Bioinformatics-based Design of a Multi-epitope Chimeric Vaccine Candidate Targeting Toxoplasma gondii

1. INTRODUCTION

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that belongs to the phylum Apicomplexa. This parasite has been reported in a wide range of hosts (warm-blooded and cold-blooded animals) [1, 2]. Tachyzoites, bradyzoites (found in tissue cysts), and sporozoites (found in oocysts) are the three main stages in the parasite's life cycle. Tachyzoites are involved in the acute phase of infection, and bradyzoites are associated with the chronic phase [3]. Infection in humans occurs through consumption of raw or undercooked meat containing cysts, ingestion of food or water contaminated with oocysts, or through the placenta during pregnancy [1, 4]. In healthy people, the infection is usually asymptomatic. However, in pregnant women, the parasite can cross the placenta and be transmitted to the fetus [5]. Infection can have dangerous consequences in patients with human immunodeficiency virus, organ transplant recipients, and cancer patients [6]. Moreover, this parasite causes significant economic losses in livestock, especially sheep and goats, worldwide [7].

The drugs used to treat toxoplasmosis are a combination of pyrimethamine and sulfonamides such as sulfadiazine [8]. However, these drugs are only effective on tachyzoites and often have various side effects such as hypersensitivity, hematotoxicity, teratogenicity, and allergic reactions [9, 10]. Given these limitations, the best option is to develop an effective vaccine to control human and animal toxoplasmosis. Recent advances in immunoinformatics have enabled the prediction of T and B-cell epitopes and the identification of potential vaccine targets. Compared to traditional vaccine design, in silico approaches offer notable advantages, including enhanced efficiency, reduced cost, and shortened time of experimental stages. Vaccine efficacy remains limited due to the complex life cycle of T. gondii, which includes tachyzoite, bradyzoite, and sporozoite stages and multiple antigens. Therefore, multi-epitope vaccine approaches that target antigens from different stages of parasite development may provide greater and more potent protection against toxoplasmosis [11]. Among the T. gondii antigens involved in protective immunity, some of the Surface Antigens (SAGs), Microneme Proteins (MICs), Rhoptry Proteins (ROPs), and Dense Granule Antigens (GRAs) are considered potential antigen targets. GRA1 is secreted by tachyzoites, bradyzoites, and sporozoites and contributes to parasite survival within the parasitophorous vacuole [12]. SAG1, a dominant surface antigen of tachyzoites, elicits both humoral and cellular immune responses [13]. ROP18, a rhoptry kinase, plays a key role in virulence and intracellular survival [14, 15], while MIC13 is involved in host cell adhesion and parasite dissemination through the intestinal epithelium [16]. Given these roles, the present study sought to design a chimeric protein vaccine candidate containing immunodominant fragments of the GRA1, SAG1, ROP18, and MIC13 antigens.

2. MATERIALS AND METHODS

3. RESULTS

3.5. Prediction and Analysis of Secondary and Tertiary Structures

The 442-residue GSRM protein contains 41.18% alpha-helices, 42.53% random coils, and 16.29% extended strands (Fig. 1A and B). Typically, a high proportion of α-helices (> 40%) and balanced coil content support proper folding and structural stability. Tertiary structure modeling yielded a C-score of -2.21 (Fig. 2A and B), which falls within the acceptable range of -5 to 2 for I-TASSER models.

3.6. Tertiary Structure Validation

Ramachandran plot analysis via SWISS-MODEL showed that 84.90% of the residues were located in the desired regions, 10.41% in the allowed regions, and 4.69% in the outlier regions (Fig. 3). When more than 90% of the residues are located in the favorable and allowed regions and less than 5% are in the outlier regions, the GSRM has acceptable stereochemical quality.

3.10. Prediction of mRNA Secondary Structure

The best-predicted structure had ΔG = −387.70 kcal/mol, and the first 10 nucleotides at the 5′ end did not form a stable hairpin or pseudoknot, supporting efficient ribosome binding and translation initiation (Fig. 4). These results indicate that the optimized GSRM transcript is suitable for robust expression in a prokaryotic system.

4. DISCUSSION

The ability of T. gondii to evade the host immune response, its complex life cycle, and its antigenic diversity remain a challenge in the development of vaccines against this parasite. Therefore, this study aimed to develop a chimeric protein vaccine containing B- and T-cell epitopes of GRA1, SAG1, ROP18, and MIC13 antigens of T. gondii and analyze various aspects of the chimeric protein using different bioinformatics tools.

