1. Introduction
Trypanosoma cruzi is a flagellated protozoan parasite, discovered by Dr. Carlos Ribeiro Justiniano das Chagas in 1909 [
1]; this finding is considered as a feat since it was the first time that the etiological agent and its vector were detected before the discovery of the disease [
2]. The transmission of this parasite is through different routes such as blood transfusion, organ transplant, congenital via, or oral route, and it is discussed as sexual transmission; however, the most common in endemic areas is vectorial transmission, caused by hematophagous bugs [
3] belonging to the Hemiptera order, Reduviidae family, and Triatominae subfamily [
4]. Chagas disease is localized in 21 Latin American countries; however, in recent years, a higher incidence has been reported in the United States of America [
5] as well as in some cases reported in Central Europe, Asia, and Oceania [
6]; this is because of the constant migratory flows [
7] and the lack of controls and screening in blood and organ donations, in which only six European countries provide the screening for Chagas disease [
8]. It is estimated that 6–8 million people are infected worldwide, causing approximately 50,000 deaths per year, while 65–100 million are at risk of being infected [
7], either because of their geographic location, socioeconomic status, or both. This disease is considered as a neglected tropical disease (NTD) by the World Health Organization (WHO) and some other organizations such as the Center for Disease Control (CDC), being especially common in rural areas [
9].
Although
T. cruzi has an asexual reproduction form, this parasite has a great heterogeneity with genotypic and phenotypic variation, and has therefore been classified into six highly related clades or taxonomic units, from TcI-TcVI, which have been divided based on their discrete typing units (DTU) [
10,
11,
12]. This genetic diversity has been related to geographic distribution, pathogenesis, clinical features, and response to therapy, all well-defined by the kind of DTU [
11,
13].
For the treatment of Chagas disease, there are only two drugs available, Nifurtimox and Benznidazole, both developed in the last century in 1965 and 1971, respectively [
14]. These treatments are not easily accessible [
14,
15], have high toxicity causing renal and hepatic dysfunction derived from the generation of highly reactive metabolites [
16], and are indicated, especially during the acute phase, congenital, reactivations, and chronic phase in patients under ideal conditions, that is, without serious comorbidities and are commonly young (<18 years), with a probability of success around 60% [
16,
17].
Currently, there is no commercial vaccine capable of counteracting or preventing the disease and its dissemination in the organism. There have been considerable efforts to develop one, either with DNA platforms, attenuated vaccines, modified viruses, and bacteria, or recombinant proteins, all of them with various formulations and adjuvants [
18]; however, none has been able to confer ideal protection against the disease. Some studies have suggested the combined use of vaccines and drugs to reduce treatment, and consequently, its toxicity [
18]. The need to obtain important advances or alternative strategies to combat Chagas disease is evident. To this purpose, reverse vaccinology has emerged as a promising alternative [
19] representing an attractive opportunity for the development of a vaccine through the analysis of proteins exposed on the surface of the parasite, thus trying to prevent infection, or at least delay its progression as well as reduce parasite congenital transmission.
Several studies have demonstrated the importance of this approach in different pathogens such as
Plasmodium falciparum and
Ascaris suum, showing a decrease in parasitemia by 80% and 60%, respectively, and
Candida albicans, with IgG1 and IgG2a immunoglobulin production [
20,
21,
22]. In a previous study, enolase from
T. cruzi was used to show their immunogenicity, identifying a transmembrane region suggesting a surface localization; epitopes for B lymphocytes and cytotoxic T lymphocytes were also predicted, suggesting the development of humoral and cellular immune responses [
23]. This protein is a multifunctional metalloenzyme with the Enzyme Commission number E.C. code 4.2.1.11, which catalyzes the reversible dehydration of D-2-phosphoglycerate to phosphoenolpyruvate [
24]; enolase as an immunogen has been capable of generating a Th1 immune response (considered fundamental for intracellular parasite elimination), Th2, or a mixture of both in response to the kind of microorganism being studied (intracellular or extracellular); therefore, there is no predominance of one of these two types of the immune response, both cell populations can be expressed [
21,
22,
25,
26]. Subsequent studies detected the presence of antibodies against the recombinant enolase from
T. cruzi (anti-rTcENO antibodies) in sera from mice experimentally immunized with rTcENO, demonstrating that the purified recombinant enolase was recognized by mouse sera. Furthermore, mice immunized with rTcENO and subsequently infected experimentally showed protection against the parasite by increasing their survival, presenting a decrease in circulating blood parasites and not presenting damage at the tissue level (cardiac and skeletal); these results indicate that rTcENO has immunogenic properties to be studied and can be proposed as a candidate for vaccine development [
27].
