Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes

https://doi.org/10.1016/j.ttbdis.2020.101445Get rights and content

Abstract

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

Introduction

Tick control methods have long relied on the use of acaricides. Nevertheless, numerous methods of tick control have been suggested and explored, among which vaccinating cattle against ticks is considered one of the most sustainable alternatives (de la Fuente and Contreras, 2015; Ghosh et al., 2007; Manjunathachar et al., 2014). Indeed, remarkable progress has been made toward identifying tick vaccine antigens (de la Fuente and Kocan, 2006; Merino et al., 2013; Nuttall et al., 2006; Valle and Guerrero, 2018). Strikingly, however, only a few antigens are reported to induce partial protection against multiple tick species (de la Fuente et al., 2013; Kumar et al., 2017; Ndawula et al., 2019; Parizi et al., 2011; Sabadin et al., 2017; Trimnell et al., 2005). For example, recombinant Bm86 (a Rhipicephalus microplus protein) (Rand et al., 1989) was shown to induce a high protection against R. microplus strains from Australia (Willadsen et al., 1995) and Cuba (Valle et al., 2004), and a lower protection against R. microplus strains from South America (Andreotti, 2006; García-García et al., 2000). The variation in protection could be attributed to differences in Bm86 amino acid sequences among different R. microplus populations (Freeman et al., 2010; García-García et al., 1999). Furthermore, phylogenetic differences within the populations of R. microplus were determined (Burger et al., 2014). Nevertheless, Bm86 was shown to induce cross-protection against Rhipicephalus annulatus (Fragoso et al., 1998), Rhipicephalus decoloratus, Hyalomma anatolicum and Hyalomma dromedarii, but not against Rhipicephalus appendiculatus and Amblyomma variegatum (De Vos et al., 2001).

Considering that multiple tick species are present in most parts of the world, it is vital to develop vaccines that can induce cross-protection against different ticks. However, based on anti-tick vaccination reports, for instance using recombinant Bm86, it seems unlikely that single-antigen tick vaccines will induce a high cross-protection against multiple tick species. Therefore, constituting cocktail vaccines present a possibility to enhance the protection range and the efficacy of anti-tick vaccines. However, how to select antigens for constituting efficacious cocktail tick vaccines is still a challenging question to researchers in the field, due to antigenic competition (Shaffer et al., 2016; Taussig et al., 1973) and the amount of non-protective epitopes exposed to the immune system (Gershoni et al., 2007; Vyas et al., 2008). Epitope-based vaccines, by contrast, do not trigger undesirable immune responses, can induce a high specific immune response and, most importantly, they could induce longer immune protection (Childs et al., 2015; Gershoni et al., 2007; Vyas et al., 2008). The concept of constituting epitope-based vaccines has been demonstrated toward tick control (Aguirre et al., 2016; Patarroyo et al., 2002). Therefore, it is plausible that replacing conventional or whole antigen-based vaccines by an epitope-based approach could enhance the potency of cocktail anti-tick vaccines. The fundamental question, however, remains how to identify and locate epitopes among the sequences of the conventional or whole antigen-based anti-tick vaccine antigens.

There are numerous methods for identifying B-cell epitopes (Ahmad et al., 2016; Gershoni et al., 2007), of which location based on protein structure is regarded as the gold standard (Gershoni et al., 2007); however, x-ray crystallographic data on the structure of tick antigens remains scanty. Nevertheless, the potential of using computer-based tools as an alternative approach to locate epitopes has been exploited (Soria-Guerra et al., 2015). Compared to crystallography, epitope prediction using in silico algorithms is cheaper, quicker and readily applicable. Indeed, numerous in silico epitope prediction tools have been reported (Potocnakova et al., 2016; Sun et al., 2019), of which the linear prediction tools are more commonly used. It is suggested that most of the sequence-based predicted epitope peptides constitute the conformational-based predicted epitopes (Van Regenmortel, 1996), but incorporate structural information on epitope prediction may include epitopes that are discontinuous. Therefore, predictions based on protein structure and linear peptide sequence are likely to give more accurate and reliable results (Assis et al., 2014).

In this work, a combination of three sequence- and conformation-based epitope prediction tools were used, which were selected among the most accurate available (Potocnakova et al., 2016; Sun et al., 2019). Additionally, CBTOPE (Ansari and Raghava, 2010) and Scratch (Cheng et al., 2005) were specifically selected to complement the structural and conformation epitope prediction tools.

Previous work by our group has demonstrated that glutathione S-transferase (GST) is a suitable candidate for a cocktail tick vaccine, inducing non-homologous cross-reaction against different tick species (Ndawula et al., 2019). Therefore, in the present study we performed in silico prediction and mapping of conserved epitopes within the GST sequence of R. appendiculatus (GST-Ra), R. decoloratus (GST-Rd), Haemaphysalis longicornis (GST-Hl), A. variegatum (GST-Av) and R. microplus (GST-Rm). Moreover, the potential epitope peptides were synthesized and screened by immunological assay using anti-GST and anti-peptide sera. The predictions can partially explain the cross-reaction phenomenon observed. Finally, the study illustrates an approach to select epitopes toward developing epitope-based tick vaccine antigens.

Section snippets

Ethics statement

Rabbits used in the experiments were housed at Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul facilities. This research was conducted in agreement with the Norms for Animal Experimentation Ethics Committee of Universidade Federal do Rio Grande do Sul (process number 38748).

GST protein sequences

The GST sequences used herein were deduced from the GSTs of R. appendiculatus (MK133338), R. decoloratus (MK133339), A. variegatum (MK133337), R. microplus (AF366931.1), H. longicornis (AY298731.1), R.

Results and discussion

Until now, vaccinating cattle against ticks is regarded as one of the most promising tick-control alternative approaches to replace the use of acaricides. Indeed, researchers have identified numerous antigens (de la Fuente and Kocan, 2006; Merino et al., 2013; Ndawula et al., 2019; Nuttall et al., 2006; Schetters et al., 2016; Valle and Guerrero, 2018), but none has so far matched the success exhibited with Bm86 under field conditions (de la Fuente et al., 2016, 2007, 1999, 1998). Combining

Conclusion

Ultimately, the data presented here support the hypothesis that GST-Ra, GST-Rd, GST-Hl, GST-Rm and GST-Av contain conserved epitopes, which may explain the cross-reaction among tick GSTs (Ndawula et al., 2019). Additionally, the study brings forward an approach to select B-cell epitopes. Most importantly, the epitopes predicted herein were found to be immunogenic. Based on these findings, our hypothesis is that the identified epitopes could be used to constitute epitope-based antigen tick

CRediT authorship contribution statement

Charles Ndawula: Conceptualization, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Marina Amaral Xavier: Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Bianca Villavicencio: Conceptualization, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Fernanda Cortez Lopes: Conceptualization, Methodology, Formal analysis, Visualization, Writing -

Declaration of Competing Interest

The authors certify that they have no affiliations with, or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.

Acknowledgements

This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES): # Procad 88881.068421/2014-01; PGCI 23038.005296/2014-37; 88881.153222/2017-01; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq): Grant # 465678/2014-9; 405763/2018-2, 302360/2018-2 and 441092/2016-0; CNPq-INCT and TWAS. Ndawula Charles Jr is a student under the TWAS-CNPq PhD Fellowship.

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