Background
Methods
Identification of conserved B cell epitopes of filovirus GP1, 2 pre-protein
-
The primary amino acids sequences of the GP1, 2 preprotein for 4 Ebolavirus species [Zaire ebolavirus (strain Eckron-76)= > sp.|P87671|, Tai Forest ebolavirus (strain Cote d’Ivoire-94)= > sp.|Q66810|, Sudan ebolavirus (strain Maleo-79)= > sp.|Q66798| and Reston ebolavirus (strain Philippines-96)= > sp.|Q91DD8|] alongside one Marburgvirus species [Marburg marburgvirus, MARV (strain Angola/2005)= > sp.|Q1PD50|] were separately fed into the interfaces of the immune epitope database analysis resource (IEDB-AR) [25] and Bebipred [26]. Four biophysical profiles (beta-turn, surface accessibility, hydrophilicity and antigenicity) were evaluated alongside the hidden Markov BeBipred propensity as per user protocols [27].
-
The same amino acids sequences were simultaneously fed into the user interface of the ClustalW software and conserved linear peptides derived according to the user protocols at default [28]. The derivative conserved epitopes were (i) queried against the National Center for Biotechnology Information (NCBI) human proteome, microbial proteome database, fungal proteome database, and protozoa proteome database and Conserved Domain Database (CDD) by BLAST-P [29, 30] and (ii) scanned against the 3-D crystal structure of Ebola GP in combination with antibodies from a human survivor (PDB entry 3CSY) using Prosite Scan [10, 31]. Details of the methodologies are available in reference [32].
-
Avialibility of Software and Databases
-
The IEDB-AR resource used in this paper is available at the following url: http://tools.immuneepitope.org/main/
-
The linear B cell epitope prediction profiles used in this study are available at the following url: http://tools.immuneepitope.org/tools/bcell/iedb_input
-
The BepiPred software used in this analysis is available at the following url: http://www.cbs.dtu.dk/services/BepiPred
-
The Clustal W multiple alignment software and algorithms is available at the following url: www.ebi.ac.uk/clustalw/
-
The NCBI database of 2107 microbial proteomes used further above, alongside its BLAST-P tool are available at the following url: http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi
-
The reference proteome of the Human genome along with its BLAST tool are available at the following url: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
-
The Protscan software and algorithms is available at the following url: http://www.expasy.org/tools/scanprosite/
-
The PDB macromolecular structure database hosting the 3- dimensional structure of EBOV GP entry 3CSY is available at the following url: http://www.ebi.ac.uk/msd-srv/msdmotif/
-
Validation of filovirus GP1, 2 antigen and host-specific antbody (IgM and IgG) detection
Results
Identity of conserved B cell epitopes of filovirus GP1,2 pre-protein
Biophysical Profile | Threshold score (Zaire; Tai forest; Sudan; Reston ebolaviruses; L.victoria marburgvirus) | Mean Threshold score |