The aim of the present work was focused on proposing a consensus sequence of the enolase protein of 429 amino acids from 15 different sequenced isolates and corresponded to different DTUs for its possible use as a vaccine. This consensus sequence was mainly evaluated for its immunogenic properties at the bioinformatic level through the analysis of epitopes, showing a high immunogenic potential with six epitopes for MHC-I and seven epitopes for MHC-II, all of them are highly representative of the American continent as well as five epitopes for B cells. Subsequently, molecular docking was performed with the membrane-associated of Toll-like receptors TLR2 and TLR4, showing a high affinity and low dissociation constant, which could lead to an innate immune response that contributes to the parasite elimination. Finally, a hypothetical chimeric construct was designed with the most representative epitopes for the Latin American population. In conclusion, the enolase consensus sequence would have the capacity to provide an ideal immune response for people at risk of infection with Chagas disease in Latin America and the world; similar properties could have chimeric proteins, which need further study and experimental evaluation to verify whether they confer protection to most of the T. cruzi strains of Chagas disease.
4. Discussion
Several studies have validated the use of bioinformatics tools in the search for new vaccines from the use of TriTrypDB for the reliable search of new antigens in
T. cruzi [
54]. Taking into account the above and based on the TriTrypDB and NCBI databases, 13 and two sequences of the enolase were obtained, respectively, belonging to almost all known DTUs except for DTU IV, which can be explained by the constant association of this variant with dogs as well as other domestic mammals in highly isolated regions such as the Paraguayan Chaco [
55], and therefore, the lack of study at the genome sequencing level. Later, the sequences were translated into amino acids, resulting in 429 amino acids for 14 sequences of 1290 bp, while the initial reference sequence corresponding to the H8 variant has 1151 bp, which codes for 384 amino acids; therefore, seeking greater reliability, the reference strain became the one corresponding to CL Brener, since it is the most studied variant worldwide [
56].
To obtain an enolase consensus sequence, the multiple alignments based on Clustal Omega showed that the Marinkellei B7 variant strain of
T. cruzi showed more point mutations, with 14 throughout the alignment (data not shown). It is also important to mention that a constant mutation was identified at the 330 position, in which seven DTUs had T and eight had M while the consensus sequence had an M of a total of 429 amino acids. The homology modeling used as a template for the crystallized structure of the
T. cruzi CL Brener enolase (PDB: 4G7F) (DTU VI) showed an identity of 99.77% at the sequence level, and QMEAN value of −0.28 and GMQE of 0.96, which indicates the degree of nativity or authenticity and quality of the global model, respectively. In this regard, the homology modeling performed by Vedamurthy G. et al., (2019) [
50] on the same platform showed the following values GMQE: 0.72, identity: 46.89%, and overall Ramachandran of 98.5%, unlike the enolase consensus obtained here, whose values were GMQE: 0.96, identity of 99.77%, and overall Ramachandran of 96.49%. These data indicate that the modelling is highly reliable, and data are confirmed by a comparison plot shown in
Supplementary Figure S1, which displayed the experimentally obtained protein structure data. There were two regions less than 0.6 (poor quality), corresponding to regions 39–42 and 260–265 with amino acids “A, S, T, G and T, F, K, S, P, E”, respectively, visualized under the 3D representation and the QMEAN coloring scheme in
Figure 3. Their absence did not seem to affect the interaction with TLRs, since no interactions have been reported for these regions, and they do not cover any of the detected epitopes. Even with this absence at the structural level, the superposition of both structures showed an RMSD of 0.068, which also validated the similarity between both models, and only one change was shown at the amino acid level corresponding to the 330 position, which did not seem to affect the alpha-helix structure and is shown in the yellow color in
Figure 3. Finally, the analysis of the structure with the Ramachandran graphs showed an overall value of 96.49% compared to an ideal of 98%; however, this value can be explained by the absence of the mentioned regions, which would be missing the amino acids of the model, among them, a glycine that due to its flexibility could contribute to a theoretical conformation, which results in a steric hindrance, and according to the MolProbity score, an overall value of 0.93 is indicated, and that the closer to zero, but lower than the template resolution (2.40 Å), would be a better value.
