Preparations, suggesting that this DNA was contained within the particles (Fig
Preparations, suggesting that this DNA was contained within the particles (Fig 7B). We utilized genomic DNA to show that DNase I digestion and EDTA inactivation were powerful (Fig 7C). Also, we detected about 150 ng of double-stranded DNA (dsDNA) per microliter and 12 ng of single-stranded DNA per microliter with the concentrated LV preparation (fig. S8A). The DNA extracted from the vector preparation appeared to be mostly composed of fragments of much less than 10 kb, whereas genomic DNA extracted from cells was of longer fragments greater than 10 kb (fig. S8B). Deep sequencing in the DNA extracted from the viral particles demonstrated that about 99 from the reads have been mapped for the human genome, whereas about only 1 from the reads had been mapped to plasmid DNA (fig. S8C). With the reads that were mapped towards the human genome, there appeared to be a random distribution across the human chromosomes (fig. S8D). These final results suggest that the DNA inside LV preparations was predominantly double-stranded, fragmented, human genomic in origin, and incorporated into the viral particles randomly. We also amplified plasmid and human DNA in HIV-1 produced from 293T cell transfection (Fig 7D). To determine regardless of whether the presence of genomic DNA in vector particles was particular towards the transfection procedure, we passaged HIV-1 in human peripheral blood mononuclear cells (PBMCs) and amplified human DNA from the cell-free HIV-1 supernatant (Fig 7E). Human DNA was not detected within the cell-free supernatant collected from uninfected PBMCs. Furthermore, a number of the DNA detected in the passaged cell-free HIV-1 supernatant was resistant to DNase I (Fig 7F), suggesting that human genomic DNA was also encapsulated inside HIV-1 particles. We next questioned regardless of whether the delivery of plasmid or genomic DNA by viral fusion would improve the DC activation generated by viral fusion itself. We treated mouse BMDCs with empty VSV-G liposomes or VSV-G liposomes carrying intact plasmid DNA or genomic DNA extracted from 293T cells. Human genomic DNA enhanced the immunogenicity with the fusogenic liposomes in wild-type BMDCs (Fig 7G), which was abrogated in STINGdeficient BMDCs (Fig 7H). The addition of intact plasmid DNA to fusogenic liposomes did not enhance BMDC activation (Fig 7G). In addition, LVs generated by either transient transfection working with plasmids or plasmid-free packaging method similarly stimulated wild-type BMDCs (Fig 7I), suggesting that plasmid DNA within the vector preparations was not probably a dominant NKp46/NCR1, Mouse (HEK293, Fc) activator of DCs. LVs generated by plasmid DNA ree cell lines capably stimulate innate and adaptive immune responses in vivo (41, 42). These findings deliver anSci Immunol. Author manuscript; readily available in PMC 2018 March 10.Kim et al.Pageexplanation for the STING and cGAS dependence observed in the innate and adaptive immune responses generated by LVs and VLPs.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptDISCUSSIONIn the present investigation, we discovered that vector-encoded protein antigen carried by vector particles via pseudotransduction sufficiently delivered antigen and stimulated the immune method. LV transduction was not inherently immunostimulatory but contributed to antigen delivery. Viral envelope ediated fusion itself induced DC activation in a PI3K-dependent but STING- and type I IFN signaling ndependent manner. Last, cellular DNA packaged from producer cells carried by particles activated the host STING and cGAS SAA1 Protein MedChemExpress pathway. Our final results sugge.
