Gating strategy for PBMC immunophenotyping analysis in acute RhCMV reinfection.

(A) Gating strategy used for the innate immune cell compartment. (B) The gating strategy used to identify T cell subsets. (C) Representative plots of side scatter high (SSChi) population following RhCMV reinfection in CMV-seropositive rhesus macaque dams. (TIF)</p

Antibodies for phenotyping and intracellular cytokine staining.

Antibodies for phenotyping and intracellular cytokine staining.</p

Improved study outcome in dams with pre-existing immunity.

The main readouts of the study are described as a frequency of total number of animals.</p

Gag-specific PCR and binding antibody assay in CD4+ T lymphocyte depleted CMV-seropositive rhesus macaque dams that received RhCMV FL-RhCMVΔRh13.1/SIV<i>gag</i>.

(A) Real time PCR for SIVgag DNA quantification performed on plasma, saliva and urine at multiple post reinfection time-points in three dams inoculated with FL-RhCMVΔRh13.1/SIVgag. Positive signal at a single time-point in KB91 saliva sample. (B) Gag-specific binding antibody assays in the three dams reinfected with FL-RhCMVΔRh13.1/SIVgag. Positive controls in this assay are SHIV-infected rhesus macaques (open symbol) were used for comparison of responses in CMV-seropositive reinfected animals (closed symbol). (TIF)</p

Early immunophenotypic changes following RhCMV reinfection in CMV-seropositive macaques.

Immunophenotyping of circulating peripheral blood mononuclear cells in acute RhCMV reinfection. Plots show the kinetics of different lymphocyte subsets in three CMV-seropositive reinfected macaques (JP01, KB91, and KK24). Paired non-parametric Wilcoxon Signed Rank test comparing baseline prereinfection values with values at time-point of maximal change in the first 7 days post reinfection was performed.</p

Evidence of reinfection in CD4+ T lymphocyte depleted FL-RhCMVΔRh13.1/SIV<i>gag</i> inoculated dams.

(A) RhCMV-specific (black line) and SIVgag-specific (grey line) real time PCR results in the saliva of one CMV-seropositive reinfected animal (KB91). All other animals were found negative for SIVgag DNA in saliva and urine. (B) Detection of SIV Gag-specific T lymphocyte responses measured longitudinally against a SIVmac239 Gag peptide pool in CMV-seropositive reinfected macaques (KB91, KK24, and JP01) inoculated with RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag. Horizontal stippled line shows negative cut-off based on pre-reinfection values.</p

CMV-specific CD8+ T lymphocyte memory responses to RhCMV immediate early (IE) proteins and exogenous SIV Gag protein in CD4+ T lymphocyte depleted RhCMV reinfected macaques.

(A) Paired IE-specific responses by CD107a expression and secretion of IFN-γ, IL-2, and TNF-α in four CMV-seropositive macaques reinfected with RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag (n = 3) or RhCMV 180.92 (n = 1). Pre-reinfection responses were compared with responses at week 8–10 post RhCMV reinfection using paired t-test. (B) Polyfunctional SPICE analysis of IE-specific responses pre vs post RhCMV reinfection showing the proportion of four-functional, three-funtional, two-functional and single function responses. CD107a (blue arc), IFN-γ (red arc), IL-2 (orange arc), and TNF-α (green arc). Four-functional responses are displayed in white, three-functional responses in dark grey, two-functional response in light grey, and mono-functional responses in black. (C) Bar graph of the polyfunctional responses pre (grey) and post (black) RhCMV reinfection (n = 4) showing the frequency of memory CD8+ T lymphocytes responding to RhCMV IE peptides. The RhCMV IE-specific response was measured by intracellular cytokine staining after stimulation with RhCMV IE1 and / or IE2 peptide pools depending on the baseline immunodominant response in individual animals. Comparison of pre- and post reinfection Boolean responses were compared with the Wilcoxon rank sum test using SPICE v6 software.</p

Ultrasound measurements over time of Biparietal Diameter (BPD) and Femur Length (FL).

(A) BPD of CMV-seropositive reinfected (green) and CMV-seropositive controls (grey) fetuses compared to reference values [37]. (B) FL of CMV-seropositive reinfected (green) and CMV-seropositive control (grey) fetuses compared to reference values [37]. (TIF)</p

Study design and kinetics of CD4+ T lymphocyte depletion in experimental groups.

(A) Schematic of study design of cCMV transmission in pregnant CMV-seropositive rhesus macaques. (B-C) Peripheral blood CD4+ T lymphocyte counts following anti-CD4 antibody administration in (B) CMV-seropositive Reinfection group (n = 5); and (C) CMV-seropositive Control group (n = 3).</p

RhCMV DNA PCR in placental and fetal tissues of CD4+ T lymphocyte-depleted dams.

RhCMV DNA PCR in placental and fetal tissues of CD4+ T lymphocyte-depleted dams.</p