HSV-1 antigens recognized by human TG-derived CD8 T-cells.

<p>Representative data from TG-TCL of 6 TG donors assayed for T-cell reactivity to proteins encoded by individual HSV-1 open reading frames (ORFs). Mean IFN-γ secretion levels, shown as arbitrary OD₄₅₀ values, by TG-TCL exposed in duplicate to Cos-7 cells that co-express the respective donor-specific HLA class I allele and the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins specifically recognized are indicated by an arrow. The HSV-1 ORFs recognized by the TG-TCL of donors TG2 and TG7 are shown in colors of the respective TG donor-specific HLA class I allele. ICP, infected cell polypeptide; VP, virion protein and in case of a viral glycoprotein (e.g. gB, glycoprotein B). Currently, no proteins names are available for HSV-1 ORFs <i>UL6</i> and <i>UL25</i>.</p

Localization and phenotype of T-cells in HSV-1 latently infected human TG.

<p>(A) Representative image of an HSV-1 latently infected TG stained by immunohistochemistry (IHC) for CD3 (red). Inset: magnification of the TG tissue showing a cluster of CD3⁺ cells in panel A. (B; left panel) Double immunofluoresence staining for CD4 (red) and CD8 (green) combined with DNA counterstaining (DAPI; blue nuclei). The white arrows signify autofluorescent cytoplasmatic granules in neurons containing lipofuscin and neuron outlines are marked with white dotted lines. (B; right panel) Consecutive TG tissue sections stained for CD8 (brown) and granzyme B (brown), CD8 (brown) and TIA-1 (brown), and CD3 (red) and CD137 (red). Sections were developed with diaminobenzidine (brown staining pattern) or 3-amino-9-ethylcarbazole (red staining pattern) and counterstained with hematoxylin (blue nuclei). Magnifications were: (A) ×20 and inset ×200, (B; left panel) ×400 and (B; right panel) ×1000. Representative images from 10 HSV-1 latently infected TG donors analyzed.</p

Comparison of T-cell cytolytic granule and cytokine transcripts to HSV-1 and VZV DNA load, and CD8β transcript levels, in human TG.

<p>(A) Scatter plot showing the mean HSV-1 and VZV genome equivalent copies (gec) per 10⁵ TG cells. (B) Comparison of HSV-1 and VZV DNA load in TG of individual donors. (C) Comparison between relative CD8β transcript levels and HSV-1 and VZV DNA load. (D) Comparison of HSV-1 DNA load with relative transcript levels of perforin, granzyme B (grB), interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α). (E) Comparison of VZV DNA load with relative transcript levels of perforin, grB, IFN-γ and TNF-α. (F) Comparison between relative CD8β transcript levels and perforin, granzyme B, IFN-γ and TNF-α. (G) Comparison of the transcript levels of CD8β, perforin, grB, IFN-γ and TNF-α between paired left and right TG of individual donors. The paired <i>T</i>-test (A), Spearman correlation (B–F) and Wilcoxon matched pairs test (G) were used for statistical analysis. Data of 26 TG specimens analyzed.</p

HSV-1 epitope-specific CD8 T-cells are localized close to sensory neuron cell bodies in the contralateral human TG.

<p>Representative image of the TG tissue of donor TG2 stained with DAPI (blue), anti-CD8 (green) and tetramers (red) that consisted of both the synthetic HSV-1 peptides ICP0_642–651 and ICP8_1096–1105 conjugated to HLA-A*0201. Inserts, lower left and upper right corner, are enlargements of areas containing tetramer-positive CD8 T-cells. The white arrows and arrowheads signify autofluorescent granules containing lipofuscin and tetramer-positive CD8 T-cells, respectively. Neuron outlines are marked with a white dashed line. Magnification was ×400.</p

Phenotype and HLA class I allele restriction of HSV-1 reactive T-cells recovered from human TG.

*<p>TG-derived T-cell lines were incubated with mock– and HSV-1–infected autologous B-cell lines and assayed by flow cytometry for intra-cellular interferon gamma (IFN-γ) expression.</p>#<p>The ratio of CD4 and CD8 T-cells of the respective TG-derived T-cell lines are indicated.</p>$<p>Patient HLA class I allele restricted HSV-1 reactive CD8 T-cell responses were defined using partially HLA class I matched BLCL. The values represent mean net percentages of live/CD3-gated IFN-γ⁺ T-cells (HSV-1 minus mock) of at least 2 separate experiments.</p><p>nd, not done.</p

HSV-1 antigens and epitopes recognized by CD8 T-cells recovered from human TG.

*<p>HSV-1 gene and protein names, and expression kinetic class, are from reference 1 and Genbank NC_001806. The amino acid (aa) location of CD8 T-cell epitopes identified are in parentheses. na, not applicable.</p>#<p>HLA allele by which the indicated proteins and peptides are recognized by the specific CD8 T-cells.</p

HSV-1 antigens recognized by human TG-derived CD4 T-cells.

