The GM-CSF genetic adjuvant increases both the magnitude and breadth of mucosal T cell responses elicited in the lungs and guts following PMED HA DNA vaccination.

<p>HA-specific T cell responses in the (<b>A</b>) lung and (<b>B</b>) gut mucosa of macaques were determined by IFN-γ ELISpot assay 4–11 weeks after the final vaccination with HA DNA (solid bars) or HA DNA+GM-CSF (hatched bars). Individual bars represent the peak number of HA-specific IFN-γ T cells detected in the jejunum and lung tissue of individual animals 4–11 weeks after the final vaccination. (<b>C</b>) Breadth of the IFN-γ T cell response in PBMC, lung, and gut. HA-specific T cell responses in the indicated tissues were measured using a standard IFN-γ ELISpot assay with 6 individual pools of overlapping peptides (11 amino acid overlaps, 103 15-mers per pool) comprising the entire amino acid sequence of the influenza A/New Caledonia/20/99 HA protein. The percent contribution of each peptide-pool specific response to the total response was determined by dividing the mean number of IFN-γ spot forming cells (SFC) measured against each individual peptide pool by the sum of the response against all peptide pools. Results represent the average of 2 time-points tested after the 3^rd DNA dose (weeks 19 and 23). *Measurement below positive threshold level for the assay.</p

Induction of mucosal antibody responses in the respiratory tract of PMED HA DNA vaccinated macaques.

<p>Bronchoalveolar lavage fluid (BALF) and tracheal swab samples were collected from macaques prior to immunization and again at 4 weeks following each boosting dose of the HA DNA vaccine, ± rhGM-CSF. IgG antibody responses in BALF (<b>A</b>) and IgA antibody responses in tracheal swabs (<b>B</b>) against the A/New Caledonia/20/99 hemagglutinin protein were detected by ELISA. Data are reported as the O.D. measured at 450 nm for each sample, diluted 1∶20 in PBS, from individual immunized animals.</p

Particle-mediated DNA vaccine delivery into the epidermis of rhesus macaques using the disposable commercial prototype ND10 device.

<p>Plasmid DNA encoding (<b>A</b>) the gene for influenza A/New Caledonia/20/99 haemagglutinin (HA) was administered alone or in combination with (<b>B</b>) a plasmid encoding the gene for rhesus macaque granulocyte-macrophage colony stimulating factor (rhGM-CSF) co-formulated onto 1–3 µM gold particles. (<b>C</b>, <b>D</b>) The ND10 delivery device consists of a cassette containing 2.0 µg plasmid (1.8µg HA DNA+0.2µg GM-CSF DNA) coated onto 1.0 mg of gold particles, a safety catch that is released when the device is held firmly against the skin surface, and an actuation button that breaks the tip off a gas microcylinder and releases helium at high pressure. Release of the helium ruptures the cassette membrane, entrains the DNA-coated gold particles into the helium jet, and propels them directly into cells in the skin. (<b>E</b>) Vaccinations were targeted to the skin located on the upper inner thigh adjacent to the inguinal lymph node. Immediately following vaccination, vaccination sites are easily visualized as red (erythema) targets in the skin. (<b>F</b>) The erythema is transient (24 hours) and vaccination sites faded but were still discernible at 8 weeks post-vaccination. (<b>G</b>) Shown is gold particle penetration into the epidermal and dermal skin layers in a representative histological cross-section of a skin biopsy collected 10 minutes after ND10 delivery.</p

Generation of multifunctional T cells in the peripheral blood and lungs of macaques following PMED HA DNA vaccination.

<p>Intracellular cytokine staining was performed on BALF collected from both vaccine groups at 4 weeks after the first boost (panels on left), as well as PBMCs from both vaccine groups collected 4 weeks after the second boost (panels on right) to measure the expression of IFN-γ, TNF-α, and IL-2 by CD4+ and CD8+ T cells after stimulation with overlapping peptides derived from the A/New Caledonia/20/99 HA protein. Bar charts show the mean total percentage (+/− SEM) of CD4+ and CD8+ T cells in the lungs (<b>A</b>) and blood (<b>B</b>) of animals from the unadjuvanted (black bars) and GM-CSF adjuvanted (hatched bars) found to express IFN-γ, TNF-α, or IL-2 following HA peptide stimulation. Indicated P values were determined using the Mann-Whitney U test (two-tailed). Stacked bar charts show the mean proportion of cells producing IFN-γ (black), TNF-α (gray), or IL-2 (hatched) to the total HA-specific CD4+ and CD8+ T cell response detected in the lung (<b>C</b>, <b>E</b>) and blood (<b>D</b>, <b>F</b>) of animals from the unadjuvanted and GM-CSF adjuvanted vaccine groups. Pie charts show the proportion of HA-specific CD4+ and CD8+ T cells in the lung (<b>G</b>) and blood (<b>H</b>) from both vaccine groups positive for the different combinations of one, two, or three cytokines.</p

LT-adjuvanted DNA vaccine increases T cells with multiple effector functions in the blood.

