Fig 1.
Plasma VL and vertical transmission events following primary maternal RhCMV infection in immunocompetent dams (n = 12).
(A) The study schedule for a new cohort of 12 RhCMV seronegative, pregnant rhesus macaques receiving intravenous inoculation of 1x106 pfu of two RhCMV strains (clinical isolate UCD52 and cloned full length, FL, 68–1 viral variants) in late first/early second trimester, followed by weekly blood draws and amniocentesis until hysterotomy near term at 20–22 weeks gestation, prior to fetal viability. A pre-infection blood draw was collected between 1–4 weeks before infection. (B) Maternal plasma VL kinetics measured by qPCR against either RhCMV gB (UL55) or the noncoding exon 1 region of the RhCMV IE gene for RhCMV-positive amniotic fluid (AF-positive, n = 5) and AF-negative (n = 7) dams. Data shown as the mean value of 6 or more technical replicates for each sample. (C) Survival curve showing the timing of detection of vertical transmission, defined as amniotic fluid RhCMV qPCR result above the limit of detection in 2 or more of 12 technical replicates. Vertical transmission was confirmed in 5/12 dams by this definition. Schematic created using Biorender.com.
Fig 2.
Plasma RhCMV-specific IgM and IgG binding responses following acute RhCMV infection during pregnancy.
In a novel cohort of 12 RhCMV seronegative, immunocompetent dams primarily infected with RhCMV at the end of first/early second trimester of pregnancy, we measured the following RhCMV-specific antibody binding responses longitudinally: (A) IgM binding to whole UCD52 RhCMV virions, (B) IgG binding to whole UCD52 virions, (C) avidity of IgG binding to whole UCD52 virions, (D) IgG binding to the ectodomain of UCD52 gB, (E) IgG binding to cell-associated UCD52 gB, and (F) IgG binding to soluble PC. AF-positive dams are shown in grey, and AF-negative dams are shown in black. Data shown as the mean of technical duplicates.
Fig 3.
The kinetics of functional RhCMV-specific antibody responses in plasma during acute maternal infection.
We measured functional antibody responses longitudinally in a novel cohort of 12 immunocompetent dams with primary RhCMV infection during pregnancy. (A) Neutralization of FL RhCMV on telomerized rhesus fibroblasts (TeloRFs), (B) neutralization of UCD52 RhCMV on monkey kidney epithelial cells, (C) monocyte-mediated antibody dependent cellular phagocytosis against free UCD52 RhCMV, (D) NK cell degranulation, a component of cell-mediated antibody dependent cellular cytotoxicity against UCD52-infected TeloRFs. Data shown as the mean of technical duplicates.
Fig 4.
Combination of RhCMV seronegative, pregnant rhesus macaque cohorts.
Treatment schedules for each of the rhesus macaque cohorts included in the combined analysis. A new group of 12 immunocompetent dams is highlighted in bold, and all other groups are from historical studies completed between 2013–2016 [15,16]. All dams were infected by intravenous inoculation of the RhCMV strains listed with 1x106 pfu for each UCD52, UCD59, (*previously reported as 1x106 TCID50 [15,16], rounded from 1.4x106 TCID50) and FL RhCMV, and 2x106 TCID50 for 180.92 (for a total of 2–3x106 total infectious virions) at week 7–9 of gestation. A total of 15 immunocompetent dams (black) included 3 dams from a previous study in which animals received a 1:1:2 combination of low passage isolates UCD52 and UCD59 and lab adapted strain 180.92 as well as 12 dams inoculated with a 1:1 combination of UCD52 and BAC-derived full length (FL) RhCMV. A total of 6 dams received a 50 mg/kg dose of recombinant rhesus anti-CD4+ T-cell–depleting antibody 1 week prior to infection. Five of these dams were infected with 1:1:2 UCD52, UCD59, and 180.92, and the remaining dam was infected with 180.92 alone. Six additional animals received CD4-depleting antibody 1 week before infection and passive infusion of a polyclonal RhCMV-positive IgG preparation before infection with 1:1:2 UCD52, UCD59, and 180.92. IgG for passive infusion was purified from RhCMV-seropositive plasma donors screened for high RhCMV binding (red) or high RhCMV neutralization on epithelial cells (blue). The high RhCMV binding IgG infusion was given at a single 100 mg/kg dose 1 hour before infection, and the high RhCMV neutralizing IgG infusion was given in a two-dose, optimized regimen with the initial 150 mg/kg dose 1 hour prior to infection and the second 100 mg/kg dose 3 days post infection. Of the immunocompetent dams, 7/15 had detectable RhCMV DNA in amniotic fluid. All 6 of the CD4-depleted dams were AF-positive, and 5 experienced spontaneous abortion. Two of the dams receiving the high RhCMV binding IgG infusion and none of the dams receiving the high RhCMV neutralizing IgG infusion were AF-positive. Figure created using Biorender.com.
