Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein

Vaccine 27 (2009) 5103–5110

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Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein夽 Xiaohui Yuan a , Zhangyi Qu a,∗ , Xiaomin Wu b , Yingchen Wang a , Lei Liu a , Fengxiang Wei a , Hong Gao a , Lei Shang a , Hongyan Zhang a , Hongbo Cui c , Yuehui Zhao c , Na Wu a , Yanhong Tang a , Le Qin a a

Department of Hygienic Microbiology, Public Health College, Harbin Medical University, Baojian Road 157, Harbin Heilongjiang 150081, PR China Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China c Microbiology Laboratory, Harbin Medical University, Harbin 150081, PR China b

a r t i c l e

i n f o

Article history: Received 21 December 2008 Received in revised form 26 April 2009 Accepted 10 June 2009 Available online 30 June 2009 Keywords: HAdV Hexon protein Epitope Mapping

a b s t r a c t The hexon protein of human adenovirus (HAdV) processes type-specific B-cell neutralizing epitopes. We developed a new effective, reliable approach to map these epitopes on hexon protein of HAdVs. A three-dimensional (3D) model of the HAdV3 hexon was obtained by homology modeling and refined by molecular mechanics and molecular dynamics simulations. A modified evolutionary trace (ET) analysis called reverse ET (RET) was used to predict the type-specific B-cell neutralizing epitopes. An epitopescreening algorithm based on analyzing the solvent accessibility surface (SAS) area from the 3D model and calculation of sites homology using RET was designed and implemented in the BioPerl script language. Five epitope polypeptide segments were predicted and mapped onto the 3D model. Finally five polypeptides were synthesized and the predicted epitopes were identified by enzyme-linked immunosorbent assay (ELISA) and Neutralization Test (NT). It was found that the type-specific neutralizing epitopes of HAdV3 are located at the top surface of hexon tower regions (residue numbers: 135–146, 169–178, 237–251, 262–272, 420–434). This work is of great significance to the molecular design of a multivalent HAdVs vaccine. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Adenoviridae viruses are nonenveloped, double-stranded DNA viruses with an icosahedral capsid comprising 240 hexons and 12 vertex capsomeres [1,2]. The human adenovirus (HAdV) can be classified into 6 species (A–F) on the basis of hemagglutination and genomic properties [3], consisting of 51 serotypes defined mainly by neutralization criteria [4,5]. HAdVs can cause a broad spectrum of human infective diseases [6–9], among which upper respiratory tract infection and pedo-pneumonia caused by serotypes 3 and 7 are particularly serious [5,10,11]. Especially in northern China, the major epidemic strains are HAdV3 and HAdV7 [12,13]. There is as yet no effective curative antiviral medicine or vaccine with which to treat these diseases.

夽 Supported by The National Natural Science Foundation of China (No. 30771909), The Doctoral Co-financing Project of Chinese Ministry of Education (No. 20070226007), The Natural Science Foundation Key Project of Heilongjiang China (No. ZJY0701), Science and Technology Project of Heilongjiang Provincial Education Department (No. 11521172). ∗ Corresponding author. Tel.: +86 451 87502965; fax: +86 451 86667248. E-mail addresses: [email protected], [email protected] (Z. Qu). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.06.041

The major coat protein of HAdV is hexon (i.e., a homotrimer protein comprising three monomers A, B and C) [14]. It has been shown that antibodies stimulated by a hexon can neutralize an HAdVmediated viral infection, and that this neutralization reaction is type-specific [15]. The tower region of this hexon homotrimer contains a large number of type-specific neutralizing epitopes (B-cell epitopes). Identifying these type-specific neutralizing epitopes is of great significance in several areas of HAdV research, including the molecular design of a HAdV vaccine [16–18], the development of a rapid HAdV diagnostic agent and preparing an antiadenovirus medicine [19,20]. But very little is currently known about the mapping of type-specific B-cell neutralizing epitopes of hexons in many serotypes of HAdV. Although it is possible to locate hypervariable regions (HVRs) [14,21] using the multiple sequence alignment (MSA) method [22], it is difficult to identify the type-specific neutralizing epitopes and to obtain the specific three-dimensional (3D) conformation of the epitope peptides for a specific serotype. The 3D conformation of hexon protein can provide relevant information about epitopes. The hexon structures of HAdV type 2 (HAdV2) and HAdV type 5 (HAdV5) [23,24] are available in the Protein Data Bank (PDB) [25], but these structures have inherent amino acid deletions and disruption of the peptide chains. In addition, related documents show that these structures are only the conserved core region

