Protein sequence retrieval
KP (accession number: ALM55777.1), EBA-175 (accession number: AAF72186.1), and LSA1 (accession number: AAC41596.1) were retrieved from the NCBI database.
Antigenicity, allergenicity, and screening for non-human homologues
The three proteins exhibited an antigenicity score above the 0.4 threshold. The antigenic scores of the LSA1, EBA-175, and KP were 0.8404, 0.6663, and 0.4234, respectively. The proteins were non-allergens and non-human homologues.
Linear B-cell epitope prediction
A total of 191 B-cell epitopes were predicted for the three proteins. The KP exhibited 36 B-cell epitopes, EBA-175 exhibited 144 B-cell epitopes, and LSA1 exhibited 11 B-cell epitopes. After screening, 45 B-cell epitopes were selected from the three proteins to construct the vaccine. The selected B-cell epitopes are presented in Tables 1–3.
Table 1. Selected linear B-cell epitopes of the Kelch Protein
Epitopes
|
Start position
|
Score
|
Antigenicity (0.9)
|
DSYIITGGENGEVLNS
|
312
|
0.97
|
1.6815
|
PRSSAMCVAFDNKIYV
|
206
|
0.86
|
1.3633
|
PRRNNCGVTSNGRIYC
|
159
|
0.73
|
1.5409
|
FRIILNFLRNPLTIPI
|
35
|
0.57
|
0.9530
|
Table 2. Selected linear B-cell epitopes of the Erythrocyte Binding Antigen 175
Epitopes
|
Start Position
|
Score
|
Antigenicity (0.9)
|
YGHLAMGNDMDFGGYS
|
242
|
0.94
|
1.359
|
HGKISEHEIKNFRKKW
|
272
|
0.93
|
1.0012
|
TPSISEGPKGNEQKER
|
777
|
0.92
|
0.9658
|
TGEIDEKLRESKESKI
|
1165
|
0.92
|
1.7611
|
SKPLSDDVRPDKKELE
|
857
|
0.91
|
1.4214
|
NFRKKWWNEFREKLWE
|
282
|
0.91
|
1.2514
|
VECKEKPCEDDNCKSK
|
650
|
0.9
|
1.2991
|
VSPENSRPETDAKDTS
|
802
|
0.89
|
1.2568
|
SGSGSATVSESSSSNT
|
899
|
0.88
|
1.1913
|
EFKEELHSDYKNKCTM
|
726
|
0.88
|
1.1138
|
YESRILKRKYKNKDDK
|
522
|
0.88
|
1.4944
|
KKLDSDLYENRNDSTT
|
1295
|
0.88
|
1.2124
|
KMKGNDTSEMSHNSSQ
|
1119
|
0.86
|
1.1346
|
HIDLDDFSKFGCDKNS
|
451
|
0.85
|
0.95
|
SQHIESDQQKNDMKTV
|
1133
|
0.84
|
1.3098
|
YEDIVLKSHMNRESDD
|
1081
|
0.83
|
1.1411
|
LSNCREKRKGMKWDCK
|
155
|
0.82
|
1.0764
|
GGNSGNVLNMRSNNNN
|
1267
|
0.82
|
1.2708
|
VSESSSSNTGLSIDDD
|
906
|
0.81
|
1.311
|
DVRPDKKELEDQNSDE
|
863
|
0.81
|
1.1517
|
EFKSIKPEVYLKKYSE
|
701
|
0.8
|
1.3007
|
DKNSVDTNTKVWECKK
|
463
|
0.79
|
1.0087
|
TTTLVKSVLNGNDNTI
|
430
|
0.79
|
0.962
|
HGEFLLERYNRSKLPK
|
328
|
0.79
|
0.9613
|
LLMIKEHILAIAIYES
|
509
|
0.78
|
0.9878
|
DVDISMPKAVIGSSPN
|
825
|
0.76
|
0.9041
|
EEIPLKTCTKEKTRNL
|
1338
|
0.76
