RESUMEN
BACKGROUND: The highly expressed surface antigen 1 (SAG1)-related sequence (SRS) proteins of T. gondii tachyzoites, as a widespread zoonotic parasite, are critical for host cell invasion and represent promising vaccine targets. In this study, we employed a computer-aided multi-method approach for in silico design and evaluation of TgVax452, an epitope-based candidate vaccine against T. gondii tachyzoite-specific SRS proteins. METHODS: Using immunoinformatics web-based tools, structural modeling, and static/dynamic molecular simulations, we identified and screened B- and T-cell immunodominant epitopes and predicted TgVax452's antigenicity, stability, safety, adjuvanticity, and physico-chemical properties. RESULTS: The designed protein possessed 452 residues, a MW of 44.07 kDa, an alkaline pI (6.7), good stability (33.20), solubility (0.498), and antigenicity (0.9639) with no allergenicity. Comprehensive molecular dynamic (MD) simulation analyses confirmed the stable interaction (average potential energy: 3.3799 × 106 KJ/mol) between the TLR4 agonist residues (RS09 peptide) of the TgVax452 in interaction with human TLR4, potentially activating innate immune responses. Also, a dramatic increase was observed in specific antibodies (IgM and IgG), cytokines (IFN-γ), and lymphocyte responses, based on C-ImmSim outputs. Finally, we optimized TgVax452's codon adaptation and mRNA secondary structure for efficient expression in E. coli BL21 expression machinery. CONCLUSION: Our findings suggest that TgVax452 is a promising candidate vaccine against T. gondii tachyzoite-specific SRS proteins and requires further experimental studies for its potential use in preclinical trials.
Asunto(s)
Antígenos de Protozoos , Biología Computacional , Epítopos de Linfocito T , Proteínas Protozoarias , Vacunas Antiprotozoos , Toxoplasma , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Toxoplasma/inmunología , Toxoplasma/genética , Toxoplasma/química , Vacunas Antiprotozoos/inmunología , Vacunas Antiprotozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/química , Animales , Ratones , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Femenino , Anticuerpos Antiprotozoarios/inmunología , Ratones Endogámicos BALB C , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/química , Humanos , Simulación de Dinámica Molecular , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/química , Toxoplasmosis/prevención & control , Toxoplasmosis/inmunología , InmunoinformáticaRESUMEN
Human toxoplasmosis is a global public health concern and a commercial vaccine is still lacking. The present in silico study was done to design a novel vaccine candidate using tachyzoite-specific SAG1-realted sequence (SRS) proteins. Overlapping B-cell and strictly-chosen human MHC-I binding epitopes were predicted and connected together using appropriate spacers. Moreover, a TLR4 agonist, human high mobility group box protein 1 (HMGB1), and His-tag were added to the N- and C-terminus of the vaccine sequence. The final vaccine had 442 residues and a molecular weight of 47.71 kDa. Physico-chemical evaluation showed a soluble, highly antigenic and non-allergen protein, with coils and helices as secondary structures. The vaccine 3D model was predicted by ITASSER server, subsequently refined and was shown to possess significant interactions with human TLR4. As well, potent stimulation of cellular and humoral immunity was demonstrated upon chimeric vaccine injection. Finally, the outputs showed that this vaccine model possesses top antigenicity, which could provoke significant cell-mediated immune profile including IFN-γ, and can be utilized towards prophylactic purposes. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00140-w.
RESUMEN
Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of "sequences related to SAG1" proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SIGNIFICANCE: To date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.
Asunto(s)
Toxoplasma , Actinas , Membrana Celular , Proteómica , Proteínas ProtozoariasRESUMEN
Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N. caninum using a novel designed plasmid. The coding sequence of NcSRS67 (without the signal peptide and the GPI anchor) was cloned and expressed constitutively instead of the ccdB system of pCR-Blunt II-TOPO. The protein was purified in a nickel sepharose column and identified by mass spectrometry (MS/MS). The constitutive expression did not affect the final bacterial growth, with a similar OD 600nm compared to the non-transformed strains. The recombinant NcSRS67 was over expressed and the native form was detected by the anti-rNcSRS67 serum on 1D western blot as a single band of approximately 38kDa as predicted. On an in vitro assay, the inhibitory effect of the polyclonal antiserum anti-rNcSRS67 was nearly 20% on adhesion/invasion of host cells. The NcSRS67 native protein was localised on part of the surface of N. caninum tachyzoite when compared to the nucleus by confocal immunofluorescence.