, range) is provided in Table 1. In conclusion, our findings suggest that rKIN 100 µg + BCG provided protective immunity in experimental VL. Optimizing antigen/adjuvant formulations to maximize Th1 and minimize Th2 responses could be crucial for developing a highly effective vaccine against human VL.

Table 1.

Summary of immune parameters (mean±standard deviation, range) measured in immunized and immunized + infected BALB/c mice groups

Group Nitrite (μM) ROS IFN-γ (pg/mL) IL-2 (pg/mL)
Before infection
  Saline 5.48±0.58 (4.9–6.2) 107.33±8.5 (98–117) 115.58±4.27 (110–121) 33.66±1.52 (32–35)
  BCG 18.72±0.80 (17.9–19.6) 277±7.9 (269–285) 201.33±0.5 (201–202) 56.66±2.08 (55–59)
  rKIN 50 (μg) 25.36±1.25 (24–27) 469.66±9.01 (460–480) 252.33±2.51 (250–255) 76.33±1.52 (75–78)
  rKIN 100 (μg) 32.91±2.1 (31–35.5) 775±5 (770–780) 381.33±7.09 (373–389) 121.66±1.52 (120–123)
  rKIN 50 μg + BCG 44.19±1.99 (42–46) 721.33±4.04 (717–726) 475.1±3.95 (470–479) 223±1 (222–224)
  rKIN 100 μg + BCG 57.14±2.51 (54–60) 883.66±0.57 (883–884.5) 675.1±3.95 (670–679) 451.5±2.29 (449–454)
After infection
  Saline 7.215±0.003 (7.21–7.22) 187±5 (182–192) 77.33±2 (75–79.3) 28.66±0.57 (28–29.2)
  BCG 22.3±1.7 (21–24.5) 277±7.9 (269–285) 405.33±4.72 (400–410) 156±1 (155–157)
  rKIN 50 (μg) 35.43±1.3 (34–37) 469.66±9.01 (460–480) 460.66±1.52 (459–462) 184±4.5 (179–188.5)
  rKIN 100 (μg) 49.33±2.8 (46–52) 775±5 (770–780) 557.33±2.08 (555–559.5) 243.33±1.52 (242–245)
  rKIN 50 μg + BCG 65.54±4.03 (61–70) 1107±7.2 (1100–1115) 822.33±1.46 (821–824) 499.33±1.52 (498–501)
  rKIN 100 μg + BCG 106.53±1.52 (105–108) 1593.45±1.27 (1592–1595) 1295.66±21.95 (1270–1320) 800.33±3.21 (797–803.5)
Group IL-4 (pg/mL) IL-10 (pg/mL) TNF (pg/mL) IL-17 (pg/mL) LDU
Before infection
  Saline 48±1 (47–49) 28.33±0.5 (28–29) 21.66±1.52 (20–23) 18.36±0.47 (17.8–19) NA
  BCG 38.1±1.1 (37–39.2) 18.2±0.6 (17.5–19) 46.33±1.52 (45–48) 21.73±1.41 (20.5–23.1) NA
  rKIN 50 (μg) 33.4±1.2 (32–35) 16.6±1.1 (15.5–18) 79±1 (78–80) 19.41±0.35 (19–20) NA
  rKIN 100 (μg) 29.8±1.3 (28.5–31) 14.5±0.9 (13.5–15.5) 139±4.58 (134–144) 24±1 (23–25) NA
  rKIN 50 μg + BCG 26.7±1.1 (25.5–28) 12.3±0.5 (11.8–12.9) 201.33±2.51 (199–204) 35±1 (34–36) NA
  rKIN 100 μg + BCG 25.2±0.8 (24.5–26) 11.8±0.4 (11.3–12.2) 307.33±1.52 (306–309) 46±1 (45–47) NA
After infection
  Saline 64.33±4.04 (60–68) 48±1 (47–49) 9.66±1.52 (8–11.2) 18±1 (17–19) 100%
  BCG 32.1±0.9 (31–33.2) 24±1 (23–25) 79±1 (78–80) 93.66±2.51 (91–96) ↓ <50%
  rKIN 50 (μg) 27.5±1.2 (26–29) 20.5±0.8 (19.5–21.3) 139±4.5 (134–143.5) 103±1 (102–104) ↓ <50%
  rKIN 100 (μg) 24.6±1.1 (23.5–25.7) 18±0.6 (17.3–18.6) 201.33±2.51 (199–204) 175±5 (170–180) ↓ <50%
  rKIN 50 μg + BCG 22.8±1.3 (21.5–24.1) 15.1±0.7 (14.3–15.8) 307±1.52 (305.5–308.5) 254±2.64 (251–257) ↓ ~80%
  rKIN 100 μg + BCG 20.1±0.6 (19.5–20.7) 13.3±0.5 (12.8–13.8) 660.33±5.50 (655–666) 362±2 (360–364) ↓ ~90%
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rKIN: Recombinant kinesin protein, ROS: Reactive oxygen species, IFN-γ: Interferon gamma, IL-2: Interleukin 2, TNF: Tumor necrosis factor, LDU: Leishman Donovan Units, BCG: Bacille Calmette–Guerin, NA: Not available, ↓: Significant reduction

Conflicts of interest

There are no conflicts of interest.

Acknowledgments

We acknowledge the technical help provided by Mr. Manoj, BD Biosciences, and BD Academy Jamia Hamdard University, New Delhi, for allowing us to perform Flow Cytometry work on LSR II. We also thank Dr. Ramu Sivakumar and Dr. Ayan Dey for performing the preliminary work and giving us a suitable vaccine candidate that was used throughout our studies. We are also thankful to the Department of Pediatrics, AIIMS, New Delhi, for providing the BCG vaccine.

Funding Statement

This study was financially supported by the Indian Council of Medical Research, New Delhi, India, through the Senior Research Fellowship (IR360) awarded to Author SSR.

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