|
0.9178
|
KESQLLLKKNDNKYNS
|
214
|
0.74
|
0.9755
|
FFASFFVLYFAKARNE
|
9
|
0.73
|
1.2622
|
QNEISVPVTGEIDEKL
|
1157
|
0.72
|
1.3005
|
MKCNISIYFFASFFVL
|
1
|
0.67
|
1.2284
|
CSNLNFEDEFKEELHS
|
718
|
0.65
|
1.1058
|
HRNKKNDKLFRDEWWK
|
580
|
0.65
|
1.7615
|
KNDNKYNSKFCNDLKN
|
222
|
0.63
|
1.3946
|
KNKNDAKVSLLLNNCD
|
403
|
0.54
|
1.1625
|
Table 3. Selected linear B-cell epitopes of the Liver Stage-Specific Antigen
Epitopes
|
Start position
|
Score
|
Antigenicity (0.9)
|
LIEHIINDDDDKKKYI
|
129
|
0.84
|
0.9624
|
LSHNSYEKTKNNENNK
|
67
|
0.83
|
1.1011
|
KKYIKGQDENRQEDLE
|
141
|
0.82
|
1.5426
|
SGSSNSRNRINEEKHE
|
47
|
0.73
|
1.2678
|
RNRINEEKHEEKHVLS
|
53
|
0.62
|
1.0914
|
T-cell epitope prediction
A total of 40 9-mer CTL epitopes were predicted for each protein. The KP exhibited 41 HTL epitopes, and the LSA1 exhibited 28 HTL epitopes. The EBA-175 exhibited 66 HTL epitopes, with 20 being strong binders and 46 being weak binders.
After screening, 13 CTL epitopes, including 5 epitopes from the KP, 3 epitopes from the EBA-175, and 5 epitopes from the LSA1, were selected for the final vaccine construct (Table 4). Additionally, an aggregate of 22 antigenic and non-allergenic HTL epitopes were selected for vaccine construction (Tables 5–7).
Table 4. Selected CTL epitopes of the retrieved proteins
Protein
|
Epitope
|
Start position
|
Score
|
Antigenicity (1.0)
|
|
DNKIYVIGG
|
216
|
0.97/0.23292199
|
1.3048
|
|
AYFGSAVLN
|
113
|
0.94/0.19808502
|
1.3332
|
KP
|
VTRDKQGRI
|
18
|
0.87/0.25791619
|
1.5446
|
|
FGGNNYDYK
|
127
|
0.94/0.18779483
|
1.3123
|
|
EARSSGAAF
|
252
|
0.63/0.46878932
|
1.2129
|
|
|
|
|
|
|
SFFVLYFAK
|
12
|
0.96/0.44967106
|
1.2967
|
EBA-175
|
AIAIYESRI
|
518
|
0.39/0.99712378
|
1.0369
|
|
REKLWEAML
|
292
|
0.05/1.2763412
|
1.4553
|
|
|
|
|
|
|
HINGKIIKN
|
26
|
0.68/0.25124408
|
1.8927
|
|
GKIIKNSEK
|
29
|
0.93/-0.19259712
|
1.5814
|
LSA1
|
KTKNNENNK
|
74
|
0.00/0.68564565
|
1.4166
|
|
SGSSNSRNR
|
47
|
0.05/0.51466315
|
1.7558
|
|
KENKLNKEG
|
119
|
0.95/-0.67246024
|
1.4036
|
KP, Kelch Protein; EBA-175, Erythrocyte Binding Antigen-175; LSA1, Liver Stage Antigen 1
Table 5. Selected HTL epitopes of the Kelch Protein
Epitope
|
Allele
|
Start/
End
|
% Rank
|
Antigenicity (1.0)
|
IFN-γ (+/-)
|
IL-4
|
IL-10
|
IILNFLRNPLTIPIP
|
HLA-DRB1*15:01,
HLA-DRB3*02:02
|
37/51
|
0.53, 0.9
|
1.1872
|
+
|
Non-inducer
|
Non-inducer
|
MCVAFDNKIYVIGGT