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of the HAdV hexon protein [15], and do not provide information of the complete tower structure, which contains the type-specific neutralizing epitopes. This study investigated two characteristics of epitopes on HAdVs hexons – namely B-cell neutralizing epitopes and type-specific epitopes – using a combination of molecular simulation technology [26] and bioinformatics evolutionary trace (ET) [27,28] analysis. The 3D structure of the HAdV type 3 (HAdV3) hexon was determined using molecular simulation/homology modeling [26,29,30], and the solvent-accessible surface area (SAS) [31] of the model was calculated. In addition, a modified ET method that we named reverse ET (RET) was employed. This involved the use of MSA, sites homology calculation, and a purpose-designed epitope-screening algorithm that combines the results from the SAS analysis and sites homology calculation. The presence of five candidate epitope segments was predicted and mapped onto the 3D model of the hexon. Finally, the predicted epitope peptides were synthesized. Two serological experiments were performed to prove the correctness of epitopes prediction: (1) enzyme-linked immunosorbent assay (ELISA) was used to detect the affinity of epitope peptides and anti-HAdV3 serum; (2) Neutralization Test (NT) was used to test the neutralizing effect to HAdV3 of antipeptides sera. 2. Materials and methods 2.1. HAdV3 and anti-HAdV3 serum The HAdV used in this study was an isolated strain obtained from clinical throat-swab specimens. In 2003 and 2004, many children in the Harbin area of China contracted fever [13], and a total of 384 throat swabs were taken from them in the Department of Pediatrics, No. 1 Subsidiary Hospital of Harbin Medical University. A strain of HAdV (namely Harbin04B) was successfully isolated in our laboratory, and cell culture, immunology, and morphological, PCR, and sequencing analyses of the hexon gene identified the virus as HAdV3, the nucleotide sequence of which has been deposited in the NCBI GenBank database (accession number: EU078562). Anti-HAdV3 serum was obtained from a 6-week-old female New Zealand rabbit that had been injected intramuscularly with 1013 HAdV3 particles and then boosted subcutaneously with 1013 viral particles emulsified in complete Freund’s adjuvant (Sigma). Blood was collected from the ear fringe vein plexus, and serum was prepared for an ELISA, with preimmune serum used as a negative control. The antibody titer and the HAdV3 specificity were detected by ELISA as described previously [32]. 2.2. Homology modeling Homology modeling, energy minimization (EM) and molecular dynamics (MD) simulations were performed using a molecular simulation software package InsightII 2005 (Accelrys Inc., San Diego, USA). The consistent-valence force field (CVFF) [33–35] was employed for EM and MD simulations. The HAdV3 hexon amino acid sequence was deduced from the corresponding nucleotide sequence derived from Harbin04B and was named by HEX3. 2.2.1. Molecular modeling and structure refinement The InsightII/Homology module was applied to build the 3D structure of the HAdV3 hexon. The web-FASTA tool [36] of the PDB (http://www.rcsb.org) was used to search for an appropriate template for the homology modeling using HEX3 as a probe. The chimpanzee adenovirus 68 (AdC68) [15] hexon (PDB code: 2obe) at 2.1 Å resolution exhibited the highest degree of homology at 85.6% higher than HAdV2 hexon (PDB code: 1p2z) [23], and

