Preparations, suggesting that this DNA was contained inside the particles (Fig
Preparations, suggesting that this DNA was contained within the particles (Fig 7B). We utilised genomic DNA to show that DNase I digestion and EDTA inactivation were successful (Fig 7C). Furthermore, 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 in the vector preparation appeared to become largely composed of fragments of less than 10 kb, whereas genomic DNA extracted from cells was of longer fragments greater than 10 kb (fig. S8B). Deep sequencing on the DNA extracted in the viral particles demonstrated that about 99 of the reads had been mapped for the human genome, whereas about only 1 of the reads were mapped to plasmid DNA (fig. S8C). On the reads that have been mapped towards the human genome, there appeared to be a random distribution across the human chromosomes (fig. S8D). These benefits suggest that the DNA within LV preparations was predominantly double-stranded, fragmented, human genomic in origin, and incorporated in to the viral particles randomly. We also amplified plasmid and human DNA in HIV-1 created from 293T cell transfection (Fig 7D). To ascertain no matter whether the presence of genomic DNA in vector particles was distinct to the transfection approach, we passaged HIV-1 in human peripheral blood mononuclear cells (PBMCs) and amplified human DNA in the cell-free HIV-1 supernatant (Fig 7E). Human DNA was not detected inside the cell-free supernatant collected from Thrombomodulin, Human (HEK293, His, solution) uninfected PBMCs. In addition, some of the DNA detected within 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 subsequent questioned regardless of whether the delivery of plasmid or genomic DNA by viral fusion would enhance 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 of 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 improve BMDC activation (Fig 7G). Additionally, LVs generated by either transient transfection making use of plasmids or plasmid-free packaging system similarly stimulated wild-type BMDCs (Fig 7I), suggesting that plasmid DNA within the vector preparations was not probably a dominant activator of DCs. LVs generated by plasmid DNA ree cell lines capably stimulate innate and adaptive immune responses in vivo (41, 42). These findings give anSci Immunol. Author manuscript; accessible in PMC 2018 March 10.Kim et al.Pageexplanation for the STING and cGAS dependence observed inside the innate and adaptive immune responses generated by LVs and VLPs.Author INPP5A Protein site manuscript Author Manuscript Author Manuscript Author ManuscriptDISCUSSIONIn the present investigation, we discovered that vector-encoded protein antigen carried by vector particles by way of pseudotransduction sufficiently delivered antigen and stimulated the immune program. 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 form I IFN signaling ndependent manner. Last, cellular DNA packaged from producer cells carried by particles activated the host STING and cGAS pathway. Our results sugge.