In the in silico approach, any antigen can be a vaccine candidate. In this study, a variety of antigens (surface antigens such as SAGs and secretory antigens such as GRAs, ROPs, and MICs) were used for vaccine design. GRA1, SAG1, ROP18, and MIC13 were selected based on their roles in parasite survival, invasion, and pathogenicity. In addition, the aim was to select antigens that are expressed in all three stages of the parasite life cycle to elicit a more comprehensive immune response, as stage-specific antigens typically provide limited protection. On the one hand, the immune response generated against the combination of epitopes of these antigens is much more effective than the immune response against a vaccine that consists of only one antigen and stimulates an immune response against that single antigen [11, 14, 24]. Recent advances in the field support the rationale behind our design. For example, Nayeri et al. (2025) performed an in silico analysis of T. gondii surface and secretory proteins. They designed a novel chimeric vaccine and demonstrated a stronger immune response elicited against a combination of multiple antigenic epitopes [25]. Ghaffari and Rahimi (2024) constructed a multi-epitope peptide vaccine based on calcium-dependent protein kinases using immunoinformatics; their analyses suggested favorable antigenicity, solubility, and safety. Therefore, it becomes necessary to conduct experimental tests [26]. Similarly, another recent in silico study used tachyzoite-specific SAG1-Related Sequence (SRS) proteins to design a peptide vaccine containing T-cell and B-cell epitopes that showed high antigenicity, non-allergenicity, and predicted interaction with human TLR4 [27]. Moreover, a genome-wide epitope vaccine study by Li et al. (2022) evaluated T. gondii membrane proteins and identified candidate epitopes for broad coverage, underscoring the power of comprehensive bioinformatics strategies [28]. In contrast, the present study introduces several methodological innovations. Rather than stitching together minimal epitopes, we selected antigenic fragments of approximately 100 amino acids from four antigens (GRA1, SAG1, ROP18, and MIC13), generated all 24 possible permutations of these fragments using the rigid A(EAAAK)A linker, and carefully screened the resulting structures for stability, folding quality, antigenicity, Ramachandran analysis, and physicochemical profile. This vaccine design strategy preserves the structural elements of the original antigens and could create a more stable and immunogenic vaccine candidate.

The selection of antigenic fragments was performed using rigorous epitope prediction analyses targeting B and T cell epitopes, ensuring that the designed construct could effectively stimulate an immune response. This is particularly important given the intracellular nature of T. gondii and the need for strong cellular immunity alongside humoral immunity. Notably, the A(EAAAK)A linker sequence used between the antigenic fragments has previously been shown to promote proper folding and functional independence of the individual domains, minimizing potential steric hindrance or epitope masking [29]. These design considerations are essential to ensure that the epitopes remain structurally accessible and immunologically active.

Our structural predictions showed that the GSRM protein has a balanced secondary structure, with a significant proportion of alpha-helices and random coils, which are favorable for stable folding and antigen presentation. Tertiary structure modeling revealed a moderate C-score (-2.21), indicating a relatively reliable Three-Dimensional (3D) conformation. Ramachandran plots confirmed the tertiary structure of the protein. The results showed that more than 90% of the residues were located in the favorable and allowed regions.

Moreover, immunoinformatic analyses predicted that the GSRM is antigenic (VaxiJen score: 0.6701) and nonallergenic, addressing two primary prerequisites for a safe and effective vaccine candidate. The pI is also one of the important physicochemical parameters that can be used to estimate the solubility of a protein at a certain pH, meaning that the protein precipitates in a solution close to the pI [24]. The pI is useful for establishing a suitable buffering system for protein purification based on pH-focused methods [24]. The presence of an acidic isoelectric point and a negative GRAVY score (-0.316) further indicates that the protein is likely to be hydrophilic and accessible to the host immune system, which is advantageous for immunogenicity. Although the instability index slightly exceeded the threshold for stability, the predicted solubility and favorable antigenicity scores suggest the protein remains a viable candidate for vaccine development. A MW (47953.46 Da) indicates that GSRM could be a suitable antigen (it has been indicated that antigens with a MW of < 5–15 kDa do not provide good immunity) [18] and an aliphatic index (80.11) indicative of good thermostability.

Codon optimization and mRNA secondary structure analysis are important steps in establishing a link between in silico vaccine design and practical protein production. High-throughput expression of antigen in E. coli facilitates purification and experimental testing. Codon optimization reduces the likelihood of ribosomal stalling, and stable mRNA folding, and the absence of inhibitory structures at the 5' end ensures translation of the chimeric protein.

This study is based exclusively on in silico analyses, which, although powerful for screening candidates, cannot fully simulate the complexity of immune responses in living systems. Structural predictions, antigenicity scores, allergenicity assessments, and solubility estimates are computational approximations that require experimental validation. Furthermore, epitope prediction tools vary in accuracy, and the selected fragments may behave differently when expressed as a recombinant protein in vitro or in vivo. The instability index suggests that the final construct may require stabilization strategies during expression and purification. Additionally, the immunogenic performance of the chimeric protein cannot be inferred without testing in animal models, including evaluation of cellular immunity, antibody responses, and assessing survival rates in mice infected with different strains. Therefore, the findings here should be considered preliminary and require experimental testing.

CONCLUSION

In conclusion, the bioinformatics-driven design of the GSRM protein represents a promising step towards the development of an effective multiepitope vaccine against T. gondii. By combining multiple antigens from different stages of the parasite's life cycle, this construct aims to overcome the limitations of single-antigen vaccines and provide broad, cross-stage protection. This work will provide a solid foundation for further experimental studies and help control toxoplasmosis in humans and animals.

REFERENCES