HAdV5 hexon (PDB code: 1p30) [24], and was thus considered to be the most appropriate template. MSA based on the Needleman and Wunsch Algorithm [37] was performed with 2obe, 1p30, 1p2z, and HEX3 to conform to the structurally conserved region (SCR). InsightII/Modeler program was used to automatically construct the initial model of HEX3. To refine the structure, the EM and MD simulations were executed in the InsightII/Discover 3 module. The entire process was divided into the following steps: first, all the hydrogen atoms and side chains were optimized in a vacuum by a 500-step steepest-descent (SD) minimization followed by conjugate gradient (CG) minimization until the final convergence was lower than 0.01 kcal mol−1 Å−1 . Second, the loop regions were optimized by fixing all atoms except for those in the tower region, and then performing 500 steps of SD and CG until the final convergence was lower than 0.01 kcal mol−1 Å−1 . Because EM cannot solve the energy-barriers problem [38], a MD simulation was performed (1000 ps at 310 K) to achieve the stable conformation for the tower region (residues: 115–310 and 400–510). Third, CG minimization of the full protein was performed until the final convergence was lower than 0.01 kcal mol−1 Å−1 . This step improved the quality of the initial model of the HEX3 homotrimer. The above procedure produced the 3D model of the HAdV3 hexon homotrimer. The structure was further checked using the InsightII/ Profiles 3D and InsightII/ProStat programs. The Profiles 3D program was used to examine the compatibility of an amino acid sequence with a known 3D protein structure [39]. The ProStat program investigated the secondary structural of the 3D model based on Kabsch Sander method [40]. 2.2.2. Solvent accessibility surface analysis (SAS) SAS analysis is commonly used to evaluate how deep a given residue is buried [31]. The SAS of the entire HEX3 homotrimer model was calculated with the InsightII/Access Surf program. A probe radius of 1.4 Å was used for all calculations. The difference in the SAS of each residue in the HEX3 model was determined. Two residue groups were created: (1) an exposed group, whose residues were greater than 25% of the maximum SAS; and (2) a buried group, whose residues were less than 10% of the maximum SAS. The SAS data were used in the subsequent epitopes screening. 2.3. Reverse evolutionary trace analysis (RET) ET analysis can extract the information obtained from the MSA of homologous proteins onto a certain 3D molecule and thereby investigate which amino acid residues are likely to be crucial for certain functions [27,28]. Because the neutralizing epitopes of HAdV3 Hexon are all type-specific, we utilized a special modified ET method to map the candidate type-specific neutralizing epitopes derived from a epitope-screening algorithm onto the 3D model of HEX3, which we called reverse evolutionary trace (RET) analysis including MSA, sites homology calculation and a designed candidate epitopes screening. 2.3.1. Multiple sequence alignment (MSA) MSA is commonly the first step of ET analysis. We obtained 23 serotypes of complete hexon amino acid sequences of HAdV including all serotypes of species A, B, C, F and a few typical serotypes of species D. The sequence data were all derived from NCBI GenBank under the following accession numbers: AC 000017 (HAdV1), AC 000007 (HAdV2), X76549 (HAdV3), NC 003266 (HAdV4), AC 000008 (HAdV5), DQ149613 (HAdV6), AC 000018 (HAdV7), DQ149615 (HAdV10), AC 000015 (HAdV11), X73487 (HAdV12), DQ149612 (HAdV14), DQ149617 (HAdV15), AY601636 (HAdV16), DQ149610 (HAdV18), AY008279 (HAdV21), DQ149611 (HAdV31),

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Table 1 Synthesized polypeptides and the location of S1–S5 and two control polypeptides P1 and P2. Segment

Locationa

Sequence

S1 S2 S3 S4 S5 P1 P2

135–146 169–178 237–251 262–272 420–434 149–160 469–482

Cb IVTAGEERAVTT C GKDITADNK C NRKVKPTTEGGVETE C GREAADAFSPE C SKDNGWEKDDNVSKS C NTFGIASMKGDN C VYKYTPTNITLPAN

a

The location is the residue number of monomer A of the hexon. Underlining indicates the added Cys(C) on the N-terminal of synthetic polypeptides. b

2.4. Epitope peptides synthesis and peptides ELISAs

Fig. 1. Phylogenetic relationships of deduced amino acid sequences of HAdV hexon proteins. The evolutionary history was inferred using the MP method. Unrooted tree reflected the distance relationship of all HAdV hexon amino acid sequences in it. Accession number of reference sequences is shown in the tree.

