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

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

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

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

Analysis of cell cycle markers suggests an age associated delay in CD4+ proliferation.

<p>We used multi-parameter flow cytometry to evaluate the proliferation kinetics of CD4+ cells in each of our cohorts. The dark lines illustrate the two old animals whereas the lighter lines illustrate the two adult animals. The cell cycling is normalized to the baseline cycling recorded during this challenge for each animal, setting this to 0%. This illustration reveals that post WNV infection CD4+ cell cycling decreased followed by a marked increase on days 7, 10 and 14 which then wanes. Of particular interest is the age associated shift in kinetics both older animals (dashed lines) had a slower increase in CD4+ cycling than the adult animals (solid lines) and a prolonged phase prior to returning to a steady state post infection.</p

Immunodeficient animals – CD8 depleted animals survive WNV infection.

<p>CD8+ Tcells from previously thymectomized and CD8 depleted animals were enumerated from total PBMC using multi-parameter flow cytometry prior to WNV infection. Twelve months prior to infection animals were thymectomized (N = 4), thymectomized and CD8 depleted (N = 4), only CD8 depleted (N = 3) or controls (N = 4) (dark filled circles) Light circles illustrate thymectomized animals, dark circles illustrate control (sham operated) animals and light filled circles represent CD8 depletion.</p

Rhesus macaque and Cynomolgus macaque cohorts.

<p>Rhesus macaque and Cynomolgus macaque cohorts.</p

Aged and adult macaques produce robust anti WNV IgM and IgG response.

<p>Panels 1–8 illustrate WNV specific antibody production from Cohort 1 begins about day 10 peaking by day 21 and maintaining in most animals out well past day 45. HI (hemaglutination) assay on the left vertical axis, and IgG and IgM specific Elisa assays are on the right vertical axis reported P/N values (positive over negative), the assay limit of detection is P/N>2 is positive.</p

Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues

<div><p>Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1–7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.</p></div

Detection of anti-ZIKV antibody responses in Rhesus plasma.

<p>Rhesus macaques infected with ZIKV were analyzed for the presence of antibodies directed against ZIKV-PRABC59 by ELISA using whole virus as capture antigen with an HRP-conjugated anti-Rhesus IgM <b>(A)</b> or IgG <b>(B)</b> secondary antibody. <b>C.</b> Sera from indicated animals obtained pre-infection (d0) or at terminal bleed (d28 or 35 pi) were tested for neutralizing activity via plaque reduction neutralization titer (PRNT) assay. <b>D.</b> Fold dilution giving 50% reduction in infectious titer for each serum sample.</p