Establishment of a Neonatal Natural Transmission Model for CMV Vaccine Development

Figures

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  Figure 1.. Study design and timeline and maternal-derived immunity. A, The 3 immunization groups consisted of group 1, QS-21 adjuvant in phosphate-buffered saline (placebo); group 2, 50 µg PC and 50 µg gB with QS-21, plus 0.2 mg pp65-2 DNA (PC/gB/pp65-2); and group 3, 50 µg PC, 50 µg gB674, and 45 µg vIL-10M2 with QS-21, plus 0.2 mg pp65-2 DNA (PC/gB/pp65-2/vIL-10M2). Various experimental procedures and their timing are indicated. Infants were cohoused with dams from birth to week 52, then released and housed with other infants (about 20 per crib). Created in BioRender (https://BioRender.com/xp6tj0h). B, Detection of anti-RhCMV gB-, PC-, and pp150-binding IgG in newborn macaques at week 0 prior to immunization. Plasma samples were serially diluted and tested against recombinant gB using a standard ELISA protocol as described in “Methods.” Each bar represents the geometric mean of titers from each group of macaques. No statistical difference between groups as determined by a 1-way ANOVA, P values were 0.853 for gB, .842 for PC, and .165 for pp150, dotted lines indicate P value > .05. C, Scatter plot of gB-binding titers of infants and respective dam. Line represents best fit calculated by…

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  Figure 2.. IgG response to vaccination and infection. A, Kinetics of IgG against vaccine antigens gB, PC, and vIL-10M2. Data are separated by vaccination group and infection status, measured by viral shedding in urine as detected by PCR. Each bar represents the geometric mean of OD450 values by ELISA and each dot represents data from an individual animal. For gB-binding IgG at week 24, titers in vaccinated, uninfected infants were significantly higher than placebo uninfected animals (P ≤ .001). At week 36 and 44, no statistical difference was observed between infected animals in group 1 and infected animals in group 2 or group 3 (P = .429 and P = .299). For PC-binding IgG at week 24, titers in vaccinated, uninfected infants were significantly higher than placebo, uninfected animals (P ≤ .001). For IgG at week 44, no statistical difference was observed between infected animals in group 1 and infected animals in group 2 (P = .581) or uninfected animals in group 3 (P = .516). For vIL10-binding IgG at week 24, no statistical difference was observed between uninfected animals in group 1 and uninfected animals in group 2 (P = .952). Animals in group 3 had statistically higher vIL10-binding IgG…

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  Figure 3.. Virus neutralization following vaccination and infection. The nAb titer of individual samples was determined using UCD52 virus in (A) fibroblasts (Telo-RF) and (B) epihelial cells (primary MKE cells) as described in “Methods.” The limit of detection of 50% neutralizing titer was 50 and 20 for the MKE cell assay and Telo-RF cell assay, respectively. Samples that did not generate a positive neutralization titer were assigned a value of 20 for Telo-RF cell assay and 50 for MKE cell assay. Each bar represents the geometric mean of ED50 from each group of macaques, and dots represent data from individual animals. Data are presented by vaccination group and infection status. A and B, Solid lines indicate P value < .05 and dotted lines indicate P value > .05 as determined by a Kruskal-Wallis test with Dunn multiple comparisons or a 1-way ANOVA followed by Dunnett comparisons with group 1 as the reference. In fibroblasts at week 28, no significant difference was observed between infected animals in group 1 and infected animals in group 2 (P = .060). At week 40, no statistical difference was observed between group 1 and group 3 uninfected animals (P = .182). At week 52, there was no…

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  Figure 4.. Functional effect of vIL-10M2 vaccination. A, Schematic representing vIL-10 inhibition assay. Created in BioRender (https://BioRender.com/e81de39). B, Percent inhibition of vIL-10 activity in animals receiving vaccines with and without vIL-10M2 (group 2 and group 3). A 1:400 dilution of plasma samples collected at the indicated times postvaccination was added to the PBMCs in media containing LPS with and without vIL-10M2. After overnight incubation, IFN-γ secretion from the LPS-stimulated human PBMCs was measured by a Luminex assay and the percentage IFN-γ produced was calculated by comparison of relative IFN-γ secretion in the presence of sera from vaccinated animals versus unvaccinated, uninfected animals, as described in “Methods.” Solid line indicates P value < .05 as determined by 2-sided 2-sample t test. Abbreviations: Ab, antibody; IFN-γ, interferon-γ; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; vIL-10M2, recombinant viral interleukin 10.

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  Figure 5.. Cellular immune response to vaccination and infection. Frequency of RhCMV pp65 (A) or IE1-specific (B) IFN-γ–producing cells was determined by ELISPOT in unvaccinated controls and vaccinated animals at week 28, 40, 52, and 78 weeks as described in the “Methods.” Data are plotted by vaccination group and infection status. Values below the LOD are shown as 1. Placebo group, n = 19; PC + gB + pp65 group, n = 18; PC + gB + pp65 + vIL-10 group, n = 20. A, Solid lines indicate P value < .05 and dotted lines indicate P value > .05 as determined by a 1-way ANOVA with group followed by Tukey multiple comparisons. For pp65 at week 28, no significant difference was observed between infected animals in group 1 and uninfected animals in group 3 (P = .062). For pp65 at week 40, no significant difference was observed between infected animals in group 1 and uninfected animals in group 2 (P = .104) or group 3 (P = .785), nor at week 52 (P = .117 and .723, respectively). Abbreviations: gB, glycoprotein B; GeoMean, geometric mean; IFN-γ, interferon-γ; LOD, limit of detection; PC, pentameric glycoprotein complex; PCR, polymerase chain reaction; RhCMV, rhesus cytomegalovirus; SFC, spot-forming cell.

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  Figure 6.. Effect of vaccination of infant macaques on CMV infection status. A, Virus shedding was measured in DNA extracted from urine using qPCR. A Ct value of 40 was deemed as negative. Y-axis indicates the percentage of PCR positive in the evaluable animals in each group, and x-axis shows the time points of sample collection in experiment weeks. In group 2, one sample was not available for evaluation. Statistical analysis was performed as described in the “Methods” section. B, CMV DNA copies in urine as measured by PCR for each animal at 52 weeks postvaccination. The geometric mean is depicted by a bar with corresponding value. Zero values are set to 1 for plotting on logarithmic axis. Dotted line indicates P value > .05 as determined by a nonparametric Kruskal-Wallis test. Abbreviations: CMV, cytomegalovirus; Ct, cycle threshold value; qPCR, quantitative polymerase chain reaction.