|
HLA-DRB1*03:01
|
211/225
|
1.8
|
1.2896
|
_
|
Inducer
|
Inducer
|
RSSAMCVAFDNKIYV
|
HLA-DRB1*03:01
|
207/221
|
1.8
|
1.4159
|
_
|
Inducer
|
Non-inducer
|
SSAMCVAFDNKIYVI
|
HLA-DRB1*03:01
|
208/222
|
1.8
|
1.3974
|
_
|
Inducer
|
Non-inducer
|
NFLRNPLTIPIPKDL
|
HLA-DRB3*02:02
|
40/54
|
2.7
|
1.0727
|
_
|
Non-inducer
|
Non-inducer
|
CVAFDNKIYVIGGTN
|
HLA-DRB1*03:01
|
212/226
|
4.2
|
1.1503
|
_
|
Inducer
|
Non-inducer
|
KAYFGSAVLNNFLYV
|
HLA-DRB3*02:02
|
112/126
|
4.9
|
1.1405
|
+
|
Non-inducer
|
Inducer
|
IFN-γ, interferon-gamma; IL-4, interleukin 4; IL-10, interleukin 10
Table 6. Selected HTL epitopes of the Erythrocyte Binding Antigen 175
Epitope
|
Allele
|
Start/
End
|
%
Rank
|
Antigenicity (1.0)
|
IFN-γ (+/-)
|
IL-4
|
IL-10
|
LYISFYFILVNLLIF
|
HLA-DRB5*01:01,
HLA-DRB1*07:01,
HLA-DRB1*15:01,
|
11/25
|
0.64,
2.2, 3.9
|
1.4308
|
-
|
Non-inducer
|
Inducer
|
ILYISFYFILVNLLI
|
HLA-DRB1*07:01,
HLA-DRB1*15:01
|
10/24
|
0.7, 2.2
|
1.4091
|
-
|
Non-inducer
|
Inducer
|
LKMKHILYISFYFIL
|
HLA-DRB1*15:01
|
5/19
|
0.98
|
1.2596
|
-
|
Inducer
|
Inducer
|
SLLLKMKHILYISFY
|
HLA-DRB4*01:01,
HLA-DRB1*15:01
|
2/16
|
1.2, 4.7
|
1.1470
|
-
|
Non-inducer
|
Inducer
|
LLLKMKHILYISFYF
|
HLA-DRB1*15:01,
HLA-DRB4*01:01
|
3/17
|
1.4, 1.7
|
1.3655
|
-
|
Non-inducer
|
Inducer
|
MKHILYISFYFILVN
|
HLA-DRB1*15:01
|
7/21
|
1.4
|
1.1342
|
-
|
Non-inducer
|
Inducer
|
KHILYISFYFILVNL
|
HLA-DRB1*15:01
|
8/22
|
1.6
|
1.1477
|
-
|
Non-inducer
|
Inducer
|
NLLIFHINGKIIKNS
|
HLA-DRB3*02:02,
HLA-DRB1*07:01
|
21/35
|
1.7, 4.4
|
1.0193
|
+
|
Inducer
|
Non-inducer
|
ILVNLLIFHINGKII
|
HLA-DRB1*15:01,
HLA-DRB1*07:01
|
18/32
|
1.8, 4.2
|
1.2744
|
_
|
Inducer
|
Non-inducer
|
LLIFHINGKIIKNSE
|
HLA-DRB3*02:02
|
22/36
|
2
|
1.1805
|
+
|
Inducer
|
Non-inducer
|
FILVNLLIFHINGKI
|
HLA-DRB1*15:01
|
17/31
|
2.2
|
1.1336
|
_
|
Inducer
|
Non-inducer
|
HILYISFYFILVNLL
|
HLA-DRB1*07:01
|
9/23
|
2.5
|
1.136
|
+
|
Non-inducer
|
Inducer
|
LIFHINGKIIKNSEK
|
HLA-DRB3*02:02
|
23/37
|
2.6
|
1.2071
|
+
|
Inducer
|
Non-inducer
|
IFN-γ, interferon-gamma; IL-4, interleukin 4; IL-10, interleukin 10
Table 7. Selected HTL epitopes of the Liver Stage-Specific Antigen 1
Epitope
|
Allele
|
Position
|
Affinity (nM)
|
% Rank
|
Antigenicity (1.0)
|
IFN-γ (+/-)
|
IL-4
|
IL-10
|
AIAIYESRILKRKYK
|
DRB1_0301
|
518
|
42.9
|
1.9
|
1.0541
|
-
|
Inducer
|
Non-inducer
|
LAIAIYESRILKRKY
|
DRB1_0301
|
517
|
44.7
|