<p>(A) Representative data from the TG-TCL of the donors TG2 and TG3 assayed for T-cell reactivity by a proliferation assay to proteins encoded by individual HSV-1 open reading frames (ORFs). Cell lysates of mock- and HSV-1 infected Cos-7 cells were used as negative and positive controls, respectively. Mean [³H]-thymidine incorporation by TG-TCL exposed in duplicate to γ-irradiated donor HLA-DQ/DR-matched allogeneic peripheral blood mononuclear cells (PBMC) pulsed with lysates generated from Cos-7 cells transfected with the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins driving the positive responses are indicated by an arrow. ICP47, infected cell polypeptide 47 and VP16, virus protein 16. (B) Proliferation assay data of the TG-TCL of donor TG2 with γ-irradiated HLA-DQ/DR TG 2-matched allogeneic PBMC pulsed with whole HSV-1 ICP47 protein (gene US12) spanning synthetic peptides (15-meric peptides with 10 amino acid (aa) overlap) as antigen presenting cell (APC). (C; left panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with the indicated recombinant HSV-1 VP16 protein (gene UL48) fragments as APC. (C; right panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with HSV-1 VP16 protein fragment (aa151–240) spanning synthetic peptides (13-meric peptides with 8 aa overlap) as APC. Data are presented as mean counts per minute of triplicate experiments. Data presented in (B) and (C) are the means ± standard error of the mean.</p

Antibody responses to codon-optimized polynucleotide vaccines encoding antigen and ubiquitinated antigen.

<p>A. Antibody titers of mice after three immunizations with 20 µg of plasmids expressing HSV-2 gD and prior to HSV-2 challenge, measured using yeast-expressed gD1 protein. B and C. Antibody titers of mice immunized as for A but not challenged, measured using yeast-expressed truncated gD1 protein and CHO cell-expressed truncated gD2 protein, respectively. Mice were immunized with pcDNA3-O-gD2 [D], a plasmid encoding a ubiquitin-tagged truncated HSV-2 gD (pcDNA3-O-Ubi-gD2_25–331 [UD]), a mixture of the two plasmids [mix], a codon de-optimized construct pcDNA3-W-gD2 [W], or empty pcDNA3 vector [-ve]. For parts A-C, * P<0.05, *** P<0.001, ns = not significant as measured by one-way ANOVA followed by Tukey’s Multiple Comparison test. Significance is shown relative to the UD construct. Log titers were compared so that the distributions were approximately Gaussian. n = 20, 8 and 8 for parts a, b and c, respectively. The geometric means are shown. The data in parts A and C have been published in patent US 2011/0287039 A1.</p

HSV-2 DNA copy number in vaginal swabs, from immunized mice, taken after HSV-2 challenge.

<p>The HSV-2 DNA copy numbers in vaginal swabs taken from mice 1, 3 and 5 days post-intravaginal challenge with 50 (1.55×10⁴ p.f.u.) or 500×LD₅₀ of live HSV-2 strain 186 are shown. 10 mice/group/challenge dose were used (with the exception of the positive control which used 5 mice/challenge dose). The geometric means and 95% confidence intervals are indicated. The differences between the HSV-copy numbers over days 1 to 5 for the mixed vaccine versus UD alone, W and empty vector control were significant at the 50× challenge level (*P<0.05 by the Kruskal-Wallis test and Dunn’s post test analysis of the area under the log10 copy number-time curves). This data has been published in patent US 2011/0287039 A1.</p

Survival, following HSV-2 challenge, of mice immunized with codon-optimized polynucleotides encoding antigen and ubiquitinated antigen.

<p>Mice sensitized with progesterone were challenged intra-vaginally with 50 (1.55×10⁴ p.f.u.) or 500×LD₅₀ of live HSV-2 strain 186, after three immunizations with 20 µg of plasmids expressing HSV-2 gD and/or ubiquitin-tagged truncated gD, and survival monitored. 10 mice/group/challenge dose were used (with the exception of the positive control which used 5 mice/challenge dose). Survival of mice immunized with a plasmid containing a codon-optimized insert encoding full length HSV-2 gD pcDNA3-O-gD2 [D], a plasmid encoding a ubiquitin-tagged truncated HSV-2 gD pcDNA3-O-Ubi-gD2_25–331 [UD], a mixture of D and UD [mix], a plasmid containing a codon de-optimized insert encoding full-length gD pcDNA3-W-gD2 [W], mouse thymidine kinase-deficient live HSV-2 strain 333 (5×10⁵ p.f.u./mouse) [+ve], or empty pcDNA3 vector [-ve] is shown. For the 500× challenge, the survival differences between UD and D and between UD and mix were significant as determined by a log-rank (Mantel-Cox) test. The survival of W and -ve was significantly different from the vaccine and +ve control groups at both challenge levels. * P<0.05. This data has been published in patent US 2011/0287039 A1.</p