ISMB2010_Poster
- 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 ¨Cshort, 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 ¨C EAIVNAQPKCNPN¡MHNQDG¨C 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 ¨CHKILTAG¨C 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 ?, 2.77 ?, 3.24 ?, 3.35 ?, and 2.90 ?. 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 ¨CDYHKILTAGL¨C 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