AB052911 (HAdV34), AB052912 (HAdV35), DQ149632 (HAdV37), X51782 (HAdV40), DQ315364 (HAdV41), EF153473 (HAdV48), DQ149643 (HAdV50). Besides the HEX3, MSA was performed using the ClustalW1.83 program [41] with the Clustal algorithm and adjusted manually to conform the optimized alignment of deduced amino acid sequences. In order to show the evolutionary relationship of these sequences, a phylogenetic tree was constructed by the maximum-parsimony (MP) method [42] using the MEGA4.0 package [43]. The phylogenetic tree is shown in Fig. 1. 2.3.2. Sites homology calculation Sites homology refers to the similarity of the same site in different sequences aligned by MSA. It reflects how a certain site is conserved in aligned homologous protein sequences. Taking the sites of HEX3 as standard sites, the sites homology in different hexon amino acid sequences were calculated according to the aligned result. The calculation method was as follows: site homology = number of conserved amino acids on same site/number of total sequences × 100%. Then a color mapping scheme was employed in InsightII environment to map the information from the sites homology calculation onto the 3D model of HEX3, we set three ranges of site homology values (≤90%, ≤60%, and ≤30%) to reflect the trend of sites homology. 2.3.3. Candidate epitopes screening Because the epitopes on the hexon protein are type-specific Bcell neutralizing epitopes [4,5], we designed an epitope-screening algorithm to screen the candidate epitopes: site with a homology of less than 45% were defined as hypervariable site. Segments fulfilling the following standards were selected as candidates: (1) length of between 6 and 15, (2) more than half of them being hypervariable sites, (3) interval between candidate sequences not shorter than three, and (4) 90% of residues belonging to the exposed group with residues greater than 25% of the maximum SAS. Finally, five candidate epitope segments were screened out and mapped onto the 3D HEX3 model using a color mapping scheme in the InsightII environment. All above of the sites homology calculation and candidate epitopes screening were implemented in programs written by the Bioperl script language [44].

2.4.1. Synthetic epitope peptides The five candidate epitope polypeptides predicted using the above-mentioned method were synthesized by the Fmoc method [45] with the solid-phase technique utilizing the Symphony Peptide Synthesizer (Tianjin Saier Biotechnology Co., Ltd. TianJin, China). In the meanwhile two control polypeptides (P1: 149–160 and P2: 469–482) were synthesized by the same method: P1 had a type-specific property but was located in the buried group in SAS analysis, while P2 was selected from the exposed group in the SAS analysis but it was conserved among the HAdV serotypes. Highperformance liquid chromatography was used to analyze the purity and correctness of the synthetic polypeptides [46]. The Cys on the N-terminal of synthetic polypeptides was added for conjugation. Each polypeptide (purity ≥85%) was then chemically linked to the carrier protein bovine serum albumin (BSA, Sigma) and keyhole limpet hemocyanin (KLH, Sigma) by the glutaraldehyde (GA) treatment method [47]. The sequences of synthesized polypeptides and their locations are listed in Table 1. 2.4.2. Peptide ELISAs The ELISAs were performed using streptavidin to coat the plates and bind biotin with casein blocker [48], with the previously prepared HAdV3 antiserum serially diluted in PBS with dilution: 1/500, 1/1000, 1/2000 and 1/4000. The ELISA plates were also coated with 100 ␮l of PBS (pH 7.4) containing the seven synthesized polypeptides (S1–S5, P1 and P2) coupled with BSA, BSA protein only as a negative control, and purified hexon protein (purity ≥85%) prepared in our lab [32] coupled with BSA by the GA methods of HAdV3 as a positive control. After incubation for 16 h at 4 ◦ C, the wells were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (Sigma) for 2 h at 37 ◦ C. After washing with PSB, 100 ␮l of the serum sample was added to the wells and incubated for 1 h at 37 ◦ C. The secondary antibody was horseradish-peroxidase-conjugated antirabbit IgG goat serum (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Shanghai, China), followed by washes and treatment with H2 O2 and o-phenylenediamine (Sigma). The optical density (OD) was measured at 490 nm after incubation for 30 min at 22 ◦ C. 2.5. Antipeptides sera and Neutralization Tests 2.5.1. Preparation of antipeptides sera Fourteen female New Zealand rabbits aged 6–8 weeks were purchased from the Department of Laboratory Animals, No. 2 Subsidiary Hospital of Harbin Medical University, and the seven synthesized polypeptides (epitope peptides: S1–S5; and two control peptides: P1 and P2) coupled with KLH were used for animal immunization (two rabbits for each coupled peptide), with preimmune serum used as a negative control.