The analysis of the physicochemical properties indicated an expected molecular weight as well as hours of half-life in different organisms and a stability of 39.77, both being ideal for purification processes [
35,
57]. Regarding the immunogenic characteristics, an expected cytoplasmic location and a Vaxign-ML score of 91.7 were obtained, indicating the probability of the enolase consensus to be a good vaccine antigen based on the data banks of the experimentally tested immunogens [
36]. The predicted epitopes for MHC-I and -II resulted in more than 1500 initial combinations for both cases using the reference HLAs for the Latin American population: initially choosing those with a
p-value ≤ 0.01, then discarding them for their representativeness in HLA supertypes, and finally, for the presence of proteasomal cleavage sites in the case of MHC-I. The results were six epitopes for MHC-I and seven epitopes for MHC-II, which are shown in linear form in
Figure 4. These results indicate that three MHC-II epitopes overlapped with regions of the MHC-I epitopes, which could help to generate a complete humoral and cellular immune response. Epitopes for B cells were also identified by linear and structural analysis, where both showed five regions with a high probability of being recognized by antibodies, and also showed splicing in three regions with one or more epitopes corresponding to HLAs. To have a better perspective and identification of the epitopes for MHC-I and -II as well as those corresponding to B cells, 3D visualizations were performed through the UCSF Chimera program, highlighting the respective regions shown in
Figure S3, where it can be appreciated that most of them are exposed in the structure of the enolase consensus, which would facilitate their recognition. The analysis by Vaxign 2.0 also showed a world map with the percentage of predicted protection for each country or region based on the reference HLAs used, the epitopes of the modeling protein that can bind to the MHC-I or -II of T cells, and the information available in the IEDB database. Regarding this, good protection was observed, mainly for a large part of Latin America, with most of its regions showing 100% predicted coverage. The analysis of the immunogenic properties and epitope prediction performed by Ong E. et al., (2020) [
58] showed a review of several SARS-CoV-2 vaccines in development through the Vaxign platform, demonstrating Vaxign-ML values >90 for three proteins, while another study carried out by Khan M. et al., (2021) [
59] showed the obtention of 18 epitopes through the IEDB platform focused on specific populations according to their HLA. These platforms are par excellence the most widely used in the design and research of new antigens; however, the recent development of the Vaxign 2.0 platform combines the two previous platforms showing values validated by both servers.
Once the regions of interest were defined, we proceeded to perform a molecular docking analysis between the modeling protein and TLR2 and TLR4, choosing the best docking based on the docking score (
Figure 6 and
Figure 7). These assays yielded values of ΔG: −18.1 and Kd: 5.0 × 10
−14 for TLR2 as well as ΔG: −18.6 and Kd: 2.5 × 10
−14 for TLR4. An analysis of molecular docking performed by Saha R., et al., (2021) [
60] reported values for ΔG of up to −19.9, similar to those obtained in this work; these data suggest a high probability of binding between the receptor and the modeling enolase protein; therefore, a probable activation of the innate immune response at the level of membrane-associated receptors could be happening, which could contribute to the elimination of the parasite in the host through the production of proinflammatory cytokines such as TNF-α and IFN-γ, which in turn can stimulate the production of reactive nitrogen species [
61,
62].
