American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Identification of B-cell Epitope Regions in Cell Surface Proteins of Streptococcus pneumoniae Serotype 19F Using Bioinformatic Tools Shirin Tarahomjoo* Division of Genomics and Genetic Engineering, Department of Biotechnology and Central Laboratory, Razi Vaccine and Serum Research Institute, Karaj 31975/148, Iran Email: starahomjoo@gmail.com Abstract Pneumococcal conjugate vaccines (PCVs) were developed through chemical coupling of polysaccharide capsules of pneumococci to immunogenic carrier proteins and World Health Organization recommends inclusion of these vaccines in national immunization programs for children. However, the PCVs implementation in developing countries is prevented by the high vaccine manufacturing costs. This issue can be resolved by construction of protein based vaccines against pneumococci. We already identified three pneumococcal surface proteins including autolysin, zinc binding lipoprotein (ZBL), and plasmid stabilization protein (PSP) as appropriate protein candidates for eliciting protection against S. pneumoniae serotype 19F. The protein protective antigenicity and the absence of autoimmunity induction were used as selection criteria. However, regarding the necessity of the antibody response for protection against pneumococci, analysis of protective B- cell epitopes of these proteins is required to elucidate their usefulness in new vaccine formulations. In the present study, therefore, we aim to identify protective B-cell epitope regions of these proteins via widely used bioinformatic tools. Both of the Bepipred and BCPreds programs were used for identification of linear B-cell epitopes. The conformational B-cell epitopes were predicted using the CBTope program. The immunoprotective abilities of epitopes were evaluated using VaxiJen. We determined the linear B-cell epitope regions, which were predicted by both Bepipred and BCPreds and have common amino acids with conformational B-cell epitopes. Our results showed that all of the three studied proteins included such protective overlapped linear B-cell epitope regions. However, a truncated form of PSP had the greatest number of the protective overlapped B-cell epitope regions. ------------------------------------------------------------------------ * Corresponding author. 107 http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 Moreover, the most probable protective epitope region resides in this protein and this epitope region is completely conserved in PSPs of serotype 19F pneumococcal strains. Therefore, the truncated PSP was an appropriate candidate for development of protein based vaccines against S. pneumoniae serotype 19F. Keywords: B-cell Epitopes; Pneumococcal serotypes; Pneumococcal vaccines; Streptococcus pneumoniae. 1. Introduction Streptococcus pneumoniae is a major pathogen that causes diseases such as pneumoniae, meningitis and sepsis [1]. World Health Organization (WHO) estimated that 476000 annual deaths among children less than 5 years of age were caused by pneumococcal infections [2]. S. pneumoniae serotype 19F is among the main pneumococcal serotypes that cause most invasive pneumococcal disease in children less than 5 years of age globally [3]. Pneumococcal vaccines have been used for protection against pneumococcal infections. The polysaccharide capsules of pneumococci are main antigenic components of these vaccines. However, the capsules are poorly immunogenic in children and are not able to induce anamnestic antibody responses upon revaccination. In order to circumvent these issues, the capsules are chemically conjugated to immunogenic carrier proteins. Therefore, the capsules are converted from T-cell independent antigens to T-cell dependent antigens, which enhance antibody responses and induce the immune memory. WHO recommends inclusion of pneumococcal conjugate vaccines (PCVs) in national immunization programs for children [2]. However, high