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. 2014 Mar 19;6(3):1346-64.
doi: 10.3390/v6031346.

HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

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HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

Martin Zydek et al. Viruses. .
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Abstract

Human cytomegalovirus (HCMV) is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs), the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs) give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs). Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A) showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb), TRL345, reactive with glycoprotein B (gB), but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

Figures

Figure 1
Figure 1
TBPCs express markers of trophoblasts and pluripotency. TBPCs from 7.3 weeks (left panel) and 15.6 weeks (right panel) of gestation were immunostained for CK7 (A, E), HMGA2 (B, F), GATA-4 (C, G) or GATA-3 (D, H). Nuclei were counterstained with DAPI. Scale bar = 100 µm.
Figure 2
Figure 2
VR1814 and AD169 replicate in TBPCs. Cells from 15.6 weeks (A) or 7.3 weeks (B) of gestation were infected (MOI 2) and immunostained for HCMV IE1, pUL112/pUL113 or pp28 at the indicated time points (green). Mock-infected controls were immunostained for CK7 (red). Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm.
Figure 3
Figure 3
(A) Infected TBPCs release progeny virions. CM from VR1814- and AD169-infected TBPCs and HFFs (MOI 0.1) were collected at the indicated time points and titrated on HFFs. (B) Progeny virions from TBPCs retain tropism. CM from VR1814- and AD169-infected TBPCs (8 dpi) was added to HFFs and ARPE-19 cells, and IE1 was immunostained (1 dpi). Nuclei were counterstained with DAPI. Scale bar = 200 µm.
Figure 4
Figure 4
HCMV pentamer complex is dispensable for virion entry into TBPCs. ARPE-19 cells (AF), HFFs (GL) and TBPCs (MS) were mock infected or infected (MOI 2) with parental TB40/E and UL131A-deficient mutant (ΔUL131A) and immunostained for IE1 proteins (2 dpi). Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm.
Figure 5
Figure 5
Infection of TBPCs is blocked by a mAb to gB but not mAbs to the pentamer complex. (A) VR1814 (MOI 0.01) was pre-incubated with medium alone, a serial log10 dilution of the indicated mAb (0.01–10 µg/mL) or HIG (0.1–100 µg/mL). Virus-antibody mixtures were then applied to ARPE-19 cells. At 2 dpi, IE1 was immunostained and numbers of infected cells were counted. Data were normalized to the no Ab control. Error bars indicate standard deviation. (B) TBPCs were incubated with virus-antibody mixtures and analyzed as described for panel A.
Figure 6
Figure 6
Neutralizing anti-gB mAb TRL345 precludes VR1814 infection and rescues TBPC differentiation. VR1814 was pretreated with 20 µg/mL of the indicated mAbs, and the mixtures were adsorbed to TBPCs. At 3 dpi, the cells were reseeded on matrigel and cultured in differentiation medium for 24 h followed by light microscopic analysis. Lower magnification photographs (upper panel), higher magnification photographs (lower panel). Scale bars = 200 µm.

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