Recombinant interferon-gamma promotes immunoglobulin G and cytokine memory responses to cathepsin L-like cysteine proteinase of Hyalomma asiaticum and the efficacy of anti-tick
Graphical abstract
Introduction
The host blood is the only source of energy and nutrients needed for survival, development and reproduction of hematophagous ticks (Sonenshine, 1995). However, hemoglobin digestion is an essential process for blood-feeding parasites to determine the peptidase-specific cleavage model of the hemoglobin molecule (Horn et al., 2009). The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C, and B) (Horn et al., 2009). The identified enzymes are potential targets to developing novel anti-tick vaccines. Cathepsin L (CPL) having potent haemoglobinase activity playing important role in the digestion of blood acquired from the host (Sojka et al., 2008; Horn et al., 2009; Franta et al., 2011), and have also been shown to have important roles in embryogenesis and tissue remodeling during insect metamorphosis, in addition to nutrient recycling and availability to the developing organism (Sojka et al., 2013). Therefore, CPL could serve as a rational target for interference for ticks growth and development.
For ticks to successfully feed, they must overcome the host defenses during blood sucking (Francischetti et al., 2009; Corral Rodriguez et al., 2009; Andersen, 2010). Their main strategy is to release anticoagulants, anti-inflammatory agents and immunosuppressants at the bite site that form a feeding lesion on the host (Aleman and Wolberg, 2013). Studies have shown different tick species preferentially induce host Th2 lymphocyte responses that have considerably less pro-inflammatory cytokines (Kovár et al., 2002; Brossard and Wikel, 2008). On the other hand, the host immune response would promote the expression of interferon-gamma (IFN-γ), which is secreted by activated T lymphocytes and NK cells, and promotes a dominant Th1 type immune response (Shtrichman et al., 2001; Schijns et al., 2000; Lowenthal et al., 1998). In previous studies, the T cell-derived macrophage-activating lymphokine, IFN-γ, is a highly pleiotropic cytokine with immunomodulatory antimicrobial, antiproliferative, and antifibrotic activities that also modulates the production or activities of several cytokines and chemokines (Schroder et al., 2004; Farrar et al., 1993; Schreiber et al., 1993; Boehm et al., 1997). IFN-γ is the most broadly acting antimicrobial-inducing and host defense-enhancing cytokine thus far identified in experimental models of infectious diseases (Murray, 1996). IFN-γ has been successfully used as an adjuvant in vaccines against other pathogens (Atrasheuskaya et al., 2004; Faulkner et al., 2003; van Slooten et al., 2001), and found to be safe in immunocompromised mice (Heath et al., 1989).
However, in vivo experiments performed with IFN-γ as an adjuvant for the anti-tick vaccine have not reported before. Therefore, in the study, we used IFN-γ as an adjuvant to recombinant H. asiaticum CPL (rHasCPL) protein vaccine. Results showed an enhanced immunoglobulin G and Th immune responses against HasCPL. Challenge with ticks in the immunized female Bagg albino C (BALB/c) mice showed protective efficacy. Therefore, CPL + IFN-γ could trigger both humoral and cellular immune responses and promotes protective immunity against H. asiaticum infestation in female BALB/c mice.
Section snippets
Ticks and animals
The larva and adult H. asiaticum were fed by experimental infestation on six-week-old female BALB/c mice purchased from the Experiment Animal Center of Xinjiang Medical University. The female BALB/c mice using to screen optimal immune conditions, and every 12 female BALB/c mice fed on commercial feed in a cage and kept at 23 ± 1℃ and 40 % relative humidity. The experiments were approved and conducted following the guidelines of the Animal Care and Use Committee of Xinjiang Agricultural
Sequence analysis of Mus-IFN-γ
The amino acid sequences of female BALB/c mice IFN-γ showed high homology to the IFN-γ of Mus musculus (100.00 %), Mus caroli (94.84 %), and Mus pahari (90.32 %). Alignment analysis also found that the four IFN-γ sequences had conserved regions within the primary structures. Further, in silico analysis show that IFN-γ contains signal peptides and transmembrane helices in 1–22 aa (Fig. 2).
Construction, expression, and purification of rMus-IFN-γ
To generate recombinant mouse (Mus-) IFN-γ, the gene was amplified from the liver and spleen, respectively (
Discussion
In the present study, we cloned IFN-γ gene from both the liver and spleen in female BALB/c mice, then the protein was expressed and purified, and identified IFN-γ using Western blotting and ELISA kit analysis. The female BALB/c mice immunized with IFN-γ as immune adjuvant and rHasCPL obtained high antibody level/titer and the efficacy of anti-tick (85.11 %). Previously, Kumar et al. reported the cross-bred animals immunized with Montanide ISA 50V2 ready-to-use adjuvant and rHa-CathL proteins
Conclusions
In conclusion, we cloned the IFN-γ from BALB/c mouse and expressed in vitro. Furthermore, IFN-γ promotes HasCPL triggered strong both humoral and cellular immune responses, and aroused more effective protective immunity against H. asiaticum larva infestation in the female BALB/c mice model. Therefore, this study suggested that IFN-γ may serve as a potential vaccine adjuvant or potentiater against ticks.
Author’s contributions
BC and RS conceived the study and designed the experiments. RS, XZ, and XF performed the experiments. RS, TG, and Hu performed the data analysis. RS wrote the original draft. All authors reviewed, edited, and contributed to final version of the manuscript and approved it for publication.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31660711). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
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