manufacturing costs of PCVs limit their implementation in developing countries [1,2]. Development of protein based vaccines against pneumococci offers a more affordable protective strategy against pneumococcal infections. Cell surface proteins are key factors in infectious processes of pathogens and have extensively been evaluated as vaccine candidates [4,5]. We already identified three pneumococcal surface proteins including autolysin, zinc binding lipoprotein (ZBL), and plasmid stabilization protein (PSP) as protein candidates for eliciting protection against S. pneumoniae serotype 19F. The candidate selection was based on the protective antigenicity, and the absence of autoimmunity induction [6]. Antibody responses are required to protect against pneumococcal infections [7]. Thus, analysis of protective B-cell epitopes in the candidates is necessary to evaluate the effectiveness of these proteins as new vaccine constituents. Compared with conventional laboratory methods, computational approaches offer the ability to undertake rapid and comprehensive epitope assessments for vaccine candidates at much lower costs [8]. In the present study, therefore, we analyzed autolysin, ZBL, and PSP via widely used bioinformatic tools for identification of immunoprotective B-cell epitope regions. To our knowledge, there is no report concerning recognition of B-cell epitope regions of these proteins for the purpose of vaccine development against S. pneumoniae serotype 19F. 2. Materials and Methods The NCBI accession numbers of autolysin, ZBL, and PSP were WP_000405240, WP_000724068, and WP_00034749 respectively. PSP was indicated in our previous work [6] as cell wall surface anchor family protein under accession number YP_002743140. Conserved domains of proteins were specified using Pfam. Protein sequences were submitted to the Bepipred and BCPreds programs for identification of linear B-cell epitopes. The immunoprotective abilities of selected B-cell epitopes were evaluated using VaxiJen. A higher 108 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 VaxiJen score refers to a higher probability for protective ability [9]. The CBTope program was used to predict conformational B-cell epitopes using amino acid sequences of the protein. COBALT alignment tool was used for the epitope region conservancy analysis in PSPs of serotype 19F pneumococcal strains. Accession numbers of PSPs were KKW85405 (S. pneumoniae CU-SPNE1-05), KKW83972 (S. pneumoniae CU-SPNE32-06), AFC95386 (S. pneumoniae ST556), and WP_000873519 (S. pneumoniae G54). PHD program was used for the secondary structure prediction. SCRATH was used for predicting the protein solubility upon overexpression in Escherichia coli. The default settings were applied to all the tools used. 3. Results and Discussion 3.1. Identification of protective B- cell epitope regions of autolysin Antibodies bind specifically to a continuous amino acid sequence of a protein known as the linear B-cell epitope or to a folded structure formed by discontinuous amino acids known as the conformational B-cell epitopes. The majority of B-cell epitopes are conformational [10]. Nevertheless, the identification of linear B-cell epitopes has demonstrated promising results for selection of the vaccine antigens [11,12]. Therefore, in this study, we determined both types of the B-cell epitopes in the proteins. Bacterial autolysins are enzymes capable of hydrolyzing peptidoglycan and have a major role in concealing this inflammatory molecule from the immune system [13]. Table 1: Protective linear B-cell epitopes of autolysin identified by Bepipred Position1 Epitope sequence VaxiJen score 13 PQVGVQPY 1.3051 24 HAHSTGNPHSTVQNEADYHWRKDPE 1.0542 66 PVDNGAWDVGGGWNAETY 0.4215 135 YCTNNQPNNHSDHVDPYPY 0.5565 285 QSADGTGW 1.4423 294 YLKPDGTLADKPEFTVEPD 1.3907 1: amino acid residue number at the epitope beginning is indicated. Bepipred and BCPreds are widely used bioinformatic tools for the linear B-cell epitope identification. The Simultaneous application of different linear B-cell epitope prediction tools results in greater accuracy of the epitope identification. Therefore, we used both of these tools to determine the B-cell epitope regions in autolysin. Nine linear B-cell epitopes were identified in the protein using the Bepipred program. VaxiJen results indicated that 6 epitopes among them were immunoprotective (Table 1). In addition, BCPreds identified 7 linear B-cell epitopes. Considering VaxiJen results, 5 epitopes among them were protective (Table 2). 