Designing a live vaccine against cutaneous leishmaniasis encoding sandfly salivary proteins and T2A peptide by immunoinformatics and homology modeling
Designing a live vaccine against cutaneous leishmaniasis encoding sandfly salivary proteins and T2A peptide by immunoinformatics and homology modeling
Mahya Sadat Lajevardi,1Tahereh Taheri,2Elham Gholami,3Negar Seyed,4Sima Rafati,5,*
1. department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran 2. department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran 3. department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran 4. department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran 5. department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran
Introduction: Intracellular parasites of the Leishmania genus are deposited in the host skin through sandfly bite along with sandfly saliva. Salivary proteins alone or in combination with parasitic antigens are promising candidates for vaccine formulation against Leishmaniasis. Herein, two immunogenic salivary proteins (PpSP15 from Ph. papatasi and PsSP9 from Ph. sergenti) were analyzed at RNA and protein level for the best combination with or without the linker to be further cloned in L. tarentolae as a live vaccine.
Methods: All possible combinations of PsSP9 and PpSP15 with or without T2A linker (PpSP15-T2A-Ppsp9, PpSP9-T2A-Ppsp15, PpSP15-Ppsp9, and PpSP9-Ppsp15) were designed at mRNA and protein levels. At the mRNA level, the transcript synthesized in L. tarentolae was estimated using trans-splicing. Then, the secondary structures of the mRNAs were predicted by the RNAfold web server. At the protein level, the 3D models of all combinations were generated by I-TASSER and refined by GalaxyRefine servers, and then validated by different parameters. Validated models were superimposed to original proteins using UCSF Chimera. The combined structures were also further analyzed for junctional T-cell epitopes and population coverage of the selected vaccine construct by immunoinformatics. The vaccine candidate was selected based on mRNA and protein stability results besides peptide analysis and physicochemical properties were qualified by ProtParam.
Results: At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSp9. At the protein level, all refined 3D models of the four combinations were structurally valid however local quality estimation showed that PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position with high superimposition quality to both original proteins. Local quality estimation of dissociated proteins followed T2A auto-cleavage (PpSP15-T2A and PsSp9-His tag) also indicated that T2A sequence or His-tag at the C-terminus of dissociated components neither affects the superimposition result nor generates new junctional epitope. The selected combination (PpSP15-T2A-PssP9) and the 2 resulting proteins (PpSP15-T2A and PpSP9-His-tag) were hydrophobe, immunogen, non-allergen and stable. Stable cloning of the selected combination in L. tarentolae resulted in successful protein production.
Conclusion: Immunogenic proteins from sandfly saliva can be successfully combined with virus-derived self-cleaving 2A peptides for live vaccine development.