Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 9;91(13):e01650-16.
doi: 10.1128/JVI.01650-16. Print 2017 Jul 1.

Impact of Antibodies and Strain Polymorphisms on Cytomegalovirus Entry and Spread in Fibroblasts and Epithelial Cells

Xiaohong Cui  1 , Daniel C Freed  2 , Dai Wang  2 , Ping Qiu  2 , Fengsheng Li  2 , Tong-Ming Fu  2 , Lawrence M Kauvar  3 , Michael A McVoy  4
Affiliations

Impact of Antibodies and Strain Polymorphisms on Cytomegalovirus Entry and Spread in Fibroblasts and Epithelial Cells

Xiaohong Cui et al. J Virol. .

Abstract

Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell to cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including glycoprotein B (gB), gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial cell entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts, the formation of syncytiumlike structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N terminus of gH, exhibited strain specificity, and neutralized both epithelial cell and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion and may assist in determining which antibodies may be most efficacious following active immunization or passive administration.IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts, (ii) demonstrate that antibodies can restrict the formation in vitro of syncytiumlike structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo, and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain and cell type dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion and may have important implications for the development of CMV vaccines and immunotherapeutics.

Keywords: antibodies; cytomegalovirus; fusion; neutralization; neutralizing antibodies; polymorphisms; spread; virus entry.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Antibodies to gB, gH/gL, gH, the pentamer, or pentamer subunits inhibit CMV spread in epithelial cells. (A) ARPE-19 epithelial cell monolayers in 96-well plates were infected with 100 PFU/well of CMV virus ABV (strain Uxc). After incubation at 37°C for 24 h, the cultures were washed and medium (∅) or medium containing serial dilutions of the indicated antibodies was added. Representative micrographs were taken on day 16 postinfection. Dilution factors on the left indicate dilutions from initial stocks of HIG (5 mg/ml), rabbit anti-UL130 or anti-UL131 antiserum (diluted 1:5), or MAbs (100 μg/ml). (B) Five hundred PFU of CMV virus ABV (strain Uxc) was incubated with medium (∅) or medium containing serial dilutions of the indicated antibodies for 1 h at 37°C and then added to ARPE-19 monolayers in 96-well plates. Representative micrographs were taken on day 6 postinfection. Dilution factors on the left indicate dilutions from initial stocks of HIG (1.25 mg/ml), rabbit anti-UL130 or anti-UL131 antiserum (diluted 1:40), or MAbs (25 μg/ml, except for TRL310 and 57.4 [2 μg/ml] and 2-25 [1 μg/ml]).
FIG 2
FIG 2
Antibody inhibition of CMV spread in epithelial cells correlates with neutralization of epithelial cell entry. GFP-based assays using CMV virus ABV (strain Uxc) were used to determine IC50s (μg/ml) for epithelial cell spread inhibition and epithelial cell entry neutralization for 14 MAbs (see Fig. 1 and Table 2). For each antibody, log-transformed IC50s for spread inhibition were plotted versus IC50s for neutralization, and linear regression was used to calculate the indicated best-fit line, correlation coefficient (r), and P value.
FIG 3
FIG 3
CMV spread in fibroblasts is insensitive to antibody inhibition. MRC-5 fibroblast monolayers in 96-well plates were infected with 100 PFU/well of CMV virus ABV (strain Uxc). After incubation at 37°C for 24 h, the cultures were washed and medium (∅) or medium containing dilutions of the indicated antibodies was added. Representative micrographs were taken on day 8 postinfection. Micrographs of neutralizing assays performed as described in the legend to Fig. 1B but using MRC-5 fibroblasts are included for comparison. Dilution factors on the left indicate dilutions from initial stocks of HIG (5 mg/ml), rabbit anti-UL130 or anti-UL131 antiserum (diluted 1:10), or MAbs prediluted to 100 μg/ml.
FIG 4
FIG 4
