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

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

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

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

CHIKV-LR persists in spleen of aged Rhesus macaques.

<p>CHIKV load was quantified in spleen tissue at necropsy (35 dpi) by virus-specific qRT-PCR from total RNA samples prepared using Trizol.</p

Macrophage and dendritic cell population changes following CHIKV infection observed in adult animals are reduced in aged Rhesus macaques.

<p>Measurement of dendritic cell and monocyte/macrophage populations following CHIKV infection. PBMC were stained with antibodies directed against CD3, CD11c, CD14, CD20, CD123 and HLA-DR (shown in panel A) and subdivided into plasmacytoid DCs (B); myeloid DCs (C); non-P/non-M DC's (D); monocyte/macrophages (E). Fold increase in numbers of cells was calculated. N = 6 for adult and N = 2 for aged animals.</p

Aged Rhesus macaques have reduced anti-virion antibody titers compared to adult animals following CHIKV infection.

<p>The production of anti-CHIKV antibodies in aged Rhesus macaques was compared to adult animals. CHIKV-specific endpoint IgG titers were measured using standard ELISA detecting either CHIKV purified virions (A & C) or CHIKV infected cell lysates (B & D) as the antigenic source. N = 6 for adult and N = 2 for aged animals. Aged animals have lower antibody titers to whole virions compared to adult animals, whereas the production of IgG responses to antigen present in CHIKV-infected cellular lysates were not age dependent.</p