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The immunization schedule was as follows: 14 rabbits were subcutaneously immunized with a 600-␮g dose of coupled peptides (1 ml) emulsified in 1 ml of complete Freund’s adjuvant (Sigma). All rabbits were boosted with the same method at 14 and 28 days after the primary immunization with a 400-␮g dose of coupled peptides (1 ml) emulsified in 1 ml of incomplete Freund’s adjuvant (Sigma). Blood samples were taken from the ear fringe vein plexus at 0 dpi (preimmune), 21 dpi, and 35 dpi, stored overnight at 4 ◦ C, and centrifuged at 1200 g relative centrifugal force to obtain clarified sera. Antibody titers were detected by ELISA using peptides coupled with BSA. The titers of the 21- and 35-dpi sera were 1:8000 and 1:10,000, respectively. The sera were then prepared for Neutralization Tests (NTs). 2.5.2. Neutralization Tests (NTs) NTs were performed to test the neutralizing effect of seven antipeptides sera to HAdV3. All of the antipeptides sera (anti-S1, anti-S2, anti-S3, anti-S4, anti-S5, anti-P1, and anti-P2), preimmune serum as a negative control, and anti-HAdV3 serum as a positive control were serially diluted in PBS, and 25-␮l aliquots of each dilution were mixed with 25 ␮l of HAdV3 with 100 TCID50 . The antibody–virus mixtures were incubated for 1 h at 37 ◦ C in the presence of 5% CO2 and then transferred to 96-well plates containing nearly confluent (85–95%) monolayers of HELA cells. Monolayers were incubated for 48 h in the presence of 5% CO2 , after which infection was monitored using fluorescence microscopy by identifying positive sera that inhibited 50% of the cytopathic effect (CPE). 3. Results 3.1. Homology modeling of HEX3 The HAdV3 hexon monomer encoded by hexon gene contains 937 amino acids, and results obtained with the web-FASTA tool have shown that the AdC68 hexon in the PDB (code: 2obe) has the highest sequence homology, at 85.6%. However, this high homology is not balanceable, since in the SCR it is more than 95%, whereas in the tower region (residues: 115–310, 400–510) it is only about 66%, and

Fig. 3. Profile 3D verification result of the HEX3 model, with residues exhibiting reasonable folding. A score >0 indicates residues are compatible, and a score <0 indicates that residues have interactions with other monomers.

the structural differences are mainly in the tower regions; therefore, our homology modeling focused on the tower region. In this study, the initial model of HEX3 was constructed using the automated homology modeling program Modeler, then MM optimization and MD simulation were performed to refined the model. The final stable structure of HEX3 and one of its monomer A are displayed in Fig. 2. The overall quality of the final structure was further evaluated using the Profiles 3D program, which is normally used to quantify the compatibility of an amino acid sequence with a 3D protein structure and especially to check the validity of a hypothetical protein structure. The result is shown in Fig. 3. Note that compatibility scores above zero are considered acceptable, and regions of the protein for which the score approaches zero or becomes negative are likely to be misfolded if a surface patch of a protein shows a low score or becomes negative, this might indicate that the surface is interacting with other proteins and should be buried internally. From Fig. 3, we can see that the negative scores were obtained in the regions of residues 186–191, 228–235 and 345–355 of each monomer. The closest approximation was to template 2obe, which

