I. Computational analysis identified novel B- and T- cell epitopes from the glycoprotein and nucleoprotein of Ebola virus, including AIGLAWIPY, YDDDDDIPF, SQDTTIPDV, VSHLTTLAT, DLVLFDLDE, LRQLANETTT, and DYHKILTAGL. II. A novel B-cell epitope was extracted from the 3D structure of Ebola glycoprotein bound to an antibody, and its sequence motifs were predicted by multiple algorithms. III. An epitope from the nucleoprotein contained a conserved protein fingerprint that defines the WW domain-containing WAC protein, and a potential WW domain was

1. Motivation:
Despite global vaccination efforts, a great need for new and improved vaccines against numerous human
pathogens still remains. Rational vaccine design (RVD) seeks to manipulate the immune system to “work
harder” by enhancing B- and T-cell recognition of epitopes –short, antigenic peptides that mediate the
cellular arm of the immune system. The first step in RVD is epitope identification and selection.
Therefore, computational analysis was completed on the glycoprotein (GP) and nucleoprotein (NP) of
Ebola, one of the deadliest viruses known.
Results:
Epitope prediction algorithms identified novel B- and T- cell peptides including AIGLAWIPY, YDDDDDIPF,
SQDTTIPDV, VSHLTTLAT, DLVLFDLDE, LRQLANETTT, and DYHKILTAGL for RVD against the Ebola virus
(EBOV). The efficiency of the algorithms in predicting biologically confirmed epitopes, suggests the novel
peptides were “rationally” selected based on their biochemical properties. A novel B-cell epitope –
EAIVNAQPKCNPN…MHNQDG– was extracted from a three-dimensional structure of the Ebola GP bound
to human antibody KZ52, and its sequence motifs were predicted by multiple algorithms. A protein
fingerprint –HKILTAG– was found in NP epitope DYHKILTAGL; this epitope is listed in the Immune Epitope
Database and Analysis Resource (IEDB). The fingerprint defines the WW domain-containing WAC protein,
and a potential WW domain was discovered in the nucleoprotein of all five EBOV species. RVD towards
the Ebola virus is thus feasible, as it is possible to discover valid B- and T- cell epitopes using
computational techniques. In addition to serving as templates for vaccine design, computationally
derived epitopes can explain viral protein conservation.
Methods
Novel Epitopes of the Ebola Virus for Rational Vaccine Design:
Therapeutic Development and Protein Conservation
Sophia Banton, Zvi Roth PhD, Mirjana Pavlovic MD/PhD
Department of Electrical Engineering (Bioengineering). Florida Atlantic University, Boca Raton, USA
Abstract Results
Introduction
Immune cells rely on epitopes to elicit antigen specific immune
responses. Epitopes are presented between cells through the major
histocompatibility (MHC) complex of which there are two types. All
nucleated cells possess MHC-1 molecules and all antigen presenting
cells (APCs) have MHC-2 molecules. The MHC complexes bind T-cell
receptors. Cytotoxic T-cells (CD8+) interact with MHC-1 and helper T-
cells (CD4+) with MHC-2. As APCs B-cells communicate directly with
CD4+ T-cells.
The Ebola virus (EBOV) is one of the deadliest viruses known to
mankind with mortality rates ranging from 23% to 90%. Five species
of EBOV have been identified and Zaire Ebola is the most virulent. The
virus’ classification as a level-4 bioterrorism agent highlights the need
to develop protective methods for quickly controlling the virus’
spread.
A vaccine is any preparation used as a preventive inoculation to confer immunity against a specific
disease. Currently, there are no licensed Ebola prophylaxes, treatments, or vaccines and traditional
vaccine design methods do not comply with the EBOV. Rational vaccine design (RVD) has two aims. The
first is to minimize the trial-and-error approach behind current vaccine design strategies, and the second
is to maximize the production of immune cells in the body post vaccination. The latter can be achieved
via the exploitation of viral properties. Successful RVD is based on the optimal selection of epitopes -
antigenic determinants, usually made of protein, that are recognized by the immune system.
