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Ebola Peptides for Diagnostics and Vaccines

Ebola Peptides for Diagnostics and Vaccines


Peptides derived from Ebola virus proteins can be used to study antigenicity and immunogenicity of Ebola proteins. In addition, these peptide epitopes can be used further to develop sensitive and accurate diagnostic tests using polyclonal or monoclonal antibodies. Another potential use for this type of peptides is for the development of unique peptide-based vaccines. In particular, succesful and potent vaccines could be developed using antigenic peptides derived from proteins of the Ebola virus or other Ebola virus strains. 
 

Figure 1: Ultra structures and models of the Ebola virus and its genome (Source: Ellis et al. 1978; CDC).  Ellis et al. in 1978 showed that electron microscopy can be used to detect and observe the ultrastructure of the Eboli virus in infected human tissue. The Ebola virus was detected in tissue samples from human liver, kidney, spleen and lung.   

Infection of a cell by a virus requires the fusion between viral and host membranes. Infection of a cell by the Ebola virus (EboV) begins with the uptake of viral particles into cellular endosomes. Experimental data suggests that the viral envelope glycoprotein (GP) catalyzes the fusion between the viral and host cell membranes. The fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike ultimately resulting in the formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Membrane fusion is mediated by fusion proteins that extrude from the viral membrane. Key components that are in contact with the host cell membrane are fusion peptides, parts of the fusion proteins. The Ebola glycoprotein (GP) is responsible for both receptor binding and membrane fusion. The GP is composed of two sub-domains, GP1 and GP2. The two domains are connected via a disulfide bond. The Ebola fusion peptide (EFP) (G524AAIGLAWIPYFGPAA539) is thought to be in direct contact with the host cell membrane. This peptide is conserved within the virus family. EFP is an internal fusion peptide located 22 residues from the N-terminus of GP2. Experimental data suggests that the EFP peptide in the presence of the membrane has a tendency to form helical structures.

Figure 2: Model of the Ebola fusion protein in its fusiogenic state as suggested by Jaskierny et al. in 2011. The globular protein GP1 is thought to initiate the binding to the host cell receptor. The GP2 domain contains a helical bundle with the fusion peptide near the N-terminus. Jaskierney et al. studied the monomeric form of the internal fusion peptide from Ebola virus in membrane bilayer and water environments using computer simulations. The wild type Ebola fusion peptide, the W8A mutant form, and an extended construct with flanking residues were examined. The researchers found that the monomeric form of wild type Ebola fusion peptide adopts a coil-helix-coil structure with a short helix from residue 8 to 11 orientate parallel to the membrane surface.

 

Using circular dichroism (CD) together with infrared (IR) spectroscopy the researchers showed that the EFP peptide has three states:

A random coil in solution and either an α–helix or a β–sheet when bound to the membrane. Furthermore, the secondary structure of the membrane-bound peptide depends on the presence of Ca2+ and in the presence of Ca2+ a β-sheet structure is preferred while in the absence of Ca2+ helical structures are dominant. A nuclear magnetic resonance (NMR) study of EFP showed that the peptide adopts a random coil structure in aqueous buffers and a more defined structure in the presence of sodium dodecyl sulfate (SDS) micelles. Tryptophan fluorescent emission data suggests that W8 enters the hydrophobic core of SDS micelles. Nuclear Overhauser effect (NOE) measurements obtained from 1H NMR suggested the presence of a short 310 helix form I9 to F12 in the middle of the peptide while the N- and C-termini appear to be less structured.


Miller et al. in 2011 performed a study using synthetic peptides of the CHR sequence region (C-peptides) to test if these peptides can inhibit the entry of the virus particles. The researchers prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, known to accumulate in endosomes, and found that this peptide specifically inhibits viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. The researchers determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Miller et al. argue that targeting C-peptides to endosomal compartments can serve as an approach to localize inhibitors to sites of membrane fusion.


To diagnose and control the endemic outbreaks of haemorrhagic fever in humans caused by filioviruses, such as the Ebola and the Marburg virus, rapid, highly sensitive, reliable, and specific assays are required. The identification and characterization of antigenic sites in viral proteins is important for the development of viral antigen detection assays.

Changula et al. in 2013 generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of the Zaire Ebola virus. The researchers divided the mABs into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. The use of synthetic peptides corresponding to the Ebola virus nucleoprotein (NP) sequence allowed to map mAb binding sites to seven antigenic regions in the C-terminal half of the NP. The mapped antigenic sites included two highly conserved regions present among all five Ebola virus species currently known. In addition, the scientists were successfully in producing species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. These results provide useful information for the development of Ebola virus antigen detection assays and potentially new vaccines for Ebola virus strains.


Table 1: Ebola virus peptides

Peptide

Sequence

Notes

 

Fusion Peptide

Jaskierny et al., 2011.

