Immunogenicity of pIX-CS_short display is higher than that of CS protein and comparable levels cannot be achieved by mixing AdV vectors and protein.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks post-immunization with 1x10¹⁰ VP/animal of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short, with 5μg of CS protein in PBS, or with 1x10¹⁰ VP/animal HAdV35.empty. n = 7 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. (B) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks after immunization with 5μg CS protein in PBS, or mixed with 1x10¹⁰ VP/animal of empty AdV-vectors (HAdV35, HAdV26, or HAdV5), animals injected with Matrix-M only serve as control. n = 6 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 5μg CS protein in PBS only, or mixed with Matrix-M, or 1x10¹⁰ VP/animal of empty Ad-vectors (HAdV35, HAdV26, or HAdV5). Horizontal bars depict mean ratios (n = 5 animals/ group).</p

pIX-CS_short display on CS-transgene expression-vectors induces strong humoral and cellular responses.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 2 and 8 weeks post-immunization with 1x10⁸, 1x10⁹, or 1x10¹⁰ VP/animal of HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short, a mix of HAdV35.empty.pIX-45-CS_short and HAdV35.CS, HAdV35.CS alone, or HAdV35.empty (10⁹ VP/ animal only). n = 5 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data by time point and dose. Black horizontal bars indicate statistical significance (p ≤ 0.05). (B) Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 1x10¹⁰ VP of the HAdV35 vectors indicated in the legend. Horizontal bars depict mean ratios (n = 5 animals/ group). (C) IFNγ ELISPOT responses to stimulation of splenocytes of Balb/C mice 8 weeks post-immunization with <i>P</i>. <i>falciparum</i> CS peptide pool (left panel; “CS”) or the H-2Kd restricted immunodominant HAdV35 hexon epitope KYTPSNVTL (right panel; “HAdV35 hexon”). n = 5 animals/ group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. ns = not significant.</p

Characterization of the pIX-CS_short display- and display-/ expression vectors.

<p>(A) pIX-CS_short capsid incorporation in purified HAdV35 vector preparations. To confirm capsid incorporation of the pIX-CS_short (~50 kDa variants) 5 x10⁹ VP/well of each purified HAdV35 vector preparation was analyzed by Western blot using anti-CS antibody. To ensure equal loading, the blots were stained with anti-fiber 4D2 antibody (~35 kDa). The pIX-fusion proteins migrate higher than their predicted size in kDa due to the NANP-repeat in the CS protein. Marker (M) is indicated with the corresponding kDa band size. An additional band (~100 kDa) is indicated with an asterisk (*). (B) pIX-CS_short capsid display by electron microscopy (EM) anti-CS staining and gold-label staining. Representative EM images of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short (upper row), HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short (middle row), HAdV35empty, and HAdV35 (bottom row) vectors. The bars represent 100 nm unless stated otherwise. (C) <i>In vitro</i> expression of CS transgene under non-replicating conditions in A549 cells. Western Blot analysis of cell lysates from A549 cells infected with 5000 VP/cell, using an anti-CS antibody. From left to right, the two controls HAdV35.empty and HAdV35.CS followed by the pIX-display vector variants with a CS transgene in E1 and without the transgene. Marker (M) is indicated with the corresponding kDa band size. (D) Heat Stability Assay showing percent (%) variation in luminescence in lysate of A549 cells infected with HAdV35.luc vector preparations subjected to 45°C temperature stress for 0 to 20 minutes. The percent variation was determined relative to the respective baseline time point (0 minutes).</p

Design of HAdV35 pIX-display-vectors

<p>Schematic representation of pIX-display HAdV35 Advac vectors containing a human CMV promoter and SV40 poly-A expression cassette in E1 and the native pIX promoter (P). The vectors either express a 376 amino acid CS protein (lacking the GPI anchor) or no transgene (empty) in the E1 region. The pIX-display-vectors, with and without the CS-transgene in E1, are genetically modified to display the 151 amino acid CSshort with or without a 3 amino acid glycine-linker (Gly) and/or 45Å-spacer.</p

An application of HOMER and ACMANT for homogenising monthly precipitation records in Ireland.

Climate change studies based only on raw long-term data are potentially flawed due to the many breaks introduced from non-climatic sources, consequently quality controlled and homogenised climate data is desirable for basing climate related decision making on. Seasonal cycles of precipitation in Ireland and the UK are projected to become more marked as the climate changes, and regional extremes in summer dry spells and winter precipitation have been recorded in recent years. Therefore to analyse and monitor the evolution of precipitation patterns across Ireland, quality controlled and homogenous climate series are needed

Differential expression of transcription factors in CD8⁺ naïve and memory T cell subsets.

<p>log₂ mean expression values were normalized to naïve cell samples.</p

Principal component analysis of transcription factor expression profiles from SIV-specific MHC tetramer-sorted CD8⁺ T cells from animals vaccinated with SIVΔnef, animals infected with wild-type SIV, and sorted CD8⁺ T cell subsets.

<p>Plot of principal components 1 and 2 for each of the expression profiles assessed from sorted naïve and memory CD8⁺ T cell subsets, SIV-specific MHC tetramer-sorted CD8⁺ T cells isolated from SIVΔnef-vaccinated animals, and MHC tetramer-sorted CD8⁺ T cells isolated from animals (n = 5) at 20 weeks following wild-type SIV infection. Principal components 1 and 2 explain 77 percent of cumulative total variance.</p

Differential expression of transcription factors in CD8⁺ T cells isolated at week 5 and week 20 post-vaccination with SIVΔnef and at week 20 post-infection with wild-type SIV.

<p>Symbols indicate log₂ expression relative to endogenous controls in cells from individual animals. Red symbols indicate Gag CM9-specific cells, blue symbols indicate Tat SL8-specific cells. Sample means are indicated by horizontal bars. Statistically significant (p≤0.05) differences in transcription factor expression between cells from week 5 and week 20 post-SIVΔnef vaccination, or between cells from week 20 post-SIVΔnef vaccination and week 20 post-wild-type SIV infection are indicated by horizontal bars with asterisks. Statistically significant differences between Gag CM9 and Tat SL8-specific cells are indicated by vertical bars with asterisks.</p

Transcription factors analyzed in bulk and SIV-specific CD8⁺ T cells.

<p>Transcription factors analyzed in bulk and SIV-specific CD8⁺ T cells.</p

Principal component analysis of transcription factor expression profiles from SIV-specific MHC tetramer-sorted CD8⁺ T cells and sorted CD8⁺ T cell subsets.

<p>(<b>A</b>) Plot of principal components 1 vs. 2, and 2 vs. 3 for each of the expression profiles assessed in sorted naïve and memory CD8⁺ T cell subsets isolated from healthy control animals (n = 5), and SIV-specific MHC tetramer-sorted CD8⁺ T cells isolated from animals (n = 4) at week 5 or week 20 following SIVΔnef vaccination. Principal components 1, 2 and 3 explain 92% of cumulative total variance. (<b>B</b>) PCA loading factors for each transcription factor.</p