1.9
|
1.1453
|
-
|
Non-inducer
|
Inducer
|
IFN-γ, interferon-gamma; IL-4, interleukin 4; IL-10, interleukin 10
Population coverage analysis
The result indicated that, on average, 31.41% of the selected HTL epitopes have appeared in 16 areas. The details are presented in Figure 1.
[Insert Figure 1]
Vaccine construction
A total of 1,448 amino acid residues was obtained. The primary sequence of the vaccine construct is displayed in Table 8. Table 9 presents the evaluation for its antigenicity, allergenicity, and physicochemical properties.
Table 8. Primary sequence of the vaccine construct
MTNVGRIKRTSIAIRNYAASMTDGGVNALSGLQGISGNSVRAQQTIEYVADATQANLFLAKEAAAKLIE
HIINDDDDKKKYIKKLSHNSYEKTKNNENNKKKKKYIKGQDENRQEDLEKKSGSSNSRNRINEEKHEKK
RNRNEEKHEEKHVLSKKYGHLAMGNDMDFGGYSKKHGKISEHEIKNFRKKWKKTPSISEGPKGNEQKE
RKKTGDEKLRESKESKIKKSKPLSDDVRPDKKELEKKNFRKKWWNEFREKLWEKKVECKEKPCEDDNC
KSKKKVSPNSRPETDAKDTSKKSGSGSATVSESSSSNTKKEFKEELHSDYKNKCTMKKYESRILKRKYK
NKDDKKKKKLDSDLYENRNDSTTKKKMKGNDTSEMSHNSSQKKHIDLDDFSKFGCDKNSKKSQHIESD
QQKNDMKTVKKYEDIVLKSHMNRESDDKKLSNCREKRKGMKWDCKKKGGNSGNVLNMRSNNNNKK
VSESSSSNTGLSIDDDKKDVRPDKKELEDQNSDEKKEFKSIKPEVYLKKYSEKKDKNSVDTNTKVWECK
KKKTTTLVKSVLNGNDNTIKKHGEFLLERYNRSKLPKKKLLMIKEHILAIAIYESKKDVDISMPKAVIGSS
PNKKEEIPLKTCTKEKTRNLKKKESQLLLKKNDNKYNSKKFFASFFVLYFAKARNEKKQNEISVPVTGEI
DEKLKKMKCNISIYFFASFFVLKKCSNLNFEDEFKEELHSKKHRNKKNDKLFRDEWWKKKKNDNKYNS
KFCNDLKNKKKNKNDAKVSLLLNNCDKKDSYIITGGENGEVLNSKKPRSSAMCVAFDNKIYVKKPRRN
NCGVTSNGRIYCKKFRIILNFLRNPLTIPIAAYHINGKIIKNAAYGKIIKNSEKAAYKTKNNENNKAAYSGS
SNSRNRAAYKENKLNKEGAAYSFFVLYFAKAAYAIAIYESRIAAYREKLWEAMLAAYDNKIYVIGGAA
YAYFGSAVLNAAYVTRDKQGRIAAYFGGNNYDYKAAYEARSSGAAFGPGPGLYISFYFILVNLLIFGPG
PGILYISFYFILVNLLIGPGPGLKMKHILYISFYFILGPGPGSLLLKMKHILYISFYGPGPGLLLKMKHILYIS
FYFGPGPGMKHILYISFYFILVNGPGPGKHILYISFYFILVNLGPGPGNLLIFHINGKIIKNSGPGPGILVNLLI
FHINGKIIGPGPGLLIFHINGKIIKNSEGPGPGFILVNLLIFHINGKIGPGPGHILYISFYFILVNLLGPGPGLIF
HINGKIIKNSEKGPGPGAIAIYESRILKRKYKGPGPGLAIAIYESRILKRKYGPGPGIILNFLRNPLTIPIPGPG
PGMCVAFDNKIYVIGGTGPGPGRSSAMCVAFDNKIYVGPGPGSSAMCVAFDNKIYVIGPGPGNFLRNPL
TIPIPKDLGPGPGCVAFDNKIYVIGGTNGPGPGKAYFGSAVLNNFLYV
|
Flagellin adjuvant is shown in blue, linker EAAAK in green, epitopes in red and the respective linkers (KK, AAY, and GPGPG) joining the each category of epitopes in black.
Table 9. Antigenicity, allergenicity, and physicochemical properties of vaccine construct
Antigenicity
|
Allergenicity
|
AA
|
MW (kDa)
