HIV infection of transduced MDDCs demonstrates functional effect on HIV infection.

<p>Five days post-transduction with lentiviral vectors ± Vpx VLP as shown, MDDCs were infected with non-pseudotyped (A) or VSV envelope-pseudotyped (B) HIV-1 engineered to express eGFP. Graph bars represent percentage of cells expressing eGFP encoded by HIV on day 3 post-infection. Error bars represent standard deviation among 9 (A) or 4 (B) donors. p values: *p ≤ 0.0286, ***p ≤ 0.0004. (C) MDDCs transduced with scrambled shRNA or CD4 targeting shRNA (but not eGFP) expressing lentiviral vectors, were infected on day 5 post-transduction with HIV engineered to express eGFP. Graph shows day 3 infection rates in MDDCs obtained from two donors.</p

Method overview.

<p>Transduction workflow as described in the Materials and Methods section.</p

MDDCs can be transduced at high efficiency in the absence of Vpx.

<p>Cells were transduced with a lentiviral vector encoding eGFP and a scrambled shRNA sequence 24 hours post-isolation from PBMCs in the absence or presence of Vpx-containing VLP, and analyzed by flow cytometry 6 days post-isolation. The top panel shows MDDC gating strategy. (A) Cells were gated based on their forward scatter (FSC) and side scatter (SSC) profile. (B) Cells gated as in (A) were subsequently gated to exclude dead cells from further analysis by setting a gate on live (propidium iodide (PI)-negative) cells. (C) Bottom dot plots show eGFP expression versus DC-SIGN-PE on MDDCs, transduced or not, as indicated. Percentages indicate fraction of eGFP expressing cells.</p

Vpx-independent, shRNA-mediated knock-down of SAMHD1 in MDDCs.

<p>(A) Histogram shows intracellular SAMHD1-Alexa 660 staining at similar time point after isolation as shown in B, without ① or with ② treatment with Vpx VLP. Control histogram represents cells stained only with secondary, goat-anti rabbit Alexa 660 antibody ③. (B) Histogram shows intracellular SAMHD1-Alexa 660 staining in MDDCs, 5 days post-transduction with scrambled shRNA (sh-scrambled) ④, or sh-SAMHD1 vectors ⑤ as indicated. Histogram depicting SAMHD1 levels in not transduced cells was overlaid for comparison ①. (C) The bar graph shows SAMHD1 down-regulation in MDDCs from 5 independent donors transduced similar to panel B with or without the addition of Vpx VLP. Error bars represent standard deviation among donors, comparison to sh-scrambled **p ≤ 0.008.</p

Transduced MDDCs do not mature per se, but retain the potential to undergo maturation.

<p>MDDCs were exposed to LPS (filled profiles) or not (open profiles) starting 4 days post-isolation. Histograms show CD80 APC, CD83 PE and CD86 APC surface staining 48h later in control non-transduced, non-transduced but exposed to Vpx VLPor shRNA-scrambled transduced cells as indicated. Numbers represent mean fluorence intensity.</p

Intra- and inter-run variation of the SG-PERT assay.

<p>(A) Standard curve composed of a pre-made six 10-fold serial dilution series of replication-competent HIV-1 containing supernatant measured in 12 independent SG-PERT experiments. For each experiment obtained Cq values are plotted versus the RT activity in each sample. RT activity values were determined by running a dilution series of recombinant HIV-1 RT in parallel. Standard deviation on the obtained crossing point values is indicated for each dilution. (B) RT activity values obtained for 8 repeated measurements of different HIV-1 samples (sample number 1 to 6) within the same run. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate intra-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). (C) RT activity values obtained for different HIV-1 samples (sample number 1 to 11) in at least 3 independent SG-PERT experiments. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate inter-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). Experiments were performed on the LightCycler® 480.</p

Sensitivity and specificity of the SG-PERT assay.

<p>(A) Melting curves of PCR products obtained by SG-PERT assay on the LightCycler® 480 when using 10¹¹ or 10⁴ pU recombinant HIV-1 RT or nuclease-free water (non-template control = <i>NTC</i>) as input for the assay, as indicated. (B, D) Amplification curves of indicated amount of (B) recombinant HIV-1 RT (pU), (D) replication competent HIV-1 (NL4-3 strain) (ng p24/mL) or nuclease-free water (<i>NTC</i>) obtained by SG-PERT on the LightCycler® 480. (C,E) Relation between input of (C) recombinant HIV-1 RT, (E) replication competent HIV-1 (<i>HIV-1</i>), HIV-1 based lentiviral vectors (<i>HIV-1 based vector</i>) or Moloney Murine Leukemia-based retroviral vectors (<i>MoMLV</i>) and obtained cycle of quantification (Cq) values by SG-PERT on the LightCycler® 480. Viral titers in the undiluted samples in (E) (value of “0” on x-axis) were 3,100 ng p24/mL for the replication competent HIV-1 virus, 1.12×10⁷ transducing units/mL (TU/mL) for the HIV-1 based viral vector and 4.9×10⁵ TU/mL for the MoMLV-based vector. Only input levels within linear range of the assay were included for correlation analysis.</p

SG-PERT assay on ABI 7300 Real-Time PCR System.

<p>(A) Amplification curves of indicated amount of replication competent HIV-1 (NL4-3 strain) (ng p24/mL) obtained by SG-PERT on the ABI 7300 instrument. The horizontal line represents the threshold line used to calculate Cq values. (B) Correlation between input levels of replication-competent HIV-1 virus and Cq values obtained by SG-PERT on the ABI 7300 instrument. p24 antigen concentration in the undiluted samples (value “0” on x-axis) was 3,100 ng/mL.</p

Evaluation of different lentiviral titration methods.

<p>Table shows SG-PERT RT activity measured on the LightCycler® 480 and p24 antigen concentration in different productions of replication-competent HIV-1 virus supernatant (HIV-1, sup 1–11) and replication-incompetent HIV-1-based MISSION® lentiviral vectors (LV, sup 1–7). TU/mL: transducing units/mL (only determined for lentiviral vectors). The number of viral particles (#VP) was calculated from p24 values by assuming 12 viral particles per fg p24. For viral particle calculation from RT activity values, an activity of 300 pU per viral particle was assumed.</p

Principle of the SG-PERT assay.

<p>Cell-free retrovirus containing supernatant is lysed and added to a reaction mix containing the MS2 RNA template, MS2 complementary primers and a SYBR Green I qPCR mastermix. During a one-step reaction, the reverse transcriptase (RT) enzymes derived from the retroviral particles will convert the MS2 RNA into cDNA and cDNA is subsequently quantified by qPCR amplification of the MS2 cDNA. The amount of synthesized cDNA represents the level of RT activity in the viral supernatant and is thereby a measure of the amount of retroviral particles.</p