Potential of Anti-CMV Immunoglobulin Cytotect CP® In Vitro and Ex Vivo in a First-Trimester Placenta Model

Abstract

Background: Congenital CMV infection is the leading cause of neonatal neurological deficit. We herein studied in vitro and ex vivo the potential of the hyperimmune globulin Cytotect CP^® (Biotest, Germany) for congenital infection prevention and treatment.

Methods: In vitro neutralization assays were conducted in fibroblasts and retinal epithelial cells on the CMV strains TB40/E and VHL/E to determine the 50% and 90% neutralizing doses (ND50 and ND90). The toxicity was assessed by measuring LDH release. Ex vivo assays were conducted in first-trimester villi explants with the TB40/E strain, namely, neutralization assays, the prevention of villi infection, and the inhibition of viral replication in infected villi. Viability was assessed by β-HCG quantification in supernatants.

Results: The in vitro neutralization tests showed that Cytotect CP^®® inhibits the development of infection foci (DN50: 0.011-0.014 U/mL for VHL/E and 0.032-0.033 U/mL for TB40E) without any toxicity. In the ex vivo neutralization assays, the DN50 were 0.011 U/mL on day 7 and 0.093 U/mL on day 14. For the prevention of villi infection, the EC50 was 0.024 U/mL on day 7. Cytotect-CP^® did not inhibit viral growth in infected villi. No impact on villi viability was observed.

Conclusions: These results sustained that Cytotect CP^® has the potential to prevent CMV congenital infection.

Keywords: Cytotect CP®; congenital cytomegalovirus; hyperimmune immunoglobulins.

Figure 1

Ex vivo assays. Three different protocols were used to evaluate the efficacy of Cytotect CP^®. For all ex vivo assays, HEF cells were seeded into 48-well plates at 10⁵ cells/well and then incubated for 5 days at 37 °C in 5% CO₂ until confluence. The villi explant model is based on infection from cell-free virus produced by infected fibroblasts. HEF cells were infected with cell-free virus stock of the TB40/E strain with a multiplicity of infection (MOI) of 1 in HEF culture medium. The addition of explants on a gelfoam (Spongostan dental™, NewPharma, Belgium) allowed infection by capillarity. We performed three types of assays, mimicking several real-life conditions in maternal blood, at the placental level, and after placental infection (A) Neutralization assay: The virus was neutralized by Cytotect CP^® before the addition of placental explants. TB40/E and Cytotect CP^® were incubated on an HEF plate for 3 h at 37 °C in 5% CO₂ before renewing the medium. After 5 days of incubation, a sponge with a placental villi explant was added in each well and was incubated at 37 °C in 5% CO₂. Day 0 was the first day of infection of the villi. (B) Prevention of villi infection assay: Cytotect CP^® and explants were added simultaneously after 5 days of infection of the cells. Day 0 was the first day of infection of the villi. (C) Efficacy assay: Villi were added after 5 days of infection of the cells. After 5 days of infection of the villi, sponges and explants were transferred on new plates without cell monolayers and Cytotect CP^® was added. Day 0 was 5 days after placental infection.

Figure 2

Neutralization activity of Cytotect CP^® in vitro. The number of foci after the neutralization assay were counted for (A) the TB40/E strain and (B) the VHL/E strain on human embryonic fibroblast HEF cells and epithelial ARPE-19 cells after 5 days of incubation (n = 3 assays).

Figure 3

Kinetics of the endotheliotropic strain TB40/E in villi explants. The mean viral loads on days 7, 14, and 21 post-infection in the villi explants. The whiskers are extended to the extreme data points.

Figure 4

Neutralization assay: ex vivo viral load in villi at 7 and 14 days post-infection. The results were compiled from three placentae. The statistical analyses were carried out using GraphPad Prism with a two-way ANOVA (GraphPad Software, San Diego, CA, USA). * p < 0.05.

Figure 5

Prevention of villi infection assays. Virus and Cytotect CP^® were added at the time of explant infection. The viral loads in the villi were measured at 7 and 14 days post-infection by comparison with the infected and non-treated villi (positive control “0”). The results were compiled from three placentae. The statistical analyses were carried out using GraphPad Prism with a two-way ANOVA (GraphPad Software, San Diego, CA, USA). The whiskers are extended to the extreme data points.

Figure 6

Efficacy assays. Viral loads were measured at 7 and 14 days post-treatment of infected villi and compared with infected and non-treated villi (positive control “0”). The results were compiled from three separate placentae. The boxplots are designed as medians (Q2), lower quartiles (Q1), and upper quartiles (Q3). The whiskers are extended to the extreme data points.

Figure 7

Placenta viability at 7 and 14 days post-infection for the “neutralization” and “prevention” assays and at 14 and 21 days post-infection for the “efficacy” assay. β-hCG units are indicated per 10⁶ cells for each concentration. In this figure, “0” corresponds to the infected villi without Cytotect CP^® (positive control). The boxplots are designed as medians (Q2), lower quartiles (Q1), and upper quartiles (Q3) from three placentae. The whiskers are extended to the extreme data points. No significant difference was found between concentrations.

Figure 8

Placenta viability. (A) Comparison of β-hCG levels between the negative control villi and villi infected with TB40E without Cytotect CP^® after 7, 14, and 21 days of infection. (B) Comparison of β-hCG levels from the negative control villi and villi with only Cytotect CP^® after 7 and 14 days of exposure to the maximum tested dose. The boxplots are designed as medians (Q2), lower quartiles (Q1), and upper quartiles (Q3). The whiskers are extended to the extreme data points.