Summary of effect of immune cell depletions on the anti-SVV response.

<p>Hallmarks of the immune response during acute SVV infection were compared between control and depleted animals. N/A indicates that comparison could not be carried out due to the depletion of the T or B cell subset in question.</p

Representative examples of varicella in control and depleted animals.

<p>Varicella rash on the trunk of a representative (A) control, (B) CD20 depleted, (C) CD8 depleted, and (D) CD4 depleted animal at 10 dpi.</p

Impact of T and B cell depletion on kinetics and magnitude of B cell proliferation and IgG/IgM production following SVV infection.

<p>Frequency of proliferating (Ki67+) B cells within marginal zone-like and memory subsets in BAL (A, B) and PBMC (C, D) was measured using FCM. Data points for B cell proliferation in CD20-depleted animals are not shown 0–14 dpi as there were no B cells in circulation during this time period. Average SVV-specific IgM (E) and IgG (F) end point titers± SEM in control, CD20 depleted, CD8 depleted, and CD4 depleted animals (n = 4/group) were determined by standard ELISA.* indicates p<0.05 as compared to control animals.</p

Efficacy of antibody-mediated depletion of immune cells during acute SVV infection.

<p>(A–C) Average frequency of CD4 T (A), CD8 T (B), and CD20 B (C) cells in bronchial alveolar lavage (BAL) of control, CD20 depleted, CD8 depleted, and CD4 depleted animals (n = 4/group) were measured using flow cytometry (FCM). Absolute numbers per µl/blood were then calculated by converting the percentage of these subsets using complete blood counts obtained at every time point (D–F).</p

Summary of rash duration, lesion number, and disease severity of control and experimental animal groups following acute SVV infection.

<p>The number of lesions for each group was determined by averaging the lesions observed on the abdomen of each animal within each group at 10dpi. The disease severity of each group was determined by averaging the assigned severity score (based on the size and duration of observed vesicles) of each animal within each group (1–2 = mild; 3–4 = moderate; 5 = severe).</p

Impact of T and B cell depletion on the kinetics and magnitude of T cell proliferation following acute SVV infection.

<p>Frequency of proliferating (Ki67+) CD4 and CD8 T cells within central and effector memory subsets was measured in BAL (A–D) and PBMC (E–H) by FCM. Data points for CD4 T cell Ki67+ frequency in CD4-depleted animals are not shown 7–21 dpi as there were no CD4 T cells in circulation during this time period. Similarly data points for Ki67+ CD8 T cell frequency are not shown 0–14 dpi as there were no CD8 T cells detected during this period. * indicates p<0.05 as compared to control animals.</p

Impact of T and B cell depletion on frequency of SVV-specific T cells.

<p>The frequency of SVV-specific T cells in BAL and PBMCs was measured by intracellular cytokine staining following stimulation with overlapping peptide pools covering ORFs 4, 31, 61 and 63. The average percentage of responding (IFNγ+ and IFNγ+TNFα+) T cells ± SEM within CD4 CM, CD4 EM, CD8 CM and CD8 EM subsets in BAL (A–D) and PBMCs (E–H) is shown. Responses detected on day 0 were on average <0.5% and were subtracted from subsequent time points. * indicates p<0.05 as compared to control animals.</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

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