Fig. 2. The final 3D structure of the HEX3 homotrimer (A), and its monomer A ribbon structure (B). The structure obtained by a series of energy minimization and MD simulation. The random coil, sheet, helix and turn are represented by green, yellow, red and blue color respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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shows that they are regions where the three hexon monomers interact with each other. The Profiles 3D verification scores were all higher in the tower region than in the base region, indicating that the conformation of the tower was acceptable. It was believed that the understanding of secondary structural elements is a foundation to investigate the large and complex structures. Four types of secondary structures were observed in the HEX3 model by ProStat program, which are denoted as random coil, sheet, helix and turn, respectively. As shown from Fig. 2, we can see that the HAdV3 hexon homotrimer protein is a stable structure comprising three interlinked hexon monomers which is consistent with some results in the literature. This trimer could be further divided into three major parts: (1) the tower region (residues 115–310 and 400–510) that forms the outer layer of homotrimer, and comprising three actual towers; (2) the base region that forms the internal layer of the capsomer, which the MSA later indicates is highly conserved among all serotypes; and (3) the neck region comprising residues between the tower and base. There are many sheets and helices in the base region, which are very important to maintaining the stability of the hexon trimer, but in the tower region the number of turns and random coils increases rapidly, especially in the region exposed on the surface of the protein, and these turns or coils might be where the neutralizing epitopes are located. The solvent accessibility surface analysis is commonly used to evaluate how deep a given residue is buried [31]. SAS of HEX3 model was calculated by performing Access Surf program. SAS analysis revealed two residue groups: the exposed group and buried group. The SAS data were be used in epitope screening in subsequent bioinformatics analysis. 3.2. Reverse evolutional trace analysis In a routine ET method [27,28], conserved regions among homologous sequences were located by MSA [22] and next mapped to a specific 3D model to investigate which amino acid residues are crucial to particular functions. However, this study utilized an especially designed RET method, focusing on variable (not conserved) regions that we called reverse evolutional trace (RET). MSA was performed with the Clustal algorithm and adjusted manually. All serotypes of species A, B, C, E, and F were included in the MSA. Some serotypes were excluded for species D because HAdVs of species D contains too many serotypes and parts of the sequence data are incomplete [14]. Then the MP tree was created using MEGA4.0 package. The evolutionary relationship of hexons (shown in Fig. 1) is in accordance with the previous studies [14,21]. A Bioperl program was written to calculate the sites homology taking the HEX3 sites as standard sites according to the MSA results. The sites homology calculation results are shown in Fig. 4. We can see that there are regions with conserved sequences and two hypervariable loops (loop 1: residues 115–310; loop 2: residues 400–510), which is consistent with some results in the literature. A color mapping scheme was employed to map three ranges of site-homology values (≤90%, ≤60%, and ≤30%) onto the 3D model of HEX3, as shown in Fig. 5. Fig. 5 indicates that the sites homology was lower closer to the tower region, the base of the hexon was highly conserved, while the tower was relatively variable. The conserved portion of the hexon is crucial for the replication and structural stability of an adenovirus. The variable portion of the hexon might contain mutations that would not affect the life cycle of the adenovirus. Such mutation lays the molecular foundation of the evolution history of the adenovirus, which results in a multitype virus family and a large type-specific antigen pool. So the tower can also be considered to be the region where the type-specific B-cell neutralizing epitopes are located. In contrast to methods used to identify HVRs based on MSA [14,21], our study adopted a new strategy based mainly on two features of

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Fig. 4. The sites homology of HEX3 amino-acid sites. Three scopes (≤90%, ≤60%, and ≤30%) of sites homology, loop 1 region (115–310) and loop 2 region (400–510) are marked.

the epitopes of the hexon protein. First, a B-cell epitope should be located on the surface of the antigen molecule [49], and the SAS of the hexon epitope sites should belong to the exposed group. Second, as a B-cell epitope, it commonly comprises 6–15 amino acids, and most importantly the neutralizing epitopes of the HAdV hexon protein are all type-specific, in that more than half of residues are hypervariable sites. We found that a homology of 45% was borderline for hypervariable sites, with this segment being type-specific when half the sites therein had a homology of lower than 45%, and that this quality was lost when the homology was higher than 50%. We therefore designed the epitope-screening algorithm described in Section 2 to determine where the epitopes are located. Type-specific analysis and SAS analysis are more important in the epitope-screening algorithm. The calculations were implemented in Bioperl script language. The screened five candidate epitope peptide segments (S1: 135–146, S2: 169–178, S3: 237–251, S4: 262–272, S5: 420–434) are displayed in Fig. 6, which indicates that these five epitopes are all located in the tower region: S1–S4 and S5 are located in loops 1 and 2 of the tower region, respectively, and they are all the surface loops that stretch to the external environment. 3.3. Peptides ELISAs Free polypeptides are difficult to coat properly in ELISAs, so all the synthesized polypeptides were coupled with BSA as a carrier protein. Two control polypeptides (P1 and P2: residues 149–160 and 469–482) were used to verify the correctness of epitopes screening, and finally identify the type-specific B-cell neutralizing epitopes. ELISAs were performed to test the antigenicity of the seven synthesized polypeptides (S1–S5, P1, and P2), a BSA as negative control, and a purified Hexon protein (coupled with BSA) of HAdV3 as positive control. The first antibody was the prepared HAdV3 antiserum with four dilution, and the secondary antibody was horseradishperoxidase-conjugated antirabbit IgG goat serum. Fig. 7 shows that synthesized peptides S1–S5 can bind to the anti-HAdV3 serum with all four dilution (from 0.35 OD value on 1:500 dilution to 0.2 OD value on 1:4000 dilution), the values were higher than that of the control peptides P1, P2 and negative control BSA with an unchanged OD value (about 0.1) at any dilution. But the binding was lower than the positive control (from 0.45 OD value on 1:500 dilution to 0.3 OD value on 1:4000 dilution).