---|---|---|
Beta turn | 1.008; 1.004; 1.005;1.022;1.039 | 1.016 |
Surface accessibility | 1.000; 1.000; 1.000; 1.000; 1.000 | 1.000 |
Hydrophilicity | 1.809; 1.569; 1.609; 1.720; 2.099 | 1.761 |
Antigenicity | 1.000;1.000; 1.000; 1.000; 1.000 | 1.000 |
Bipred | 0.350; 0.350; 0.350; 0.350; 0.350 | 0.350 |
EBOV/MBGV spp
| Peptide | Start position | End Position | Length |
---|---|---|---|---|
Zaire, EBOV
| HHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITN | 407 | 484 | 78 |
Tai Forest, TAFV
| DHSTTQPAKTTSQPTNSTESTTLNPTSEPSSRGTGPSSPTVPNTTESHAELGKTTPTTLPEQHTAASAIPRAVHPDELSGPGFLTNTIRGV | 399 | 489 | 91 |
Sudan, SUDV
| TMAPSPETQTSTTYTPKLPVMTTEEPTTPPRNSPGSTTEAPTLTTPENITTAVKTVWA | 416 | 483 | 58 |
Reston, RESTV
| STQGLTNGETITGFTANPMTTTIAPSPTMTSEVDNNVPSEQPNNTASIEDSPPSASNETIDHSEMNSIQG | 364 | 433 | 70 |
Marburg, MARV
| STKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDEDLTTSGSGSGEQEPYTTSDAATKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQGVVTEPGKTNTTAQPSMPPHNTTTISTNNTSKHNLSTPSVPIQNATNYNTQSTAPENEQTSAPSKTTLLPTENPTTAKSTNSTKSPTTTVPNTTNKYSTSPSPTPNSTAQHLVYFR | 220 | 432 | 213 |
Validation of filovirus GP1, 2 antigen and host-specific antbody (IgM and IgG) detection
Synthetic epitopes
Cloning and expression of recombinant EBOV GP1, 2 protein
IgM detection EIA
Host Specific IgM | |||
---|---|---|---|
UG-Filo-Peptide 1 | UG-Filo-Peptide 2 | UG-Filo-Peptide 3 | |
Best-fit values | |||
• YIntercept | 0.03191 | 0.02953 | 0.03235 |
• Slope | -1.111e-005 | 0.0004278 | 5.556e-005 |
Std. Error | |||
• YIntercept | 0.0007379 | 0.0005631 | 0.0008208 |
• Slope | 0.0001311 | 0.0001001 | 0.0001459 |
95% Confidence Intervals | |||
• YIntercept | 0.03039 to 0.03343 | 0.02837 to 0.03069 | 0.03066 to 0.03404 |
• Slope | −0.0002812 to 0.0002590 | 0.0002217 to 0.0006339 | −0.0002449 to 0.0003560 |
Goodness of Fit | |||
• Degrees of Freedom | 25 | 25 | 25 |
• R square | 0.0002871 | 0.4223 | 0.005769 |
• Absolute Sum of Squares | 7.739e-005 | 4.506e-005 | 9.574e-005 |
• Sy.x | 0.001759 | 0.001343 | 0.001957 |
Number of points | |||
• Analyzed | 27 | 27 | 27 |
IgG detection EIA
Host Specific IgM | |||
---|---|---|---|
UG-Filo-Peptide 1 | UG-Filo-Peptide 2 | UG-Filo-Peptide 3 | |
Best-fit values | |||
• YIntercept | 0.03076 | 0.03876 | 0.02914 |
• Slope | 0.0001031 | −2.404e-005 | 0.0001580 |
Std. Error | |||
• YIntercept | 0.0004409 | 0.0007923 | 0.001033 |
• Slope | 9.769e-006 | 1.756e-005 | 2.302e-005 |
95% Confidence Intervals | |||
• YIntercept | 0.02989 to 0.03163 | 0.03720 to 0.04033 | 0.02710 to 0.03118 |
• Slope | 8.382e-005 to 0.0001224 | −5.870e-005 to 1.061e-005 | 0.0001125 to 0.0002034 |
Goodness of Fit | |||
• Degrees of Freedom | 169 | 169 | 168 |
• R square | 0.3973 | 0.01098 | 0.2189 |
• Absolute Sum of Squares | 0.001312 | 0.004239 | 0.007126 |
• Sy.x | 0.002787 | 0.005008 | 0.006513 |
Number of points | |||
• Analyzed | 171 | 171 | 171 |