Fig 5.
Kinetics of plasma VL and RhCMV-specific antibody responses for all treatment groups through 3 weeks post RhCMV infection.
(A) Maternal plasma VL measured by qPCR for the UL55 or IE genes; (B) IgM and (C) IgG binding to whole UCD52 RhCMV virions; (D) avidity of IgG binding against whole UCD52 virions; IgG binding to (E) PC, (F) soluble gB ectodomain by ELISA, and (G) cell-associated gB via transfected cell binding assay; antibody mediated RhCMV neutralization on (H) fibroblasts and (I) epithelial cells; (J) ADCP; and (K) ADCC; (L) Survival curves showing detection of RhCMV in amniotic fluid. Treatment groups are represented by the color of each line. Solid lines represent AF-negative dams, and dashed lines represent AF-positive dams. Previously published data for historical studies was used for soluble gB- and PC-binding IgG responses and fibroblast and epithelial cell neutralization [15,16], and available samples from those studies were utilized for measurement of remaining assays.
Fig 6.
Univariate comparison of maternal VL and humoral responses by vertical transmission status in immunocompetent dams.
The area-under-the-curve (AUC) of each response over 0–3 weeks post RhCMV infection was used to distill the magnitude and kinetics of each response into a single value in a critical window for vertical transmission. Amniotic fluid (AF) was assessed for RhCMV DNA by qPCR and a positive result (AF-positive) was defined by 2 or more of 12 replicates with detectable amplification between 2 and 12 weeks after maternal infection. Pairwise comparisons between AF-positive and AF-negative dams were performed using Wilcoxon Rank Sum Tests (Table 1). VL: viral load; ADCP: antibody dependent cellular phagocytosis; ADCC: antibody dependent cellular cytotoxicity.
Fig 7.
Univariate comparison of maternal VL and humoral responses by vertical transmission status in CD4-depleted dams.
The area-under-the-curve (AUC) of each response over 0–3 weeks post RhCMV infection was used to distill the magnitude and kinetics of each response into a single value in a critical window for vertical transmission. Amniotic fluid (AF) was assessed for RhCMV DNA by qPCR and a positive result (AF-positive) was defined by 2 or more of 12 replicates with detectable amplification between 2 and 12 weeks after maternal infection. Pairwise comparisons between AF-positive and AF-negative dams were performed using Wilcoxon Rank Sum Tests (Table 1). VL: viral load; ADCP: antibody dependent cellular phagocytosis; ADCC: antibody dependent cellular cytotoxicity.
Table 1.
Univariate analysis comparing AF-positive versus AF-negative dams within subgroups.
Fig 8.
RhCMV-specific antibody responses negatively associate with maternal VL.
(A) Correlation matrix showing Spearman correlation coefficients for pairwise correlations between the AUC of each RhCMV-specific antibody response over the first 3 weeks post infection. (B) Principal component analysis (PCA) using the AUC of each response over the first 3 weeks post infection, showing clustering by AF-negative (magenta) versus AF-positive (green) with treatment groups represented by the symbol shape. Loading vectors for each measured response demonstrate the influence of each response. (C) The same PCA with group represented by color, transmission status represented by shape, and animal IDs labeled to better demonstrate the effect of treatment group. VL: viral load; NT: neutralization; PC: pentameric complex; ELISA: enzyme-linked immunosorbent assay; ADCP: antibody dependent cellular phagocytosis; ADCC: antibody dependent cellular cytotoxicity.