109 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 Table 2: Protective linear B-cell epitopes of autolysin identified by BCPreds Position1 Epitope sequence Vaxijen score 12 LPQVGVQPYRQVHAHSTGNP 1.161 134 EYCTNNQPNNHSDHVDPYPY 0.4469 221 RKHTDGNWYWFDNSGEMATG 0.6321 251 FNEEGAMKTGWVKYKDTWYY 0.7268 283 FIQSADGTGWYYLKPDGTLA 1.0224 1: The amino acid residue number at the epitope beginning is indicated. The CBTope program uses the protein amino acid sequence for the prediction of conformational B-cell epitopes. Using this program, 8 conformational B-cell epitope regions were identified in the protein (Table 3). The epitope regions beginning at the amino acid residue 97 and 172 were exclusively conformational, whereas the rest of the conformational B-cell epitope regions overlapped with the identified linear B-cell epitopes. Table 3: Conformational B-cell epitope regions of autolysin Position 1 No. of epitope residues Epitope region sequence 2 1 20 MEINVSKLRT DLPQVGVQPYRQ 36 7 QNEADYH 97 7 EFMT DYRLYI 131 22 KTHEYCTNNQPNNHSDHVDPYPYLAKWG 172 13 LTIETGWQK NDTGYW 207 31 YYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGW 254 21 EGAMKTGWVKYKDTWYYLDAKEGAMVSNAFIQSA 300 4 TLAD 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 2: Amino acid residues of conformational B-cell epitopes in each region are shown underlined. The linear B-cell epitopes determined by Bepipred and BCPreds were overlapped at three regions, which included amino acids common with the conformational B-cell epitopes (Table 4). Regarding the VaxiJen scores, the epitope region beginning at the amino acid 283 was the most probable protective linear B-cell epitope region of autolysin. 110 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 Table 4: Protective overlapped linear B-cell epitope regions of autolysin Position 1 B-cell epitope region 2,3 VaxiJen score 12 LPQVGVQPYRQVHAHSTGNP 1.1610 134 EYCTNNQPNNHSDHVDPYPY 0.4469 283 FIQSADGTGWYYLKPDGTLADKPEFTVEP 1.2326 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 2: Amino acids common with the conformational B-cell epitope regions are in italics. 3: The most probable linear B-cell epitope region is in bold. 3.2. Identification of protective B- cell epitope regions of ZBL ZBL is a lipoprotein, which is involved in the bacterial zinc uptake. The signal peptide of ZBL is cleaved between amino acids 18 and 19 [14]. The mature protein obtained following cleavage of the signal peptide was used in the B-cell epitope analysis. Fifteen linear B-cell epitopes were identified in the protein using Bepipred. Thirteen epitopes among them were immunoprotective as indicated by VaxiJen results (Table 5). Eight linear B- cell epitopes were recognized in the protein using BCPreds. Considering VaxiJen scores, four epitopes among them were protective (Table 6). Table 5: Immunoprotective linear B-cell epitopes of mature ZBL identified by Bepipred Position1 Epitope sequence VaxiJen score 1 CSNQKQADG 2.2763 26 QVAGDTA 1.3983 41 GTEPHEYEPSAKAVA 0..6642 100 GGEEEEGDHDHGEEGHHHEF 2.0596 168 KAYTEGLSQAKQ 0.5478 205 GLSPDAEPSAA 0.8943 247 SKEAGVK 2.3193 262 SLTEEDTKAGE 2.0618 287 QTTDQEGPAIEPEKAEDTKTVQNGYFEDAAVK 1.1307 354 KMTQAEYKAY 0.6471 365 TKGYQTDV 1.4374 385 VQGGQSKKY 2.6108 461 DNWPTYYPDNLSGQEI 1.0051 1: The amino acid residue number at the epitope start is indicated. 