|
pI
|
½ Life (hrs) in
E. coli
|
Instability Index
|
Aliphatic Index
|
GRAVY
|
Antigenic (0.9144)
|
Allergen
|
1448
|
163.3
|
9.77
|
> 10
|
33.37
|
74.71
|
−0.705
|
AA, Amino acid length; MW, Molecular weight; pI, Theoretical Isoelectric point; GRAVY, Grand average hydropathicity
3D modeling, refinement, and validation of the vaccine construct
The selected model exhibited a confidence score of −1.01 (Figure 2). The Ramachandran plot analyses of the 3D model before and after refinement are reported in Figures 3a and b, respectively. For the refined structure, analysis revealed that there were 30 outliers, with 91.3% amino acids in the favored regions and 97.9% amino acids in the allowed regions, as opposed to 115 outliers, with 77.7% in the favored regions and 92% in the allowed regions for the unrefined model.
[Insert Figure 2]
[Insert Figure 3]
Codon optimization and in silico cloning
Upon optimization, the vaccine construct exhibited a nucleotide sequence of 4,344 bp, CAI of 0.9891, and GC content of 43.36%. Upon cloning, the recombinant exhibited a total nucleotide sequence of 9,568 bp (Figure 4).
[Insert Figure 4]
Molecular docking
The selected vaccine-TLR5 complex exhibited 28 hydrogen bonds, with 9 hydrophobic interactions for the ligand, 8 hydrophobic interactions for the receptor, and a binding energy of −11.6 kcal/mol. The 3D and 2D structures of the complex are shown in Figure 5. The selected vaccine-TLR8 complex exhibited 30 hydrogen bonds, with 10 hydrophobic interactions each for the ligand and the receptor, and a binding energy of −11.1 kcal/mol. The 3D and 2D structures of the complex are shown in Figure 6.
[Insert Figure 5]
[Insert Figure 6]
Molecular dynamics simulation
Figure 7 depicts the RMSD and RMSF plots of the vaccine-TLR8 complex. The RMSD plot revealed an initial mean deviation of 2–4 Å within the first 55 ns. However, the standard deviation got stable afterwards and remained consistent throughout the remaining simulation period. The RMSF plot revealed high fluctuations at the N-terminus, indicating high flexibility, with little fluctuations towards the C-terminus.
[Insert Figure 7]
Simulating immune response
The induced secondary and tertiary responses were observed at substantially higher levels than the primary response. Figure 8 presents the immune response simulation results.
[Insert Figure 8]