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Background <p><i>Leishmania</i> parasites are transmitted through the bite of infected female sand flies. The sand fly inoculum contains both the parasite and the salivary proteins, which can modulate the immune system’s function. Some of these salivary proteins have the potential to be used as a vaccine candidate. Since there have been fewer studies investigating the salivary proteins of <i>Phlebotomus</i> (<i>Ph.</i>) <i>sergenti</i>, this prompted us to select among the three protein members of <i>Ph. sergenti</i> apyrase family (PsSP40, PsSP41, and PsSP42) and measure its effectiveness as vaccine candidate against <i>Leishmania</i> (<i>L.</i>)<i> tropica</i>.</p> Methods <p>To select among the three family members as the candidate for immunization, different parameters including the physicochemical characters, three-dimensional structure, virtual immune stimulatory potential, and human leukocyte antigen (HLA) class II-binding epitope content were considered. To investigate the effect of immunization with the selected antigen through immunoinformatics analysis (PsSP42) against <i>L. tropica</i> infection, we immunized BALB/c mice with two distinct recombinant plasmids (conventional VR1020 and new-generation NTC9385R) two times at 3-week intervals followed by immediate electroporation. Eight weeks post-challenge, the parasite load in draining lymph nodes was measured by quantitative real-time polymerase chain reaction (PCR). The interferon (IFN)-γ and interleukin (IL)-4 cytokines before (against recombinant <i>Leishmania tarentolae</i> expressing PsSP42) and after (against parasite frozen/thawed antigens) <i>L. tropica</i> infection (2 × 10<sup>7</sup> parasite per footpad plus <i>Ph. sergenti</i> salivary gland homogenate (SGH)) were measured by enzyme-linked immunosorbent assay (ELISA).</p> Results <p>On the basis of immunoinformatics analysis of three apyrase salivary proteins from <i>Ph. sergenti</i>, PsSP42 demonstrated superior HLA class II-binding peptides compared with the other two proteins (PsSP40 and PsSP41) and was selected for immunization studies. Our findings indicated that NTC-PsSP42 and not VR1020-PsSP42 plasmid immunization relatively reduced the parasite load in the draining lymph nodes. This was assigned to a significant higher IFN-γ to IL-4 ratio induced by NTC-PsSP42 immunization in comparison with pertinent controls.</p> Conclusions <p>In our study, although the expected protective response was not achieved by any of the recombinant plasmids, the NTC-PsSP42 platform induced a weak Th1-polarized immune response, which partially influenced the parasite load. Since the new generation of plasmids are worth evaluating owing to the lack of antibiotic resistance genes on the backbone, we recommend further assessment of NTC-PsSP42 potential adjuvnated with immunostimulatory sequences such as as CpG motifs or even in heterologous prime-boost regimens.</p> Graphical Abstract <p></p>

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Evaluating the effect of immunization with DNA encoding Phlebotomus sergenti apyrase protein (PsSP42) against Leishmania tropica infection in BALB/c mouse model

  • 期刊论文

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  • 发表日期：

    2026年3月9日

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  • Samira Hosseinpour Jahednia,
  • Hossein Rezvan,
  • Hamzeh Sarvnaz,
  • Sima Habibzadeh,
  • Alireza Nourian,
  • Tahereh Taheri,
  • Negar Seyed,
  • Elham Gholami,
  • Sima Rafati

摘要

Background

Leishmania parasites are transmitted through the bite of infected female sand flies. The sand fly inoculum contains both the parasite and the salivary proteins, which can modulate the immune system’s function. Some of these salivary proteins have the potential to be used as a vaccine candidate. Since there have been fewer studies investigating the salivary proteins of Phlebotomus (Ph.) sergenti, this prompted us to select among the three protein members of Ph. sergenti apyrase family (PsSP40, PsSP41, and PsSP42) and measure its effectiveness as vaccine candidate against Leishmania (L.) tropica.

Methods

To select among the three family members as the candidate for immunization, different parameters including the physicochemical characters, three-dimensional structure, virtual immune stimulatory potential, and human leukocyte antigen (HLA) class II-binding epitope content were considered. To investigate the effect of immunization with the selected antigen through immunoinformatics analysis (PsSP42) against L. tropica infection, we immunized BALB/c mice with two distinct recombinant plasmids (conventional VR1020 and new-generation NTC9385R) two times at 3-week intervals followed by immediate electroporation. Eight weeks post-challenge, the parasite load in draining lymph nodes was measured by quantitative real-time polymerase chain reaction (PCR). The interferon (IFN)-γ and interleukin (IL)-4 cytokines before (against recombinant Leishmania tarentolae expressing PsSP42) and after (against parasite frozen/thawed antigens) L. tropica infection (2 × 107 parasite per footpad plus Ph. sergenti salivary gland homogenate (SGH)) were measured by enzyme-linked immunosorbent assay (ELISA).

Results

On the basis of immunoinformatics analysis of three apyrase salivary proteins from Ph. sergenti, PsSP42 demonstrated superior HLA class II-binding peptides compared with the other two proteins (PsSP40 and PsSP41) and was selected for immunization studies. Our findings indicated that NTC-PsSP42 and not VR1020-PsSP42 plasmid immunization relatively reduced the parasite load in the draining lymph nodes. This was assigned to a significant higher IFN-γ to IL-4 ratio induced by NTC-PsSP42 immunization in comparison with pertinent controls.

Conclusions

In our study, although the expected protective response was not achieved by any of the recombinant plasmids, the NTC-PsSP42 platform induced a weak Th1-polarized immune response, which partially influenced the parasite load. Since the new generation of plasmids are worth evaluating owing to the lack of antibiotic resistance genes on the backbone, we recommend further assessment of NTC-PsSP42 potential adjuvnated with immunostimulatory sequences such as as CpG motifs or even in heterologous prime-boost regimens.

Graphical Abstract