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Fig. 5. Conserved sites of the HEX3 sequence mapping of the 3D model of HEX3, with the red region representing ≤90% (A), ≤60% (B), and ≤30% (C) of sites homology.

These results show that the candidate epitope polypeptides are well recognized by the type-specific neutralization anti-HAdV3 serum and that the entire epitopes of the hexon protein maybe complex and multiple epitopes might comprise the entire antigen of the hexon. As a comparison, the P1 control polypeptide exhibited a type-specific property but was located at a buried group, and the P2 control was extracted from the exposed group but conserved among HAdV serotypes, it was found that control polypeptides P1 and P2 hardly reacted with the serum, which are almost the same as the negative control. These results indicate that the S1–S5 polypeptides with exposed features and type-specific characteristics are indeed the type-specific neutralizing epitopes of HAdV3 hexon protein.

3.4. Neutralization Tests The serum-neutralizing antibody titer is the maximum serum dilution that can protect 50% of the cell culture from the CPE. NTs were performed with seven serially diluted antipeptides sera (anti-S1, anti-S2, anti-S3, anti-S4, anti-S5, anti-P1, and anti-P2), preimmune serum and anti-HAdV3 serum neutralizing HAdV3 cultured in HELA cells. After continuous observation for 48 h, all the wells of preimmune serum and anti-P1 and anti-P2 CPE were positive, with the cells becoming round to present typical grape-like lesion. The anti-S1, anti-S2, anti-S3, anti-S4, anti-S5, and anti-HAdV3 sera could protect HELA cells from the CPE at serum-

Fig. 6. Predicted epitopes in the three towers region of hexon protein of HAdV-3 for S1–S5 colored by black, violent, green, red and blue respectively (side view: A, top view: B) and their corresponding locations on the primary amino acid sequence of HEX3 hexon (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Table 2 Comparison of different anti-HAdV3 neutralizing titers for different antisera. Dilution

1:50 1:100 1:200 1:300 1:400 1:600 1:800 1:1600

Neutralization of different antisera with HAdV3 isolated strain (Harbin04B) Antisera

Anti-P1

Anti-P2

Anti-S1

Anti-S2

Anti-S3

Anti-S4

Anti-S5

Anti-HAdV3

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

− − − − + + + +

− − − − + + + +

− − − − − + + +

− − − − + + + +

− − − − + + + +

− − − − − − − +

Fig. 7. Optical densities (ODs) of peptides ELISAs using synthesized polypeptides (S1–S5, P1 and P2), BSA as the negative control and purified HAdV-3 hexon protein as the positive control with the prepared HAdV-3 antiserum (dilution: 1/500, 1/1000, 1/2000 and 1/4000). The OD value of S1–S5 are all higher than that of P1, P2 and BSA in any dilution but less than that of the positive control.