Finally, the analysis performed with PDBsum showed more detailed information and diagrams of the interactions between both proteins, showing only nine salt-bridge interactions between chain A (enolase consensus) and chain C (TLR2). In the second analysis, three salt-bridges, eight hydrogen-bridges, and 257 weak bonds between chain A and chain B (TLR4) were shown. This detailed analysis of the bonds in the docking quantitatively and qualitatively describes the binding, being especially high for TLR4, with numerous electrostatic and van der Waals type interactions. This information reinforces the probable interaction necessary to activate the receptors since these interactions are fundamental for the stabilization of the protein complex, which leads to an innate immune response that can activate an immediate adaptive immune response [
63,
64].
Of the two protein chimeras that could be proposed as potential mRNA vaccines, the first one, with epitopes for MHC-I, can be seen in
Figure 8, consisting mainly of a type 0 cap that helps to increase immunogenicity and contains a Kozak sequence to facilitate recognition and thus translation initiation. Then, the coding DNA sequence or CDS region contains the epitopes separated by a GGGS connector indicated by its flexibility in epitopes of up to nine amino acids. There are also two UTR regions flanking the chimera with the coding genes for β-globin and α-globin, which helps to generate adequate stability and increase their translation [
65,
66,
67]. In the case of the second chimera, the same structure was proposed, except for the epitopes; in this case, they corresponded to MHC-II, but due to their length, some of them overlapped with epitopes for MHC-I, as shown in
Figure 9, separated by GPGPG connectors that provide greater flexibility, which is necessary in longer epitopes. The design of both chimeras was intended to be in a general way, since further in silico evaluations are needed to propose a more detailed structure; however, the epitopes proposed here show considerable potential for further study and proposal as a vaccine. The design of both protein chimeras containing the most representative epitopes of the enolase consensus was performed manually, since it is a proposal based on structures designed and validated by different authors such as Saadi M. et al., (2017) [
68]; Srivastava S. et al., (2018) [
69]; Michel-Todó L. et al., (2019) [
54]; He J. et al., (2020) [
70]; Khan M. et al., (2021) [
59]; and Saha R. et al., (2021) [
60] following recommendations focused on the use of fusion peptides, stabilizing genes, and translation enhancers as well as the use of the zero-type cap to enhance the immune response. The proposed MHC-I/MHC-II proteins’ chimeric constructs proved to be antigenic and not allergenic or toxic, so its use in assays as immunogens is possible. On the other hand, the modeling of these protein chimeric constructs was carried out with the AlphaFold server, which has proven to be the best method for modeling compared to others and generates results very similar to those obtained by crystallography [
51].
Although the analyses with bioinformatics tools by these different research groups are not exclusive to T. cruzi, they cover other models and validate their use; this is an important approach in the development of vaccines and the search for new antigens, which need to be complemented through research and validation in vitro and in vivo.
The ideal type of immune response for
T. cruzi elimination in humans is not yet fully elucidated, but several studies have suggested a Th1 immune response as fundamental for parasite elimination [
71] as well as a humoral immune response that contributes to the production of proinflammatory cytokines such as TNF-α, IL-2, and IFN-γ [
72], which in turn stimulate the production of reactive oxygen species [
71] as well as the production of antibodies for the elimination of the parasite in the bloodstream [
72]. The results obtained in this work identified the selected epitopes and in particular, the modeling consensus of enolase, as serious candidates to provide protection against Chagas disease through the activation of a Th1/Th2 immune response, presenting so far, the ideal theoretical immunogenicity against almost all known strains of
T. cruzi. However, further complementary in silico, in vitro, and in vivo studies are required to confirm that the proposed enolase consensus or chimeric peptides can confer immunity against
T. cruzi infection.