Host Specific IgG | |||
---|---|---|---|
UG-Filo-Peptide 1 | UG-Filo-Peptide 2 | UG-Filo-Peptide 3 | |
Best-fit values | |||
• YIntercept | 0.4165 | 0.4743 | −0.1944 |
• Slope | −0.04364 | −0.02456 | 0.2225 |
Std. Error | |||
• YIntercept | 0.2465 | 0.03052 | 0.2744 |
• Slope | 0.04380 | 0.005423 | 0.04877 |
95% Confidence Intervals | |||
• YIntercept | 0.1125 to 0.7205 | 0.4115 to 0.5372 | −0.7596 to 0.3708 |
• Slope | −0.1339 to 0.04657 | −0.03573 to − 0.01339 | 0.1220 to 0.3229 |
Goodness of Fit | |||
• Degrees of Freedom | 25 | 25 | 25 |
• R square | 0.03819 | 0.4507 | 0.4542 |
• Absolute Sum of Squares | 8.635 | 0.1324 | 10.70 |
• Sy.x | 0.5877 | 0.07276 | 0.6543 |
Number of points | |||
• Analyzed | 27 | 27 | 27 |
Host Specific IgG | |||
---|---|---|---|
UG-Filo-Peptide 1 | UG-Filo-Peptide 2 | UG-Filo-Peptide 3 | |
Best-fit values | |||
• YIntercept | 1.7181 | 1.8197 | 1.7197 |
• Slope | 0.01042 | 0.02079 | 0.01126 |
Std. Error | |||
• YIntercept | 0.08487 | 0.09550 | 0.07882 |
• Slope | 0.001047 | 0.001192 | 0.0009768 |
95% Confidence Intervals | |||
• YIntercept | 0.3010 to 3.1352 | 0.3268 to 3.3125 | 0.4145 to 3.0248 |
• Slope | 0.008361 to 0.01248 | 0.01844 to 0.02314 | 0.009336 to 0.01318 |
Goodness of Fit | |||
• Degrees of Freedom | 274 | 271 | 277 |
• R square | 0.4655 | 0.5286 | 0.4242 |
• Absolute Sum of Squares | 163.2 | 201.8 | 144.5 |
• Sy.x | 0.7719 | 0.8629 | 0.7222 |
Number of points | |||
• Analyzed | 276 | 276 | 276 |
PEPTIDE-1 | PEPTIDE-2 | PEPTIDE-3 | TOTALS | AVERAGES | ||
---|---|---|---|---|---|---|
# | Statistical Function | Value(s) | ||||
1 | Sample size (N) | 94 | 94 | 94 | 282 |
94
|
2 | SUM (Σ) OD | 65.54183297 | 69.40603 | 88.86964 | 223.8175 |
74.60583
|
3 | Average OD | 0.697253542 | 0.738362 | 0.955587 | 2.391203 |
0.797068
|
4 | VAR | 0.808399425 | 1.582093 | 0.720303 | 3.110795 |
1.036932
|
4 | Σ (Deviance)2 | 76.34237 | 214.0516 | 71.75194 | 362.1459 |
120.7153
|
5 | Σ (Deviance)2/(N-1) | 0.820886 | 2.30163 | 0.771526 | 3.894042 |
1.298014
|
6 | STD | 0.906027 | 1.517112 | 0.878366 | 3.301505 |
1.100502
|
7 | MAX | 2.586364 | 2.397667 | 2.869666 | 7.853697 |
2.617899
|
8 | MIN | −0.45733 | −0.71833 | −0.07133 | −1.24699 |
−0.41566
|
9 | RANGE | 3.043698 | 3.116 | 2.941 | 9.100698 |
3.033566
|
10 | MEDIAN | 1.293182 | 1.178334 | 1.227666 | 3.699182 |
1.233061
|
Filovirus GP1, 2 Antigen | ||
---|---|---|
PAb 1 | PAb-2 | |
Best-fit values | ||
• YIntercept | 0.1108 | 0.2391 |
• Slope | −0.001581 | −0.006704 |
Std. Error | ||
• YIntercept | 0.008089 | 0.02180 |
• Slope | 0.0004032 | 0.001087 |
95% Confidence Intervals | ||
• YIntercept | 0.09479 to 0.1269 | 0.1958 to 0.2823 |
• Slope | −0.002381 to −0.0007811 | −0.008860 to − 0.004548 |
Goodness of Fit | ||
• Degrees of Freedom | 100 | 100 |
• R square | 0.1333 | 0.2756 |
• Absolute Sum of Squares | 0.1596 | 1.160 |
• Sy.x | 0.03995 | 0.1077 |
Number of points | ||
• Analyzed | 102 | 102 |