Antibodies to gB, gH/gL, and the pentamer inhibit the formation of syncytiumlike structures in CMV-infected fibroblasts. (A) MRC-5 fibroblast monolayers in 96-well plates were infected with 50 PFU/well of virus BADr. After incubation at 37°C for 24 h, the cultures were washed and medium (∅) or medium containing HIG (5 mg/ml) or MAb (100 μg/ml for TRL345, 25 μg/ml for all others) was added. Representative micrographs were taken on day 7, 10, or 13 postinfection. (B) MRC-5 fibroblast monolayers in 96-well plates were infected at the indicated MOIs with a pentamer-negative (HB15) or pentamer-positive (BADr) variant of strain AD169 or a pentamer-negative (TS15) or pentamer-positive (TS15-rN) variant of strain Towne. Representative micrographs were taken on days 3 and 6 postinfection.
FIG 5
FIG 5
Four rabbit MAbs recognize linear epitopes in CMV gH. (A) ARPE-19 cells were infected with viruses representing strain AD169 (BADr), Towne (TS15-rN), or Uxc (ABV), and infected-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the indicated rabbit MAbs or with a rabbit antipeptide antiserum specific for gH (anti-gH antiserum). Locations of molecular mass markers are indicated. (B) A fluorescence-based peptide array identified residues 27 to 48 in gH from AD169 as binding selectively to all four MAbs. Within this region, ClustalW alignment of 71 gH peptide sequences in the NCBI database identified five sequence variants that could be assigned to two groups represented by AD169 or Towne. Shown is a ClustalW alignment of the five variants assigned to the AD169 or Towne group. Frequencies within the database are indicated for each group and for individual variants. Signal intensities (fluorescence units) from the peptide array indicate binding of MAb 15.1 to each variant peptide. Similar results (not shown) were obtained for antibodies 58.5 and 223.4. Dashed box indicates the AP86 epitope (residues 34 to 43) that is reported to react with murine MAb AP86-SA4 (59). (C) Two synthetic peptides comprised of gH residues 27 to 48 from AD169 or 27 to 47 from Towne were used as substrates in ELISAs to confirm binding specificities of the four rabbit MAbs. The data shown are representative of two independent experiments.
FIG 6
FIG 6
Fibroblast and epithelial cell neutralizing activities of antibodies to linear epitopes in gH are strain specific. (A) MAbs recognizing linear epitopes in gH were assayed as described in the legends to Fig. 1B and 3 for neutralization of viruses representing CMV strain Uxc (ABV) or Towne (TS15-rN) using MRC-5 fibroblasts or ARPE-19 epithelial cells. Representative micrographs were taken on day 3 (TS15-rN/MRC-5), 4 (ABV/MRC-5 and TS15-rN/ARPE-19), or 6 (ABV/ARPE-19) postinfection. For ABV-based assays, the dilution factors on the left indicate dilutions from initial stocks of HIG (1.25 mg/ml) or MAbs (25 μg/ml). For TS15-rN-based assays, the dilution factors on the left indicate dilutions from initial stocks of HIG (2.5 mg/ml) or MAbs (50 μg/ml). (B) The indicated MAbs were assayed for neutralization of viruses representing CMV strains AD169 (BADr), Towne (TS15-rR), NR, and TB40/E. Neutralizing titers are inverse IC50s (μg/ml); error bars indicate 95% confidence limits.
FIG 7
FIG 7
Fibroblast but not epithelial cell neutralizing activities of MAbs to discontinuous epitopes in gH/gL are strain specific. (A) Antibodies recognizing discontinuous epitopes formed by gH/gL were assayed as described in the legend to Fig. 6A. (B) The indicated MAbs were assayed as described in the legend to Fig. 6B.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Yeager AS, Grumet FC, Hafleigh EB, Arvin AM, Bradley JS, Prober CG. 1981. Prevention of transfusion-acquired cytomegalovirus infections in newborn infants. J Pediatr 98:281–287. doi:10.1016/S0022-3476(81)80662-2. - DOI - PubMed
    1. Adler SP, Chandrika T, Lawrence L, Baggett J. 1983. Cytomegalovirus infections in neonates acquired by blood transfusions. Pediatr Infect Dis 2:114–118. doi:10.1097/00006454-198303000-00009. - DOI - PubMed
    1. Adler SP, Starr SE, Plotkin SA, Hempfling SH, Buis J, Manning ML, Best AM. 1995. Immunity induced by primary human cytomegalovirus infection protects against secondary infection among women of childbearing age. J Infect Dis 171:26–32. doi:10.1093/infdis/171.1.26 (Erratum, 171: 1080, doi:10.1093/infdis/171.4.1080-a.) - DOI - DOI - PubMed
    1. Nigro G, Adler SP, La Torre R, Best AM. 2005. Passive immunization during pregnancy for congenital cytomegalovirus infection. N Engl J Med 353:1350–1362. doi:10.1056/NEJMoa043337. - DOI - PubMed
    1. Addo MM, Rosenberg ES. 2002. Cellular immune responses in transplantation-associated chronic viral infections. Transpl Infect Dis 4:31–40. doi:10.1034/j.1399-3062.2002.00006.x. - DOI - PubMed

Publication types

Substances

LinkOut - more resources