111 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 In the CBTope program, 11 conformational B-cell epitope regions were recognized in the mature ZBL (Table 7). The epitope region beginning at the amino acid residue 430 was exclusively conformational. The rest of the conformational epitope regions were overlapped with the identified linear B-cell epitopes. Table 6: Immunoprotective linear B-cell epitopes of mature ZBL identified by BCPreds Position1 Epitope sequence VaxiJen score 58 QDADTFVYENENMETWVPKL 0.5112 99 PGGEEEEGDHDHGEEGHHHE 2.4164 285 LKQTTDQEGPAIEPEKAEDT 1.4277 348 KAKLTGKMTQAEYKAYYTKG 0.7859 1: The amino acid residue number at the epitope start is indicated. Table 7: Conformational B-cell epitope regions of mature ZBL Position1 No. of epitope residues Epitope region sequence2 1 13 CSNQKQADGKLNI 26 12 QVAGDTANVELLIG 49 20 PSAKAVAKIQDADTFVYENENMETWV 141 23 LSADYPDKKE TFEKNAAAYIEKLQALDKAYTEGL 196 7 YGLKQVAISG 214 6 AARLAELTEYVKKN 249 8 EAGVKTDVLNPLESLT 286 40 KQTTDQEGPAIEPEKAEDTKTVQNGYFEDAAVKDRTLSDYAGNWQSVYPF 369 19 QTDVTKINITDNTMEFVQGGQ 430 5 QFSDH 459 20 EMDNWPTYYPDNLSGQEIAQEMLA 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 2: Amino acid residues of conformational B-cell epitopes in each region are shown underlined. The linear B-cell epitopes identified in the mature ZBL by Bepipred and BCPreds were overlapped at two regions, which included amino acids common with the identified conformational B-cell epitopes (Table 8). The epitope region beginning at the amino acid 285 showed the highest VaxiJen score and was therefore the most probable protective linear B-cell epitope region of the protein. 112 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 Table 8: Overlapped linear B-cell epitope regions of mature ZBL Position1 Epitope region2,3 Vaxijen score 285 LKQTTDQEGPAIEPEKAEDTKTVQNGYFEDAAV 1.0701 348 KAKLTGKMTQAEYKAYYTKGYQTDV 0.8959 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 2: Amino acids common with the conformational B-cell epitope regions are in italics. 3: The most probable protective B-cell epitope region is in bold. 3.3. Identification of protective B- cell epitope regions of PSP PSP contains an YSIRK type family signal peptide, which is cleaved between the amino acids 36 and 37. Moreover, it contains an LPxTG motif beginning at the amino acid residue 658 [6]. We used the truncated PSP containing the amino acids 37-657 for the B-cell epitope analysis. The linear B-cell epitope analysis using Bepipred showed the presence of 15 linear B- cell epitopes in the protein. VaxiJen results indicated that all of the epitopes were protective (Table 9). BCPreds analysis identified 17 linear B-cell epitopes in the truncated PSP. Fourteen epitopes among them were protective as indicated by VaxiJen results (Table 10). Table 9: Protective linear B-cell epitopes of truncated PSP identified by Bepipred Position1 Sequence VaxiJen score 2 EVVGETSSSKDNM 1.1265 24 STSLSAEPHSDTLSLD 0.9694 41 NSSLSTDNNGGVSINQNKPDITND 1.0703 94 YGAIGDGVNDDRQAIQ 0.7286 112 IDAAAQGLGGGNVYFP 1.1389 173 TDDGAQVEWGPTEDISYSGGTID 0.9767 199 ALNEEGTKAK 2.1738 290 YALNDDGKKSENVT 1.6011 309 FGKSDKSGE 3.0686 328 TLSTQNPS 1.0665 367 FDKKVKGESVHYRE 1.7546 444 INNNSKETEQP 1.7294 509 NANEKEPVIRDSDGN 0.9024 540 LSDKNEKEKNKEEKQSNSNNVIDSNQKNGEF NSSKDNRQMNDKIDNKQDNKTEEV 1.6322 599 VGDGRETENHIN 2.1816 1: The amino acid residue number at the epitope beginning is indicated. 113 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 Table 10: Protective linear B-cell epitopes of truncated PSP identified by BCPreds Position1 Sequence VaxiJen score 1 NEVVGETSSSKDNMQMNSLL 1.062 22 STSTSLSAEPHSDTLSLDNN 0.8067 55 NQNKPDITNDTMMGNISGME 0.7343 90 NVKDYGAIGDGVNDDRQAIQ 0.499 113 DAAAQGLGGGNVYFPEGTYL 0.4168 173 TDDGAQVEWGPTEDISYSGG 0.9974 217 GAFAIGNSNNVTIKNVTFKD 0.9995 293 NDDGKKSENVTIQNSYFGKS 1.2743 364 GNRFDKKVKGESVHYRESGA 1.3528 439 VTKNVINNNSKETEQPNIEL 1.0637 511 NEKEPVIRDSDGNFNIVTEN 0.6281 534 KIVTNNLSDKNEKEKNKEEK 1.6349 555 SNSNNVIDSNQKNGEFNSSK 0.7679 585 NKQDNKTEEVNYKIVGDGRE 1.5427 1: The amino acid residue number at the