<p>SIV-specific T cells with 1–4 cytokine (IFN-γ, TNF-α, IL-2) or cytolytic (CD107a) effector functions were measured in PBMC 14 weeks after ART was withdrawn (week 58) by flow cytometry following <i>in vitro</i> stimulation with overlapping peptide pools derived from genes included in the DNA vaccine (RT, Nef, Gag, Env). Boolean gating was performed to identify the total frequency of CD4+ and CD8+ T cells in the blood of ART responders with any one of 1–4 effector functions. (<b>A</b>) Cumulative mean (±SEM) frequency of SIV-specific CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, IL-2 or CD107a. Cumulative mean frequency (± SEM) of SIV-specific (<b>B</b>) CD4+ and (<b>C</b>) CD8+ T cells co-expressing any combination of 2–4 effector functions. Mean (± SEM) frequency of SIV-specific (<b>D</b>) CD4+ and (<b>E</b>) CD8+ T cells expressing the indicated combination of dual effector functions.</p

Therapeutic DNA vaccination protects from AIDS.

<p>Maintenance of CD4+ T cell counts as a marker of clinical disease progression was measured by flow cytometry. Shown is % CD4+ T cell count relative to baseline. Animals that maintained their CD4+ T cell counts above 50% of their baseline level (hashed line) level remained clinically healthy throughout the study.</p

Therapeutic DNA vaccination reduces viral load and protects from viral rebound after drug is stopped.

<p>Plasma viral RNA load was determined by real time RT-PCR. Boxed areas (ART) indicate period of antiretroviral drug therapy. Arrows indicate DNA vaccine doses. (<b>A</b>) Response to ART (weeks 0–18). Shown are viral loads in 41 SIV-infected macaques before starting ART (weeks 0–6) and during the first 12 weeks of ART only (weeks 6–18). ART responders (black) had declining viral loads during ART only. ART low responders (red) had little or no decline in viral loads during ART only and maintained ≥5×10⁴ viral RNA copies/ml (dotted line). (<b>B</b>) Log-fold reduction in viral load during ART only. The change in viral load during ART in each animal was determined by dividing the mean viral load prior to initiating ART (weeks 4–6) by the mean viral load during the first 12 weeks of ART but prior to initiating the therapeutic vaccinations (weeks 8–18). Shown is the log-fold reduction in mean viral load during ART for each animal. ART low responders had <1 log-fold reduction. ART responders exhibited 1.6–4.9 log-fold reductions in viral load during ART. (<b>C</b>) Mean plasma viral loads in each group prior to initiating ART (week 6, Pre-ART) and after 12 weeks on ART but before starting therapeutic vaccinations (week 18, Pre-vaccine). (<b>D</b>) Mean plasma viral loads during vaccinations and after withdrawing ART. Differences in mean viral loads after withdrawing ART are significant starting at weeks 68 and 48 in the DNA (P = 0.019) and DNA+LT (P = 0.002) groups, respectively, and remained significant thereafter when compared to controls (Wilcoxon test). (<b>E</b>) Viral loads in each animal. Protection from viral rebound was defined as containment of viral load ≤100 viral RNA copies/ml (hashed line) for a period of at least 5 months after drug was withdrawn (weeks 45–66).</p

Relationship between <i>TRIM5</i> haplotype and outcome.

1<p><u>Rebound</u>: Plasma virus load rebounded to >1000 copies/ml within 26 weeks after stopping ART.</p>2<p><u>Elite Controller</u>: plasma virus load is contained post-ART at ≤100 copies/ml for duration of study (44 after stopping ART).</p>3<p><u>Late Rebound</u>: Plasma virus load was contained for at least 26 weeks after stopping ART but rebounded to >1000 copies/ml before end of study.</p>4<p><u>TFP/Q and Q/CypA</u>: associated with intermediate susceptibility to SIVmac251 and SIVsmE660 infection (34, 35).</p>5<p><u>TFP/TFP and TFP/CypA</u>: associated with resistance to SIVmac251 and SIVsmE660 infection (34, 35).</p

Statistical Analysis of Trim5 haplotype versus outcome.

1<p>2-sided Fisher's Exact Test.</p

Therapeutic DNA vaccination increases mucosal SIV-specific T cell responses in the gut.

<p>(<b>A</b>) The magnitude of the SIV-specific IFN-γ T cell response in the gut was determined by ELISPOT analysis of lamina propria lymphocytes (LPL) isolated from jejunal resections. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033715#s2" target="_blank">Results</a> represent the mean number of IFN-γ spot forming cells (± SEM) for each group from 2 separate experiments performed at 2 time-points post-infection (weeks 42 and 48). (<b>B</b>) Breadth of the T cell response in each macaque in the blood (top panels, PBMC) and gut (lower panels, GALT). SIV-specific T cell responses in PBMC and LPL isolated from the jejunum were measured by IFN-γ ELISPOT assay against 11 separate pools of overlapping peptides (15-mers overlapping by 11 amino acids) comprising the indicated amino acid sequences from SIV Gag, Env, Pol, and Nef. The percent contribution of each peptide-pool specific response to the total response was determined by dividing the mean number of IFN-γ spot forming cells (SFC) measured against each individual peptide pool by the sum of the response against all peptide pools. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033715#s2" target="_blank">Results</a> represent the average of 2 time-points tested at weeks 42 and 48. (<b>C</b>) Mean absolute GALT CD4+ T cell counts measured by flow cytometric analysis of mononuclear cells isolated from the jejunal lamina propria at weeks 12, 42, and 48 post-infection were similar in animals that controlled vs. failed to control viral rebound after ART was stopped. Controllers are animals that contained virus at ≤100 copies for at least 5 months after stopping ART. Non-controllers are animals that exhibited viral rebound within the same timeframe.</p