P. falciparum, the most lethal form of malaria, is the leading cause of infection-related death. Malaria mortality is a global health and economic concern, despite effective treatments. This is related to an increase in P. falciparum's resistance to first-line antimalarial medicines . As a result, a successful vaccination would be a helpful tool for controlling or even eradicating malaria. The sporozoite stage targets two of the most promising malaria vaccine candidates, although sporozoites are only exposed to the host's immune system for a few minutes. Each cycle in the RBC lasts 2-3 days, but the trophozoite remains undetectable since it is concealed within the cell. The trophozoite exports antigens to the RBC membrane's outer surface and these antigens could be identified . Plasmodium conducts numerous cycles of RBC infection, giving infected people time to develop antibodies that identify these proteins. They are temporarily exposed to the host immune response at this phase, making them accessible to antibodies that target their antigens on the surface [30, 31]. Because the parasite is directly exposed to the host humoral immune response during the erythrocytic stage, making it an appealing vaccine target. The PfGARP gene is only produced during the early stages of the parasite life cycle, and immunolocalization experiments revealed that PfGARP is found on the outer surface of trophozoite-infected RBC. In the absence of immune effector cells or complements, Anti-PfGARP inhibits parasite growth by blocking and destroying trophozoite-infected RBCs . Vaccine development and production are often lengthy, challenging, and costly processes. Researchers can benefit from in silico methodologies for the rational design of vaccines, particularly for pathogens, because of advancements in molecular immunology and the identification of immune-dominant epitopes, and progress in bioinformatics and immune informatics approaches . Accordingly, in the present study, we use bioinformatics methods to design a potential candidate epitope-based vaccine against malaria based on the PfGARP protein, one of the major antigenic proteins of blood-stage malaria . For this purpose, we first selected PfGARP as the target antigenic protein for further analysis. T helper cells, which are necessary for almost all adaptive immune responses, are the principal mediators of cell-mediated immunity. They aid in activating B cells, which release antibodies and macrophages, and cytotoxic T lymphocytes, which kill infected target cells . Additionally, in the following section, we predicted probable B-cell and T helper cell epitopes from the PfGARP protein in order to develop a multi-epitope vaccine (MEV) capable of inducing a humoral response. Then, to generate the MEV, the predicted epitopes (B-cell and HTL epitopes) were fused using suitable linkers (AAY and GPGPG linkers) as specialized spacer sequences. The AAY linkers play a role in increasing epitope presentation and removing junctional epitopes. In the about of GPGPG linkers, these types of linkers can cause stimulate T-helper responses and conformational dependent immunogenicity of helper and antibody epitopes [34–36]. In this study, to compensate for these vaccines' low immunogenicity, the D0/D1 domains of the flagellin protein from S. Typhimurium bacteria were utilized as an adjuvant to boost the effectiveness of immune system stimulation . Also, The EAAAK linker was employed to connect the D0/D1 domains at the N-terminal region of the multi-epitope sequence. For effectively separating the adjuvant interference with protein segments, the EAAAK linker helps to reduce interruption and increases the degree of expression and bioactivity of the target fusion protein. Finally, a candidate vaccine with a length of 414 amino acids, including some linear B-cell and HTL epitopes fused to the adjuvant sequence, was constructed. MEV was predicted to be antigenic with the probability of antigenicity 0.926395 and non-allergenic. This means that our MEV potentially produces a strong immune response without an allergic reaction, making it a potent vaccine. The structure's physicochemical properties were examined: The MEV construct has a molecular weight of 46 kDa. MEV has a theoretical pI value of 5.03, indicating that it is acidic. An extinction coefficient index for a chemical can be explained as the amount of light absorbed at a specific wavelength. The build has an extinction coefficient of 35995M-1 cm-1. The candidate vaccine protein had an instability index (II) value of 40.11, indicating that our protein construct is relatively a stable protein (II >40 implies instability). The aliphatic index measures how much space aliphatic amino acids (alanine, valine, isoleucine, and leucine) take up in the side chains of proteins. It could be viewed as a beneficial factor in increasing globular protein thermostability. The construct had an aliphatic index of 65.12, indicating that it was a thermostable protein. The GRAVY (Grand Average of Hydropathy) value for a peptide or protein is calculated by adding the sum of the hydropathy values of all amino acids by the number of residues in the sequence. Positive and negative values, respectively, represent the hydrophobic and hydrophilic qualities of a substance. The GRAVY value of our suggested construct was -1.110, indicating that it is a hydrophilic protein . The half-life is a forecast of how long it takes for half of the protein in a cell to vanish after production. ProtParam tool predicted the half-life of our construct in the following; 30 hours (mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo) and >10 hours (Escherichia coli, in vivo). For many biochemical and functional evaluations of recombinant proteins, the solubility overexpression in the E. coli host is one of the requirements that help in the efficient purification process in later stages . Here, the solubility upon overexpression of our multi-epitope protein construct predicted (by SOLpro server) with probability 0.759117, which indicates the overexpression of our multi-epitope protein in E. coli, insoluble form.
The PSIPRED method was used to analyze the secondary structure of our protein construct, which revealed that it mainly was alpha-helical (60 percent), 39 percent coils, and 0.4 percent of the amino acids in strand formation. Various servers got the vaccine constructs tertiary structure (SWISS-MODEL, phyre2, and I-TASSER). ProSA-web, RAMPAGE, and ERRAT servers were used in the validation process to identify potential faults and improve the quality of the projected 3D model. Based on validation data, the selected model had a high validation score and did not require further refinement. According to the Ramachandran plot, most residues are favored (96.0%) and 4.0% in permitted regions. This proposal used the Cluspro server to test the immunological interaction between the designed MEV construct and the TLR5 receptor. Cluspro presented dozens of docked models scored based on protein surface hydrophobicity, geometry, and electrostatic complementarity. Between the hydrophobicity models, we choose the best possible docked model. As a result, the best-docked complex was selected as the docked structure with the lowest energy score (-1353.6).
In this research, CABS-Flex 2.0 software was used for MD simulation. CABS-Flex presents the stable arrangement of the TLR5-designed vaccine complex. The individual amino acid residue's root means square fluctuation (RMSF) values (using CABS Flex 2.0) were described. The highest and lowest RMSF values show that our complicated structure fluctuates more and less during the simulation process, respectively. The MEV's structure fluctuates, indicating its considerable flexibility and validating it as a suitable vaccine structure.
To achieve high-level production and translation efficacy of our multi-epitope protein in E. coli, we used a codon optimization technique (strain K12). The CAI value (0.85) and GC content (46.32%) data were obtained in this study, indicating that the protein vaccine may be expressed more strongly in the E. coli K-12 system [40, 41]. Finally, the MEV sequence was included in the pET-28a vector, which efficiently and effectively encoded the MEV protein in E. coli cells. We urge that validation experiments containing in vitro and in vivo studies be undertaken in the future to develop our candidate vaccine against malaria, based on the findings of this study.