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

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Highlights

  • Interferon-gamma from BALB/c mouse was cloned and expressed in vitro.

  • Recombinant interferon-gamma promotes immunoglobulin G and Th immune responses to cathepsin L-like cysteine proteinase of Hyalomma asiaticum.

  • Interferon-gamma as a potential vaccine adjuvant or potentiater promotes HasCPL triggered strong the efficacy of anti-tick.

Abstract

Among bloodsucking arthropods, hard tick is a vector of transmitting the most diverse human and animal pathogens, leading to an increasing number of manifestations worldwide. The development of the anti-tick vaccine has the potential to be an environmentally friendly and cost-effective option for tick management. We have previously demonstrated the induction of both humoral and cellular response against Hyalomma asiaticum (H. asiaticum) following immunization with recombinant cathepsin L-like cysteine protease from H. asiaticum tick (rHasCPL), and could control tick infestations. Interferon-gamma (IFN-γ), is an immunomodulatory factor that plays an important role in the regulation of adaptive immunity against infection. In the present study, recombinant BALB/c mouse IFN-γ (rMus-IFN-γ) was cloned and expressed using a prokaryotic expression system, and verified by Western blotting and IFN-γ-ELISA kit analysis. Female BALB/c mice (n = 12) were used for immunization using rHasCPL (100 μg) plus IFN-γ as adjuvant (10 μg). In immunized female BALB/c mice, the levels of anti-CPL antibodies as well as cytokines were determined using ELISA analysis. Protective efficacy of immunization was evaluated by larvae H. asiaticum challenge of immunized female BALB/c mice. Using rMus-IFN-γ as an adjuvant to rHasCPL vaccine (CPL + IFN-γ) promoted specific antibody IgG (IgG1 > IgG2a) and increased production of IFN-γ and IL-4 compared to immune rHasCPL group (CPL). The protected rate of immunized mice from tick challenge was significantly higher after immunization with CPL + IFN-γ (85.11 %) than with CPL (63.28 %). Immunization using CPL + IFN-γ promoted the activation of anti-HasCPL humoral and cellular immune responses, and could provide better protection against H. asiaticum infestation. This approach may could help develop a candidate vaccine for control tick infestations.

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.

References (40)

  • A.A. Smith et al.

    Cross-species interferon signaling boosts microbicidal activity within the tick vector

    Cell Host Microbe

    (2016)
  • D. Sojka et al.

    New insights into the machinery of blood digestion by ticks

    Trends Parasitol.

    (2013)
  • M.L. van Slooten et al.

    Immunoadjuvant activity of interferon-gamma-liposomes co-administered with influenza vaccines

    Biochim. Biophys. Acta

    (2001)
  • M.M. Aleman et al.

    Tick spit shines a light on the initiation of coagulation

    Circulation

    (2013)
  • A.V. Atrasheuskaya et al.

    Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice

    Clin. Exp. Immunol.

    (2004)
  • U. Boehm et al.

    Cellular responses to interferon-g

    Annu. Rev. Immunol.

    (1997)
  • M. Brossard et al.

    Immunology of interactions between ticks and hosts

    Med. Vet. Entomol.

    (1997)
  • M. Christe et al.

    Influence of genetic background and parasite load of mice on immune response developed against nymphs of Ixodes ricinus

    Parasitol. Res.

    (1999)
  • K.V. Clemons et al.

    Efficacy of recombinant gamma interferon for treatment of systemic cryptococcosis in SCID mice

    Antimicrob. Agents

    (2001)
  • M.A. Farrar et al.

    The molecular cell biology of interferon-gamma and its receptor

    Annu. Rev. Immunol.

    (1993)
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