Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec 2;13(12):2883-2893.
doi: 10.1080/21645515.2017.1347740. Epub 2017 Jul 12.

Epitope Mapping of Ebola Virus Dominant and Subdominant Glycoprotein Epitopes Facilitates Construction of an Epitope-Based DNA Vaccine Able to Focus the Antibody Response in Mice

Affiliations
Free PMC article

Epitope Mapping of Ebola Virus Dominant and Subdominant Glycoprotein Epitopes Facilitates Construction of an Epitope-Based DNA Vaccine Able to Focus the Antibody Response in Mice

Daniel A J Mitchell et al. Hum Vaccin Immunother. .
Free PMC article

Abstract

We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.

Keywords: DNA vaccine; Ebola virus; antibody; epitope mapping; filovirus; focusing; mice.

Figures

Figure 1.
Figure 1.
Antibody binding after full substitution mutagenesis of 391TPVYKLDISEATQVEQHHRRTDNDS415. Substituted amino acids are shown on the right side of each heat map. Green indicates significantly reduced binding. (A) The core of the mAb 13F6 epitope was determined to be 406QHHRRTD412, with 406QXXRXT411 being most essential for binding. (B) The core of mAb 6E3 was also 406QHHRRTD412, with 406QXHRR410 most essential to binding. (C) The core of the epitope for mAb 6D8 was determined to be 394YKLDI398, with 395KLD397 as the most essential amino acids. (D) Polyclonal sera from mice vaccinated with WT GP of EBOV showed strongest binding to 397DISEAT402, with 398ISXXT402 as the most crucial amino acids.
Figure 2.
Figure 2.
Full substitution mutagenesis of mAbs 13C6 and 6D8. The letter plots depict pepscan results for each peptide on the X axis with recorded intensities (optical density, OD) expressed in arbitrary units (AU) plotted on the Y axis. (A) Binding of mAb 6D8 to a full series of substitutions of 391TPVYKLDISEATQVEQHHRRTDNDS415 indicates decreased binding upon mutations to 393VYKLD397 (boxed), which is thought to be the epitope core. (B) Binding of mAb 6D8 to a full substitution series of 470GEESASSGKLGLITNTIAGVAGLIT494 indicates no binding to this stretch of amino acids. (C) Binding of mAb 13C6 to a full substitution series of 470GEESASSGKLGLITNTIAGVAGLIT494 indicates decreased binding upon mutations to 487GVAGLIT493 (boxed) and appears to be generally sensitive to introduction of negative charges into the peptides. (D) Binding of mAb 13C6 to a full substitution series of 391TPVYKLDISEATQVEQHHRRTDNDS415 indicates no binding to this stretch of amino acids.
Figure 3.
Figure 3.
Relative binding of serum samples from mice, guinea pigs and rhesus macaques to amino acids in CLIPS. Blue indicates strong binding and tan indicates weaker binding. No color indicates no measureable binding with that particular sample.
Figure 4.
Figure 4.
EBOV GP structural modeling. A surface model representation of the EBOV GP trimer is shown in white. The 3D representation omits the mucin-like domain in GP, which is considered unstructured. Smooth regions of the surface highlight the parts of the structure that were determined through X-ray crystallography. Other portions of the structure that were not resolved experimentally have been modeled “de novo” and are shown as collections of dots to indicate the approximate location of the missing fragments. Epitopes M1 to M4 identified in the CLIPS study and included in the multi-epitope constructs described in Table 1 are highlighted in purple, blue, magenta and red, respectively.
Figure 5.
Figure 5.
Mep 1 and Mep2 expression. (A) Cells were transfected with the Mep1 or Mep2 DNA vaccine constructs or with the WT GP DNA vaccine and stained with DAPI (top panels). Immunofluorescent antibody staining was performed with EBOV mouse mAb 6D8 (middle panels). Lower panels show a merge of the top and middle panels. (B) ELISA was performed on cell supernatants of mock-transfected cells or cells transfected with the Mep1 or Mep2 constructs. (C) Groups of mice (N = 10) were vaccinated 3 times with empty plasmid vector, the WT GP DNA vaccine, or the Mep1 or Mep2 constructs. ELISA was performed on serum samples obtained 3 weeks after each vaccination using inactivated EBOV virions as antigen.
Figure 6.
Figure 6.
ELISA using linear peptides containing epitopes included in Mep2. Linear peptides are as defined in Table 1 and Table S1. The epitopes contained in part or in whole in each of the peptides are identified beneath the peptide numbers. (A) Samples from mice vaccinated once with the WT GP DNA vaccine followed by 2 vaccinations with an empty plasmid vector control; (B) samples from mice vaccinated 3 times with the WT GP DNA vaccine; (C) samples from mice vaccinated 3 times with the Mep2 DNA vaccine; and, (D) samples from mice vaccinated once with the WT GP DNA vaccine followed by 2 vaccinations with the Mep2 DNA vaccine.

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Grant-Klein RJ, Altamura LA, Badger CV, Bounds CE, Van Deusen NM, Kwilas SA, Vu HA, Warfield KL, Hooper JW, Hannaman D, et al. Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges. Hum Vaccines Immunotherapeutics. 2015;11:1991-2004. doi:10.1080/21645515.2015.1039757 - DOI - PMC - PubMed
    1. Grant-Klein RJ, Van Deusen NM, Badger CV, Hannaman D, Dupuy LC, Schmaljohn CS. A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge. Hum Vaccines Immunotherapeutics. 2012;8:1703-6. doi:10.4161/hv.21873 - DOI - PMC - PubMed
    1. Mellquist-Riemenschneider JL, Garrison AR, Geisbert JB, Saikh KU, Heidebrink KD, Jahrling PB, Ulrich RG, Schmaljohn CS. Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs. Virus Res. 2003;92:187-93. doi:10.1016/S0168-1702(02)00338-6. PMID:12686428 - DOI - PubMed
    1. Corti D, Voss J, Gamblin SJ, Codoni G, Macagno A, Jarrossay D, Vachieri SG, Pinna D, Minola A, Vanzetta F, et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science. 2011;333:850-6. doi:10.1126/science.1205669. PMID:21798894 - DOI - PubMed
    1. Timmerman P, Puijk WC, Meloen RH. Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology. J Mol Recognition: JMR. 2007;20:283-99. doi:10.1002/jmr.846. PMID:18074397 - DOI - PubMed

Publication types

MeSH terms

Feedback