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
. 2011 Nov;204 Suppl 3(Suppl 3):S825-32.
doi: 10.1093/infdis/jir295.

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles

Osvaldo Martinez  1 , Lee Tantral, Nirupama Mulherkar, Kartik Chandran, Christopher F Basler
Affiliations

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles

Osvaldo Martinez et al. J Infect Dis. 2011 Nov.

Abstract

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Production of virus-like particles. (A) Produced virus-like particles (VLPs) expressed 1 of 4 different glycoproteins (GPs): wild-type Ebola virus GP (EBGP), Ebola virus GP with its mucin-like domain removed (ΔMucGP), Ebola virus GP with its mucin-like domain replaced with Crimean–Congo hemorrhagic fever virus mucin-like domain (CMsubGP), and vesicular stomatitis virus GP (VSV-G). Spent supernatant from virion protein 40 (VP40) and EBOV GP plasmid transfected cells were purified, and (B) the GP were identified by size by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and GelCode Blue staining, and (C) antibody specificity with Western blot analysis. VP40 is detectable by total protein stain on ΔMucGP, EBGP, and CMsubGP VLP, but not VSV-G VLP. Significant bands present in GelCode Blue stained total protein gels are denoted by brackets, which correspond to the ▵mucin GP1 subunit at ∼60kDa, wild-type Ebola virus GP1 at ∼130 kDa, and CMsubGP smear at ∼120 kDa. Polyclonal rabbit αN-terminus of EBGP antibody was used to stain GP1. VSV-G is present on the stained protein gel at 60 kDa, and is bound by monoclonal αVSV-G antibody. The 50k Da band detected in the CMsubGP lane (*) is likely to be a degradation product. Anti-EBOV mucin-like domain antibody (9c11) was used to stain EBOV mucin-like domain. (D) Beta-lactamase equivalents of purified beta-lactamase VP40 (BLAVP40), BLAVP40 + EBGP, BLAVP40 + CMsubGP, and BLAVP40 + ΔMucGP VLPs were tested for their ability to infect dendritic cells. The region shows the percentage of cells infected.
Figure 2.
Figure 2.
Antibody titration of virion protein 40 glycoprotein–immunized mouse sera by Western blot. A, Mice were immunized with virus-like particles (VLPs) according to the schedule shown, and sera from the final bleed were used in experiments as shown. B, Sera from mice treated with phosphate-buffered saline (PBS sera) and (C) sera from mice immunized with vesicular stomatitis virus glycoproteins (VSV-G) VLPs (VSV-G Sera) blots show no binding to any of the 3 Ebola virus glycoprotein (EBGP) constructs at dilutions of 1:250. D, EBGP sera, (E) Ebola virus glycoprotein without its mucin-like domain (ΔMucGP) sera, and (F) Ebola virus glycoprotein with a Crimean–Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP) sera were diluted 1:1000 to 1:30,000 and used to stain Western blots of VLPs.
Figure 3.
Figure 3.
Serum antibody binding against cell surface–expressed glycoproteins. (A) CHO-K1 cells were cotransfected with green fluorescent protein (GFP) and limiting amounts of Ebola virus glycoprotein (EBGP), Ebola virus glycoprotein without its mucin-like domain (ΔMucGP), or Ebola virus glycoprotein with a Crimean–Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP) expression plasmids and approximately 48 h later were harvested, incubated with mouse sera, and stained with phycoerythrin-conjugated donkey αMouse immunoglobulin G. Antibody binding is shown as a percentage of phycoerythrin-positive cells, initially gated on GFP-positive cells by flow cytometry. Binding that is significantly different by 1-tailed t test is indicated by brackets labeled with either 2 asterisks (**) for P < .01 or 1 asterisk (*) for P < .05. Enzyme-linked immunosorbent assay (ELISA ) performed with EBGP, ΔMucGP, CMsubGP and phosphate-buffered saline (PBS) sera at indicated dilutions. ELISA plates were coated with either (B) recombinant vesicular stomatitis virus (rVSV)-EBGP or (C) rVSV-ΔMucGP. The hatched line denotes the average optical density (OD) + 2 standard deviation calculated using the PBS sera binding.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sanchez AG, Thomas W, Heinz F. Filoviridae: Marburg and Ebola viruses. In: Knipe DM, editor. Fields virology. Philadelphia: Lippincott Williams and Wilkins, 2007.
    1. Takada A, Robison C, Goto H, et al. A system for functional analysis of Ebola virus glycoprotein. Proc Natl Acad Sci U S A. 1997;94:14764–9. - PMC - PubMed
    1. Wool-Lewis RJ, Bates P. Characterization of Ebola virus entry by using pseudotyped viruses: Identification of receptor-deficient cell lines. J Virol. 1998;72:3155–60. - PMC - PubMed
    1. Sanchez A, Yang ZY, Xu L, Nabel GJ, Crews T, Peters CJ. Biochemical analysis of the secreted and virion glycoproteins of Ebola virus. J Virol. 1998;72:6442–7. - PMC - PubMed
    1. Kuhn JH, Radoshitzky SR, Guth AC, et al. Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavirus bind a common receptor. J Biol Chem. 2006;281:15951–8. - PubMed

Publication types

-