EFP

G524AAIGLAWIPYFGPAA539

Chain A fusion peptide in SDS micelles at pH 7

 

 

 

 

C-Peptide Study

Miller et al. 2011

Tat-Ebo

YGRKKRRQRRR-GSG-IEPHDWTKNITDKIDQIIHDFVDK

Ebola virus chain A fusion peptide

Lys-Ebo

       KKKK-GSG-IEPHDWTKNITDKIDQIIHDFVDK

Ebola virus chain A fusion peptide

Tat-only

YGRKKRRQRRR

 

Tat-Scram

YGRKKRRQRRR-GSG-HTEHINFQDDTIKIWPDVIKIKDD

 

Tat-ASLV

YGRKKRRQRRR-GSG-FNLSDHSESIQKKFQLMKEHVNKIG

 

 

 

 

 

Peptide epitopes of mABs against EBOV NP

Changula et al. 2013

ZNP31-1-8

ZNP41-2-4

YDDDDDIPFP, aa 421–430

NP protein

ZNP74-7

YDDDDDIPFPGPINDDDNPG, aa 421–440

NP protein

ZNP24-4-2

QTQFRPIQNVPGPHRTIHHA, aa 521–540

TPTVAPPAPVYRDHSEKKEL, aa 601–620

NP protein

ZNP106-9

DTTIPDVVVD, aa 451–460a

NP protein

ZNP98-7

MLTPINEEADPLDDADDETS, aa 561–580

NP protein

ZNP35-16-3-5

DDEDTKPVPNRSTKGGQQKN, aa 491–510

NP protein

ZNP62-7

YRDHSEKKELPQDEQQDQDH, aa 611–630

NP protein

 

Ebola virus NucleoProtein (NP) sequence

>gi|158341892|gb|ABW34756.1| nucleoprotein, partial [Zaire ebolavirus]
RQIQVHAEQGLIQYPTAWQSVGHMMVIFRMMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSG
LLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLF
PQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKK
NEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQDTT
IPDVVVDPDDGSYGEYQSYSENGMNAPDDLVLFDLDEDDEDTKPVPNRLTKGGQQKNSQKGHHTEGRQTQ
SRPTQNVPGPRRTIHHASAPLTDNDRGNEPSGSTSPRMLTPINEEADPLDDADDETSSLPPLESDDEEQD
RDETSNRTPTVAPPAPVYRDHSEKKELPQDEQQDQDHTQEARNQDSDNTQPEHSFEEMYRHIL


The location of the Zaire envelope protein (ZNP) peptides are highlighted in red and magenta within the amino acid sequence of Ebola virus nucleoprotein.

Table 2: Observed mutations for the QTQFRPIQNVPGPHRTIHHA, aa 521–540, peptide.


Models of Ebola virus peptides and proteins

Figure 3: NMR structure of the Ebola virus chain A fusion peptide, GAAIGLAWIPYFGPAA.


Figure 4: Crystal structure models of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain.

Table 3: Peptides used for the production of rabbit antisera by Changula et al. 2013.


Virus Protein

Peptide

Amino Acids

EBOV NP

QDHTQEARNQD

628-638

SUDV NP

QGSESEALPINSKK

631-644

TAFV NP

NQVSGSENTDNKPH

630-643

BDBV NP

QSNQTNNEDNVRNN

628-641

RESTV NP

TSQLNEDPDIGQSK

630-643

MARV NP

RVVTKKGRTFLYPNDLLQ

635-652

 

Legend: BDBV = Bundibugyo virus; EBOV = Ebola virus; MATV = Marburg virus; RESTV = Reston virus; SUDV = Sudan virus; TAFV = Tai Forest Ebola virus.


The membrane proximal external region (MPER) peptide


Regula et al. in 2013 investigated the role of the membrane proximal external region (MPER) that precedes the transmembrane domain of glycoprotein 2 (GP2) of Ebola virus strains. Earlier research indicated that an infection by a filovirus requires membrane fusion between the host and the virus. The fusion process is facilitated by the two subunits of the envelope glycoprotein, the surface subunit GP1and the transmembrane subunit GP2. A sequence region called the membrane proximal external region (MPER) is a tryptophan (Trp, W) rich peptide segment located immediately in front of the transmembrane domain of GP2. In the human immunodeficiency virus 1 (HIV-1) glycoprotein gp41, the MPER is known to be critical for membrane fusion. In addition, this amino acid sequence was also identified as a target for several neutralizing antibodies. Regula et al. characterized the properties of GP MPER segment peptides of Ebola virus and Sudan virus. The study used  micelle-forming surfactants and lipids, at pH 7 and pH 4.6. The researchers employed circular dichroism (CD) spectroscopy and tryptophan fluorescence to determine if GP2 MPER peptides bind to micelles of sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC). Nuclear magnetic resonance (NMR) spectroscopy was used to reveal that residues 644 to 651 of the Sudan virus MPER peptide interacted directly with DPC. This interaction enhanced the helical conformation of the peptide. The scientists found that the Sudan virus MPER peptide moderately inhibited cell entry by a GP-pseudotyped vesicular stomatitis virus. However, it did not induce leakage of a fluorescent molecule from large unilamellar vesicle comprised of 1-palmitoyl-2-oleoylphostatidyl choline (POPC) or cause hemolysis. The analysis performed by this research group suggested that the filovirus GP MPER binds and inserts shallowly into lipid membranes.


