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. 2024 Jan 11:14:1294397.
doi: 10.3389/fimmu.2023.1294397. eCollection 2023.

Immunogenicity and protective efficacy of tuzin protein as a vaccine candidate in Leishmania donovani-infected BALB/c mice

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Immunogenicity and protective efficacy of tuzin protein as a vaccine candidate in Leishmania donovani-infected BALB/c mice

Moodu Devender et al. Front Immunol. .

Abstract

Visceral leishmaniasis (VL) is referred to as the most severe and fatal type of leishmaniasis basically caused by Leishmania donovani and L. infantum. The most effective method for preventing the spread of the disease is vaccination. Till today, there is no promising licensed vaccination for human VL. Hence, investigation for vaccines is necessary to enrich the therapeutic repertoire against leishmaniasis. Tuzin is a rare trans-membrane protein that has been reported in Trypanosoma cruzi with unknown function. However, tuzin is not characterized in Leishmania parasites. In this study, we for the first time demonstrated that tuzin protein was expressed in both stages (promastigote and amastigote) of L. donovani parasites. In-silico studies revealed that tuzin has potent antigenic properties. Therefore, we analyzed the immunogenicity of tuzin protein and immune response in BALB/c mice challenged with the L. donovani parasite. We observed that tuzin-vaccinated mice have significantly reduced parasite burden in the spleen and liver compared with the control. The number of granulomas in the liver was also significantly decreased compared with the control groups. We further measured the IgG2a antibody level, a marker of Th1 immune response in VL, which was significantly higher in the serum of immunized mice when compared with the control. Splenocytes stimulated with soluble Leishmania antigen (SLA) displayed a significant increase in NO and ROS levels compared with the control groups. Tuzin-immunized and parasite-challenged mice exhibit a notable rise in the IFN-γ/IL-10 ratio by significantly suppressing IL-10 expression level, an immunosuppressive cytokine that inhibits leishmanicidal immune function and encourages disease progression. In conclusion, tuzin immunizations substantially increase the protective immune response in L. donovani-challenged mice groups compared with control.

Keywords: Leishmaniasis; Tuzin; antibodies; antigenic; cytokine; immunogenicity; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression, purification, and confirmation of tuzin protein in Leishmania donovani parasites: (A) SDS-PAGE; expression of recombinant tuzin protein in Escherichia coli. Rosetta (DE3) strain and confirmation of expression by Western blot using anti-His tag antibody. (B) SDS-PAGE of purified recombinant tuzin protein and confirmation of tuzin protein by Western blot using anti-tuzin primary antibody raised in rabbit. (C) Confirmation of tuzin protein expression in L. donovani parasites after probing with anti-tuzin antibody by western blot. (D) Tuzin protein localization by immunofluorescence assay: panel 1—bright field; panel 2—anti-tuzin probed with FITC fluorescence; panel 3—DAPI fluorescence; panel 4—merged. (A, B) Promastigote control (without anti-tuzin antibody) and test (with anti-tuzin antibody), respectively; (C, D) amastigote control and test, respectively.
Figure 2
Figure 2
In-silico structural and antigenic analysis of tuzin protein. (A) PSIPRED-based transmembrane prediction report for the tuzin protein shows the color coding of the sequence nature of the tuzin protein: the gray shading indicates the transmembrane helix, the colorless portion represented the cytoplasmic portion, and the orange color indicates the extracellular helix sequence of the tuzin protein. (B) The figure illustrates the detailed visualization of the N-terminal cytosolic helix and C-terminal extracellular helix. (C) The antigenicity propensity scores are generated by IMED prediction; the average antigenicity propensity score ≥1 is considered a potent antigen. The average antigenicity propensity score for the tuzin protein is 1.0437, considered a potent antigen.
Figure 3
Figure 3
Measurement of spleen size and parasite load. (A) Spleens from all the experimental groups of mice were collected and measured using a scale. (B) Intracellular amastigotes were counted in the spleen using Giemsa staining: the graph shows the amastigote numbers for 100 fields. The values showed significant differences between the infected control and the tuzin-immunized group and were calculated using Student’s t-test (***P < 0.05). The data shown here are a representative dataset from one of the independent animal studies. *p ≤ 0.05, **p ≤ 0.005.
Figure 4
Figure 4
Histological analysis of liver tissue of various groups of mice 8 weeks after infection/challenged. (A) Healthy control, infected control, adjuvant-immunized + challenged, and tuzin-immunized + challenged groups. (B) Granuloma counts; infected control, adjuvant-immunized + challenged, and tuzin-immunized + challenged group. (C) Standard curve plotted with various dilutions of genomic DNA of L. donovani parasites. (D) Parasite load in liver tissue quantified by qRT-PCR. The significant difference between the infected control and the tuzin-immunized + challenged group was calculated using Student’s t-test (*P < 0.05).
Figure 5
Figure 5
The titer of IgG1 and IgG2a antibodies measured by ELISA. The sera of all mice groups were collected after 8 weeks of infection/challenged. (A) Level of IgG1. (B) Level of IgG2a. The data are presented as the mean ± SD for each group. Significant differences between the infected control and the tuzin-immunized + challenged group (***P < 0.005) were calculated using Student’s t-test. (C) The IgG2a/IgG1 ratios were measured and calculated using Student’s t-test. (D) Nitric oxide level was measured after 8 weeks of infection/challenged; the nitrite level of the sample was calculated using standard curves made from various concentrations of NaNO2. (E) ROS was also measured after 8 weeks of infection/challenged. The green fluorescence of H2DCFDA was measured using flow cytometry, and mean fluorescence intensity (MFI) was represented as a bar graph. *p ≤ 0.05, **p ≤ 0.005.
Figure 6
Figure 6
The relative gene expression analysis in the spleen of mice using RT-qPCR. RT-qPCR analysis of Th1- and Th2-specific cytokine relative expression in the spleen of mice. Healthy, infected groups, adjuvant-immunized, and tuzin-immunized + challenged groups and Th1/Th2 cytokines such as IL-4, TGF-β, and IFN-γ levels were unchanged in all groups of mice (A–C), whereas IL-10 cytokine was significantly downregulated in tuzin-immunized + L. donovani-challenged groups (D). However, the IFN-γ/IL-10 ratio was significantly high in tuzin-immunized and challenged mice compared with the other groups (E). Estimation of cytokines (pg/mL) released in the serum of various groups of mice by the ELISA method. Healthy, infected control, adjuvant-immunized + challenged, and tuzin-immunized + challenged groups. There were no significant changes in IFN-γ level (F); however, the most interesting point is that the IL-10 cytokine was found significantly lower in tuzin-immunized + challenged groups compared with the control (G). Total IFN-γ/IL-10 ratio was also significantly higher in tuzin-immunized challenged groups compared with the control (H). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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