Figure 1: TEM of the Ebola virus
(Scott Camazine/Photo Researchers Inc.)
Figure 2: B cell Antigen presentation to a
CD4+ T cell
Computational tools were used to identify epitopes of the two main Ebola antigens: the glycoprotein and
the nucleoprotein.
Sequence Analysis
(1.) Epitope prediction servers were used to identify B- and T-cell epitopes for the Zaire EBOV antigens.
To ensure data integrity, servers with established credibility were included: IEDB (ESAP, KTA), Biopred,
NETM_ANN, IEDB_ANN, PROPRED, and NETHMHCII.
(2.) Zaire EBOV sequences were tested in the OMA and PROSITE databases to identify protein
fingerprints and patterns.
(3.) Sequence alignments on the amino acid sequences of the Zaire, Sudan, Reston, Ivory Coast, and
Bundibugyo EBOV nucleoproteins (NCBI accession no. NC_002549, NC_006432, and NC_004161;
GenBank accession no. ACI28629.1 and ACI28620.1) were completed using ClustalW.
Structural Analysis
(1.) PDB structure 3CSY of the Zaire EBOV glycoprotein bound to human antibody KZ52 was visualized
and analyzed in Pymol for noncovalent protein binding interactions.
(2.) Three-dimensional protein structure prediction was completed using the (PS)2: protein prediction
server (http://ps2.life.nctu.edu.tw/) and PDB Structures 1I5H, 2YSB, and 1K9R.
Antigen Type Length (aa) Virulence
Glycoprotein Polypeptide 676 Host cell attachment & Entry
Nucleoprotein Polypeptide 739 Virus Replication
II. EBOV Glycoprotein contains novel three-dimensional epitopes
III. EBOV Nucleoprotein contains the WAC protein fingerprint and a
WW domain
Figure 5. Structural prediction of EBOV WW domains
(A) Predicted structure of ZEBOV nucleoprotein WW domain based on SAV1 (orange), NEDD4 (green), and YAP65 (purple). (B)
NEDD4 WW domain (PDB 1I5H). (C) SAV1 WW domain (PDB 2YSB). (D)YAP65 WW domain (PDB 1K9R). Tryptophan (W)
residues shown in red and indicate the start and finish of each WW domain.
Results
I. Epitope Prediction Servers identified Novel EBOV epitopes
Conclusions
Acknowledgments
Federation of American Societies for Experimental Biology (FASEB)
Department of Biological Sciences, FAU
FASEB
Figure 3. Structural analysis of the Zaire EBOV glycoprotein bound to human antibody KZ52
(A) The trimeric Ebola Virus glycoprotein (silver) interacts with the light (orange) and heavy (green) chains of human antibody
KZ52. Purple boxes indicate sites of interactions, all of which are identical. (B) The EBOV GP epitope (purple) interacts with
antibody loops at GP slits. (C) Salt bridges stabilize epitope-antibody binding. Salt-bridge measurements from top to bottom
are 3.41 Å, 2.77 Å, 3.24 Å, 3.35 Å, and 2.90 Å.
Figure 4. EBOV WAC fingerprint and WW domain sequence conservation
(A) Epitope NP20-29 is conserved in all five Ebola virus species: Bundibugyo (BEBOV), Ivory Coast (CIEBOV), Zaire (ZEBOV),
Reston (REBOV), and Sudan (SEBOV). (B) WW domains in EBOV species, WAC proteins, and mammalian proteins: PIN1, NEDD4,
YAP65, and SAV1. (h, human; r, rat; m, mouse)
Computational methods used in this study discovered the first concrete epitope of the Ebola virus,
completing the first step of RVD. Epitope EAIVNAQPKCNPN…MHNQDG was extracted from the first
solved three-dimensional structure of a human antibody bound to the EBOV glycoprotein, and its
variants were predicted as B-cell and MHC-2 epitopes. This peptide is the best vaccine candidate, as it is
the only epitope “physically” shown to interact with the EBOV inside a human body. In addition to the
primary epitope, multiple B- and T-cell peptides were selected as the best candidates for RVD. Some
peptides were novel, many were promiscuous, and others have been validated by in vivo studies. The
novel peptide motifs included AIGLAWIPY, YDDDDDIPF, SQDTTIPDV, VSHLTTLAT, DLVLFDLDE,
LRQLANETTT, DYHKILTAGL and their variants.