epitope beginning is indicated. CBTope analysis revealed the presence of 10 conformational B-cell epitope regions in the protein (Table 11). The epitope region beginning at the amino acid 615 was exclusively conformational. However, the rest of the conformational B-cell epitope regions were overlapped with the linear B-cell epitopes. Table 11: Conformational B-cell epitope regions of truncated PSP Position1 No. of epitope residues Epitope region sequence2 51 11 GVSINQNKPDIT 89 4 LNVKD 126 5 FPEGTYLVK 187 5 ISYSGGTIDMNGA 224 13 SNNVTIKNVTFKDSYQGHAIQIAGSKNVLVDNSR 272 45 IISKESIQIEPLTRKGFPYALNDDGKKSENVTIQNSYFGKSD KSGELVTAIGTHYQTLSTQNPSNIKIL NNHF 375 8 SVHYRESGAALVN 457 27 ELLRVSDNLVVSENSIFGGKEGIVIEDSKGKITVLNNQF 520 10 SDGNFNIVTENGLYKIV 587 8 QDNKTEEVNYKIVGDGRE 615 7 IVDVKQK 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 114 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 2: The amino acid residues of conformational B-cell epitopes in each region are shown underlined. The linear B-cell epitopes identified in the truncated PSP by Bepipred and BCPreds were overlapped at 8 regions, which included amino acids common with the identified conformational B-cell epitopes (Table 12). Considering VaxiJen scores, the epitope region beginning at the amino acid 534 was the most probable protective linear B-cell epitope region of the protein. Moreover, the VaxiJen score of this overlapped B-cell epitope region was higher than those identified in autolysin and the mature ZBL. The conservancy of this region in PSPs of pneumococcal serotype19F strains was analyzed using the COBALT multiple protein sequences alignment tool. The region was fully conserved in the pneumococcal strains. This result indicated that the immunological response elicited against this region is protective against serotype 19F pneumococcal strains. The secondary structures predicted for the truncated PSP using the PHD program were α helix, extended strand and random coil with the predominance of random coil. The overlapped B-cell epitope region beginning at the amino acid residue 534 was located in random coil and extended strand. SCRATCH predicted 80% solubility for the truncated PSP upon overexpression in E. coli. Therefore, the protein can be recovered from the cellular soluble fraction. Table 12: Overlapped linear B-cell epitope regions of truncated PSP Position1 Epitope sequence2,3 VaxiJen score 41 NSSLSTDNNGGVSINQNKPDITNDTMMGNISGME 0.9445 112 IDAAAQGLGGGNVYFPEGTYL 0.5299 173 TDDGAQVEWGPTEDISYSGGTID 0.9767 290 YALNDDGKKSENVTIQNSYFGKSDKSGE 1.4416 364 GNRFDKKVKGESVHYRESGA 1.3528 439 VTKNVINNNSKETEQPNIEL 1.0637 509 NANEKEPVIRDSDGNFNIVTEN 0.6279 534 KIVTNNLSDKNEKEKNKEEKSDKNEKEKNKEEKQSNSNNV IDSNQKNGEFNSSKDNRQMNDKIDNKQDNKTEEVNYKIVGDGRETENHIN 1.6046 1: Amino acid residue numbers at the beginning of epitope regions are indicated. 2: Amino acids common with the conformational B-cell epitope regions are in italics. 3: The most probable protective B-cell epitope region is in bold. 4. Conclusions and Recommendations Among three proteins including autolysin, the mature ZBL, and the truncated PSP of S. pneumoniae serotype 19F, the truncated PSP possesses the greatest number of protective overlapped linear B-cell epitope regions, 115 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2015) Volume14, No 3, pp 107-117 which were predicted by both Bepipred and BCPreds programs and have amino acids common with the protein conformational B-cell epitopes. In addition, the overlapped B--cell epitope region with the most probability of eliciting protection resides in this protein and this region is fully conserved in PSPs of serotype 19F pneumococcal strains. Therefore, the truncated PSP is considered as an appropriate candidate for development of protein based vaccines against S. pneumoniae serotype 19F. In future, the protein ability in eliciting protection against pneumococci will be assessed experimentally. 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