Construction Of MS2 Virus-like Particle Packaging HIV-1 Gag-encoding MRNA For Studying Gene Expresion And Function In Vitro And Immune Response In Vivo

BackgroundGene transfer vehicles based on mRNA have emerged as attractive alternatives to vehicles made of DNA for gene therapy or vaccine, as RNA-mediated gene expression retains the advantages of DNA vaccines without the risk of integration into the host genome or severe side effects such as the generation of autoimmune disease or anti-DNA antibodies. The major limitations on the potency of RNA vaccines are their instability and inability to spread in vivo. Virus-like particles (VLPs) based on the bacteriophage MS2 have demonstrated remarkably high stability and may provide an improved platform for RNA-based genetic vaccination. However, no in vivo study of an MS2 VLP-mediated RNA vaccine has been reported.ObjectiveWe undertook the current study to investigate the potential of MS2 VLPs packaging HIV-1 gag-encoding RNA produced in Saccharomyces cerevisiae as a delivery vehicle for RNA vaccination in mice.Methods1. The construction of ten one-plasmid double-expression systems. The plasmid pMS was constructed by inserting the cDNA sequence encoding MS2 coat protein and maturase into one cloning site of vector pESC-Ura. Then HIV-1 gag encoding sequences with two pac sites was inserted in-frame into the plasmid pMS to generate the recombinant plasmid pMS-GAG. The capsid mRNA packaging vector pMS2C was constructed as controls for pMS-GAG.2. Virus-like particles expression.The recombinant plasmids were transformed into competent Saccharomyces cerevisiae strain YPH499. To produce large stocks of VLPs, individual clones were amplified on synthetic dextrose minimal medium (SD) plates at 30℃, and then cultured in SG medium to induce the expression of the virus-like particles. 3. Virus-like particles purification. The cells were pelleted and then resuspended in sonication buffer and the virus-like particles were extracted with the sonication. The supernatant was incubated overnight with the DNaseI and RNase A at 37℃. Then the virus-like particles were purified by resin exclusion chromatography.4. The verification of the virus-like particles. The purified virus-like particles were analyzed by 1% agarose gel electrophoresis with ethidium bromide staining and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 18% gels to identify pMS-GAG VLPs. The particles were also identified by transmission electron microscopy at 100 kV and 59,000×screen magnification.5. Durability of pMS-GAG VLPs. Resistance to nucleases, plasma stability and freeze-thaw cycles.6. Expression of VLP mRNA products in mammalian cell lines. The purified RNA of pMS-GAG VLPs was transfected into mammalian cell lines by using the transfection reagent DOTAP. After 24 h, the cells were harvested, and cell lysates were subjected to ELISA using the Genescreen Plus HIV Ag-Ab assay.7. Six to eight week-old female BALB/c mice were intramuscularly vaccinated with either 15μg of total protein of purified pMS2C VLPs (as a control) or 15μg of total protein of purified pMS-GAG VLPs (5×1012 RNA copies) resuspended in sterile phosphate-buffered saline (PBS) and emulsified 1:1 in Freund's adjuvant. The mice received 2 boosts with the same amount of MS2 VLPs in Freund's incomplete adjuvant at 3 and 5 weeks after initial vaccination. Mice injected with Freund's adjuvant alone were used as controls. Eight mice were used per group. Seven days after the final immunization, serum was recovered from the mice by tail bleeding and detected anti-Gag antibodies by an end-point dilution ELISA assay.ResultsThe HIV-1 gag mRNA were packaged successfully into VLPs using one-plasmid double-expression systems in Saccharomyces cerevisiae. The MS2 VLPs were completely resistant to DNase and RNase, rather stable in normal human EDTA-preserved plasma at 4℃, and were largely intact after 5 freeze-thaw cycles. The RNA extracted from pMS-GAG VLPs was efficiently translated in all the 3 mammalian cell lines (HeLa, HEK-293, and CHO). Our results revealed that in mice, the MS2 VLP-mediated mRNA delivery system can generate strong antibodies specific for the mRNA-encoded antigens.Conclusions 1. The recombinant plasmid pMS-GAG was successfully constructed by molecular cloning method.The MS2 VLPs, which produced in the Saccharomyces cerevisiae expression system, were successfully identified by 1% agarose gel electrophoresis with ethidium bromide staining, denaturing SDS-polyacrylamide gels and transmission electron microscopy.2. The bacteriophage capsid proteins effectively protected RNAs from RNase. The pMS-GAG VLPs also survived 5 freeze-thaw cycles and were stable at 4℃for at least 4 months. Therefore, the MS2 VLPs produced in Saccharomyces cerevisiae fulfill the stability requirements for a new mRNA transfection vector.3. We successfully produced MS2 VLPs packaging functional HIV-1 gag mRNAs in Saccharomyces cerevisiae. Functionality in eukaryotic systems of packaged mRNAs was confirmed by showing that mRNAs purified from VLPs can be efficiently translated in vitro and in cell cultures. The MS2 VLP-mediated mRNA delivery system can generate strong antibodies specific for the mRNA-encoded antigens.