As demonstrated here, the computational prediction of epitopes for RVD is an efficient three step
process: sequence analysis, selection of promiscuous motifs, and structural analysis. With step one of
RVD completed, the next step in vaccine development is to reconstitute the selected epitopes as
immunogenic compounds and implement then as vaccine deliverables.
The biological role of epitopes appears multifunctional. An Ebola nucleoprotein epitope –DYHKILTAGL–
contains a conserved eukaryotic protein fingerprint, which led to the identification of its WW domain.
Biological discovery is often accidental and it turns out that computationally derived epitopes not only
hold promise for vaccine design and therapeutic development, but can also provide insight on viral
protein conservation.
Glycoprotein Nucleoprotein
Epitope B-cell MHC1:CD8+ MHC2:CD4+ B-cell MHC1:CD8+ MHC2:CD4+
_DDDIPF_ DDDIPF YDDDDDIPFGP YDDDDDIPFP
_DTTIP_ *DTTIP SQDTTIPDV DTTIPDVVVD
_WIPYFGPAA_ AIGLAWIPFYGP *WIPYFGPAAEGIYTE
WIPYFGPAA
IPYFGPAAE
AIGLAWIPF
WIPYFGPAA
WIPYFGPAAE
IGLAWIPFY
_EAIVNAQPK_ IVNAQPKCNPNLHYW
EAIVNAQPKCNPN
REAIVNAQPK
IVNAQPKCN
REAIVNAQ
_FKAALSSL_ SFKAALSSLAKHGEYAP FKAALSSLA
*SFKAALSSL
FKAALSSLA
KAALSSLAKH
VNSFKAALS
_RLMRTNFL_ MVIFRLMRTNFL
*RLMRTNFL
VIFRLMRTN
MVIFRLMRTNFL
FRLMRTNFL
FRLMRTNFLI
_YHKILTAGL_ DYHKILTAG
HKILTAGLS…
YHKILTAGL
YHKILTAGLS
_VSHLTTLAT_ EAAVSHLTTLATISTSPQSLT VSHLTTLAT
AVSHLTTLAT
_FLSFASL _ GQFLSFASLFLP *FLSFASLFL
*GQFLSFASL
ANAGQFLSF
LSFASLFLPK
_MHNQDG _ MHNQDG PAAEGIYIEGLMHNQ
AEGIYIEGLMHNQDG
DLVLFDLD_ DLVLFDLD DLVLFDLDE
KTLEAITAA LAKLTEAITAASLPKTS *KLTEAITAA
LTEAITAASLPK
TEAITAASL
_DAVLYYHMM_ FDAVLYYH *DAVLYYHMM
_LRQLANETT_ LRQLANETT
GLRQLANETTQA
LRQLANETT
Table 1. Best Computationally Derived Epitopes
Numerous epitopes were found for receptors on B-cells, CD4+ helper T-cells, and CD8+ cytotoxic T-cells. Shared motifs indicate the
epitopes are promiscuous among immune cells and are the best choices for vaccine design. Motifs extracted from PDB structure
3CSY are shown in green. *Biologically confirmed epitopes
protected.F1000
Posters.C
opyrightprotected.
ers.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Poster
d.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightp
ightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightp
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Poste
1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrightprotected.
.C
opyrightprotected.F1000
Posters.C
opyrightprotected.F1000
Posters.C
opyrig
rotected.F1000
Posters.C
opyrightprotected.F1000
Po
00
Posters.C
opyrightprotec