Quantitative evaluation of PpSP15-LmSTI1 fusion gene expression following transfection with an alphavirus-derived self-amplifying mRNA and conventional DNA vaccine platforms.

New vaccine platforms are crucial to address complex parasitic infections such as cutaneous leishmaniasis. Self-amplifying mRNA (SAM) based vaccines represent the next generation nucleic acid-based platform. In the present study, we compared the expression levels of PpSP15-LmSTI1 fusion gene in BHK-21 cells following transfection with Semliki Forest virus (SFV)-derived SAM, SFV-derived plasmid DNA (pSFV-PD) and conventional plasmid DNA (pcDNA3.1+). PpSP15-LmSTI1 fusion gene expression levels were evaluated at different time points, using quantitative Real-time PCR. All data were validated and normalized by two internal control genes. According to the results, mean values of relative expression were significantly higher for SFV-PD SAM/fusion than pcDNA/fusion and pSFV-PD/fusion at all concentrations and time points. Our results showed that higher levels of PpSp15-LmSTI1 antigen expression could be achieved using a SAM vector than pcDNA and pSFV-PD, making it a valuable and efficient alternative to conventional plasmid DNA-based vaccines against leishmaniasis.

Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector

Background: Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies. Methods: pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR). Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR. Conclusion: Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.

An alphavirus-derived self-amplifying mRNA encoding PpSP15-LmSTI1 fusion protein for the design of a vaccine against leishmaniasis.

The main aims of the present study were to design a fusion protein of Leishmania major stress-inducible protein 1 (LmSTI1) and Phlebotomus papatasi SP15 (PpSP15), and to express it in the form of alphavirus packaged Self-amplifying mRNA (SAM). Two combinations, PpSP15-LmSTI1 and LmSTI1-PpSP15 fusion forms, were analyzed for folding and minimum free energies of the mRNA. Conformational studies on 3D modeled fusion and native forms were performed, and the Root-Mean-Square-distance (RMSD) of the Cα atomic coordinates were calculated. Antigenicity and stability were predicted using bioinformatics tools. The coding sequences of PpSP15-LmSTI1 fusion, PpSP15, and LmSTI1 were cloned into an alphavirus-based vector and used to produce the SAM constructs. All the subcloned constructs were then subjected to packaging in the form of viral replicon particles (VRPs),and were evaluated for their ability to infect BHK-21 cells and express the recombinant fusion proteins. The in-silico analysis indicated that the PpSP15-LmSTI1 combination could be a promising candidate based on lower folding ΔG of mRNA, higher protein antigenicity and lower instability indexes, and less conformational changes compared to the native proteins and the LmSTI1-PpSP15 fusion form. Packaged SAM encoding fusion and native antigens are used for infection of mammalian cells and for recombinant protein expression. This is the first study on in silico designing and successful packaging of an alphavirus-derived SAM in the form of the VRPs to target leishmaniasis.

In silico study of ligand binding site of toll-like receptor 5.

BACKGROUND: Toll-like receptor-5 (TLR-5) is a member of TLRs family and responsible for bacterial flagellin recognition. The activation of TLR-5 with flagellin leads to initiation of signaling cascades, which in turn results in transcription of pro-inflammatory cytokines. Regarding the critical role of TLR-5 agonists and antagonists in activation of innate immune responses, an increasing number of studies have focused on their therapeutic applications in drug and vaccine design. In this study, to identify the most critical region and residues of TLR-5 for interaction with flagellin, different truncated forms of TLR-5 were designed and subjected to protein-protein interaction studies. MATERIALS AND METHODS: The interactions of the full native TLR-5 and its truncated forms with bacterial flagellin (FliC) were evaluated using Hex docking server and molecular interaction analysis was performed using Dimplot analysis. RESULTS: According to our in silico results, truncated form C (an amino acid sequence containing residues 174-401 of TLR-5) has the most suitable interaction with FliC and seven amino acids within this region were found to be crucial for the interaction with flagellin. CONCLUSIONS: These results provide new insights in to potential drug target sites of TLR-5, which may guide future TLR-5 targeting studies.

In silico and in vivo studies of truncated forms of flagellin (FliC) of enteroaggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections.

The new generation of vaccines against infectious diseases is based on recombinant fusion proteins. Flagellin (FliC) of enteroaggregative Escherichia coli (EAEC) could be considered as a potent adjuvant in designing new vaccines. However, because of its large size, incorporation of this protein with a vaccine antigen might negatively influence recognition of the vaccine epitopes by the immune system. Designing the truncated forms of FliC, capable of inducing innate immune response, enhances the immune responses to the target antigen. We have previously shown that two truncated forms of FliC are able to induce Interleukine-8 production in HT-29 epithelial cell line. In this study we designed recombinant vaccine against urinary tract infections (UTIs) using truncated forms of FliC and type 1 fimbrial FimH adhesin from uropathogenic Escherichia coli (UPEC) and studied their in silico interactions with Toll-like receptor 5 (TLR-5) via docking protocols. The best fusion protein was subjected to cloning and expression. The ability of the recombinant vaccine and the truncated forms in inducing immune responses was investigated. Our results showed that truncated forms are capable of inducing Th1 (forms A and B) and Th2 (form A) responses and fusion vaccine induced strong cellular and humoral immune responses.

Antigenicity and immunogenicity of fused B-subunit of heat labile toxin of Escherichia coli and colonization factor antigen I polyepitopes.

Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I-positive strain which may be due to solvent inaccessibility of epitope residues on intact fimbriae.

In Silico and In Vitro Studies of Truncated Forms of Flagellin (FliC) of Enteroaggregative Escherichia coli (EAEC).

Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and chronic diarrhea worldwide. It has been shown that flagellin (FliC), a major bacterial surface protein of EAEC, causes IL-8 release from certain epithelial cell lines via activation of TLR-5. Based on the ability of this protein to activate innate immunity, flagellin can be considered as a potent adjuvant in new vaccines and adjuvant effects of native or recombinant forms of flagellin have been demonstrated. In the current study we designed various truncated forms of FliC-EAEC based on its interaction site with TLR-5 and assessed the interactions via docking protocols. Then, the most appropriate truncated forms were PCR amplified, cloned in to pGEX-5X-1 plasmid and expressed. Finally, the expressed proteins were tested for pro-inflammatory properties. Our in silico and in vitro results indicated that two truncated forms of FliC- EAEC (amino acids 79-117 and 477-508) effectively interact with TLR-5, thus could be capable of inducing in vivo immune responses.