neutralizing antibody titers of 1:300, 1:300, 1:400, 1:300, 1:300, and 1:800, respectively, as indicated in Table 2. It was found that five antiepitopes (anti-S1, anti-S2, anti-S3, antiS4 and anti-S5,) sera can neutralize the infection caused by HAdV3, and protect HELA cells from the CPE, although the antibody titer is lower than that for anti-HAdV3 serum. The preimmune, anti-P1, and anti-P2 sera were unable to protect the HELA cells from infection at the titers tested. The NT results indicated the correctness of the mapping of the five epitopes. 4. Discussion B-cell epitopes on antigens were initially studied by investigating special structures using X-ray crystal diffraction [24]. This method was effective at predicting epitopes, but both X-ray crystal diffraction and nuclear magnetic resonance (NMR) methods require large and expensive equipment [26]. In the 1980s, Hopp and Woods reported that a hydrophilicity parameter could be used to predict B-cell epitopes [50]. Developments in bioinformation technologies for determining solvent accessibility, secondary structure and flexibility etc. [51–53] have also been used in recent years to predict B-cell epitopes on antigens. These methods are all based on the prediction from primary structure of proteins, making their accuracy and reliability questionable. Homology modeling [26,29] is the most powerful method for predicting the structure of unknown proteins, and represents a new direction for structure-based epitope-prediction technology. No previous study has identified the homotrimer of HAdV3 hexon by homology modeling and MM or MD simulation. In this study we developed a new approach for epitopes mapping of the HAdV hexon protein by combining molecular modeling [26] technology and bioinformatics ET [27,28] analysis based on

two important features of the epitopes of the HAdV hexon protein: (1) all epitopes are B-cell epitopes, and are located on the surface of the hexon homotrimer; and (2) the neutralizing epitopes of the HAdV hexon protein are all type-specific. Firstly, molecular modeling technology was used to construct the complete 3D model of the HAdV3 hexon homotrimer molecule based on the sequence of the hexon protein of HAdV3 isolated from clinical specimens in our laboratory, homology modeling was used to initially model HEX3; then EM and MD simulations were performed to refine the hexon homotrimer. The refined HEX3 model was used to calculate the SAS. Solvent accessibility is regarded the most important feature, and it is generally acknowledged that B-cell epitopes are located on the surface of the antigen protein [49]. Secondly, a special RET method was designed for predicting the HEX3 epitopes that differed from the routine ET method. By performing MSA we looked for the variable region rather than the conserved region to locate type-specific sites. Then the two B-cell epitope features were taken together, and an epitope-screening algorithm mentioned previously was implemented by Bioperl script language, then five candidate epitope peptides were screened out and mapped onto the 3D model of HEX3. It was found that the five candidate type-specific B-cell epitopes were not only type-specific among different serotypes but also located on the surface where was possible exposure to the external solvent environment, increasing the probability of the epitopes contacting the immune system. Finally, in ELISAs and NTs, the five candidate type-specific neutralizing epitope polypeptides and two control polypeptides on the HAdV hexon protein were synthesized and coupled with KLH (for immunization) and BSA (for ELISA, preventing the emergence of anti-KLH antibodies and interference between the experiments). The peptide ELISAs showed that the affinity for the anti-HAdV3 serum was higher for the five predicted epitope peptides than for the BSA and P1 and P2 controls. Moreover, the NTs showed that the antiepitopes (anti-S1, anti-S2, anti-S3, anti-S4 and anti-S5) sera can neutralize the infection caused by HAdV3 and protect HELA cells from the CPE. The two serological experiments identified the correctness of predicting epitopes from molecular modeling and bioinformatics analysis. The type-specific neutralizing epitopes of hexon protein of HAdV3 were then mapped accurately. We have not only predicted and identified the precise location and amino acid sequences type-specific B-cell neutralizing epitopes of HAdV3 hexon, but also developed a new effective, reliable approach for epitopes mapping of HAdVs hexon and at the same time we also have obtained the conformation of five type-specific B-cell neutralizing epitopes of HAdV3 hexon, in next step which can be used for MD simulation and molecular docking study with the corresponding monoclonal antibody molecules. Epitopes mapping of HAdVs is very important to the molecular design of a HAdVs vaccine, developing rapid HAdVs diagnostic agents, preparing anti-HAdV drugs, and studying the mechanism of immunity deduced by hexon. However, further study is needed to better understand the relationship of the antigenicity of hexon and its epitopes conformation.

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Acknowledgments [27]

We thank Zhiwei Yang, Cheng Xing of Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, for helping with the homology modeling and the MM and MD simulations. We also thank Weijun Lu and Changqing Ying of our laboratory for help with ELISAs and NTs experiments.

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