GP2 MPER Peptides


Table 4: Alignment of GP2 MPER peptides from different viruses.

Virus Strain

GP2 MPER Peptide

Amino Acids

EBOV

    DKTLPDQGDNDNWWTGWRQW

632 to 651

BDBV

    DKPLPDQTDNDNWWTGWRQW

632 to 651

SUDV

    DNPLPNQDNDDNWWTGWRQW

632 to 651

TAFV

    DNNLPNQNDGSNWWTGWKQW

632 to 651

RESTV

    DNPLPDHGDDLNNWTGWRQW

633 to 652

FIV

    LQKWEDWVGWIGNIPQYLKG

767 to 786

HIV-1

LLELDKWASLWNWFDITNWLWYIK

660 to 683

 

Table 4 shows the amino acid alignment of GP2 MPER regions from different members of the five Ebola virus species. Many residues that are identical in at least four of the viruses. For comparison, the MPER segments of FIV and HIV-1 gp41 are included.

 

Legend: BDBV Bundbuyo virus, EBOV Ebola virus, FIV filio virus, HIV-1 human immunodeficiency virus 1, RESTV Reston virus, SUDV Sudan virus, TAFV Thai Forest virus.


Alignments of GP2 MPER peptides from various virus strains.


Location of the GP2 MPER peptides within the GP2 protein of the Ebola virus

Figure 5: The location of the MPER peptides is highlighted in yellow in the crystal structure of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain. The amino acid of the peptide shown in gray where not observed in the crystal indicating that this part of the peptide may take up a random coil structure in the crystal.


Regula et al. used EBOV and SUDV MPER peptides for their study because both viruses are the most prevalent and pathogenic among the ebolaviruses. Synthetic peptides corresponding to the MPER region for EBOV and SUDV were used. The N-termini were blocked with an acetyl group and the C-termini contained an amide group.

 

The study revealed three characteristics of the GP2 MPER peptides:

  • As a peptide, the GP2 MPER binds to micelle-forming surfactants in a pH-independent manner with higher affinity for zwitterionic micelles; 
  • A large conformational change to a more predominantly helical state occurs for the tryptophan-rich region of this peptide upon micelle-binding;
  • These peptides have modest viral entry inhibitory activity but do not induce leakage from LUVs.

 

The study observed inhibitory activity for the S-MPER peptide which suggests that addition of this peptide may interfer with the viral entry process. For the FIV MPER peptide it was observed that a WX2WX2W motif is required for the membrane interaction responsible for its inhibitory activity.

This peptide motif, WTGWRQW, is strictly conserved among all species.

Results of the study indicated that the MPER peptide segments of EBOV and SUDV bind membrane surfaces which induces a conformational change in the Trp-rich peptide segment. This behavior suggests a role for the EBOV and SUDV MPER in membrane fusion.

 

Reference

http://www.cdc.gov/vhf/ebola/

D. S. ELLIS, D. I. H. SIMPSON, D. P. FRANCIS, J. KNOBLOCH, E. T. W. BOWEN, PACIFICO LOLIK, AND ISAIAH MAYOM DENG; Ultrastructure of Ebola virus particles in human Liver. Journal of Clinical Pathology, 1978, 31, 201-208.

Katendi Changula
, Reiko Yoshidac, Osamu Noyoric, Andrea Marzid, Hiroko Miyamotoc, Mari Ishijimac, Ayaka Yokoyamac, Masahiro Kajiharac,Heinz Feldmannd, Aaron S. Mweenea, Ayato Takadaa; Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein.  Virus Research 176  (2013) 83– 90.

Thomas Hoenen, Allison Groseth, and Heinz Feldmann; Current Ebola vaccines. Expert Opin Biol Ther. 2012 July; 12(7): 859–872.  oi:10.1517/14712598.2012.685152.

Adam J. Jaskierny
, Afra Panahi, and Michael Feig; Effect of flanking residues on the conformational sampling of the internal fusion peptide from Ebola virus. Proteins. 2011 April ; 79(4): 1109–1117. doi:10.1002/prot.22947.

Emily Happy Miller, Joseph S. Harrison, Sheli R. Radoshitzky, Chelsea D. Higgins, Xiaoli Chi, Lian Dong, Jens H. Kuhn, Sina Bavari, Jonathan R. Lai, and Kartik Chandran; Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosome J Biol Chem. May 6, 2011; 286(18): 15854–15861. Published online Mar 16, 2011. doi:  10.1074/jbc.M110.207084. PMCID: PMC3091195.

Lauren K. Regula, Richard Harris, Fang Wang, Chelsea D. Higgins, Jayne F. Koellhoffer, Yue Zhao, Kartik Chandran, Jianmin Gao, Mark E. Girvin, and Jonathan R. Lai; Conformational Properties of Peptides Corresponding to the Ebolavirus GP2 Membrane-Proximal External Region in the Presence of Micelle-Forming Surfactants and Lipids. Biochemistry. 2013 May 21; 52(20): . doi:10.1021/bi400040v.