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

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

Study outline of animal groups.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of neurologic defects in newborns with particularly severe sequelae in the setting of primary CMV infection in the first trimester of pregnancy. The majority of cCMV cases worldwide occur after non-primary infection in CMV-seropositive women; yet the extent to which pre-existing natural CMV-specific immunity protects against CMV reinfection or reactivation during pregnancy remains ill-defined. We previously reported on a novel nonhuman primate model of cCMV in rhesus macaques where 100% placental transmission and 83% fetal loss were seen in CD4+ T lymphocyte-depleted rhesus CMV (RhCMV)-seronegative dams after primary RhCMV infection. To investigate the protective effect of preconception maternal immunity, we performed reinfection studies in CD4+ T lymphocyte-depleted RhCMV-seropositive dams inoculated in late first / early second trimester gestation with RhCMV strains 180.92 (n = 2), or RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgag inserted as an immunological marker, administered separately (n = 3). An early transient increase in circulating monocytes followed by boosting of the pre-existing RhCMV-specific CD8+ T lymphocyte and antibody response was observed in the reinfected dams but not in control CD4+ T lymphocyte-depleted dams. Emergence of SIV Gag-specific CD8+ T lymphocyte responses in macaques inoculated with the FL-RhCMVΔRh13.1/SIVgag virus confirmed reinfection. Placental transmission was detected in only one of five reinfected dams and there were no adverse fetal sequelae. Viral whole genome, short-read, deep sequencing analysis confirmed transmission of both reinfection RhCMV strains across the placenta with ~30% corresponding to FL-RhCMVΔRh13.1/SIVgag and ~70% to RhCMV UCD52, consistent with the mixed human CMV infections reported in infants with cCMV. Our data showing reduced placental transmission and absence of fetal loss after non-primary as opposed to primary infection in CD4+ T lymphocyte-depleted dams indicates that preconception maternal CMV-specific CD8+ T lymphocyte and/or humoral immunity can protect against cCMV infection.</div

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