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Page 1: Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein

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Vaccine 27 (2009) 5103–5110

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olecular modeling and epitopes mapping of human adenovirusype 3 hexon protein�

iaohui Yuana, Zhangyi Qua,∗, Xiaomin Wub, Yingchen Wanga, Lei Liua, Fengxiang Weia,ong Gaoa, Lei Shanga, Hongyan Zhanga, Hongbo Cuic, Yuehui Zhaoc, Na Wua,anhong Tanga, Le Qina

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

r t i c l e i n f o

rticle history:eceived 21 December 2008eceived in revised form 26 April 2009ccepted 10 June 2009vailable online 30 June 2009

eywords:

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. Athree-dimensional (3D) model of the HAdV3 hexon was obtained by homology modeling and refined bymolecular mechanics and molecular dynamics simulations. A modified evolutionary trace (ET) analysiscalled reverse ET (RET) was used to predict the type-specific B-cell neutralizing epitopes. An epitope-screening algorithm based on analyzing the solvent accessibility surface (SAS) area from the 3D model and

AdVexon proteinpitope Mapping

calculation of sites homology using RET was designed and implemented in the BioPerl script language. Fiveepitope polypeptide segments were predicted and mapped onto the 3D model. Finally five polypeptideswere 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 HAdV3are located at the top surface of hexon tower regions (residue numbers: 135–146, 169–178, 237–251,

work

262–272, 420–434). Thisvaccine.

. Introduction

Adenoviridae viruses are nonenveloped, double-stranded DNAiruses with an icosahedral capsid comprising 240 hexons and 12ertex capsomeres [1,2]. The human adenovirus (HAdV) can be clas-ified into 6 species (A–F) on the basis of hemagglutination andenomic properties [3], consisting of 51 serotypes defined mainlyy neutralization criteria [4,5]. HAdVs can cause a broad spectrumf human infective diseases [6–9], among which upper respiratoryract infection and pedo-pneumonia caused by serotypes 3 and 7

re particularly serious [5,10,11]. Especially in northern China, theajor epidemic strains are HAdV3 and HAdV7 [12,13]. There is as

et no effective curative antiviral medicine or vaccine with whicho treat these diseases.

� Supported by The National Natural Science Foundation of China (No.0771909), The Doctoral Co-financing Project of Chinese Ministry of Education (No.0070226007), The Natural Science Foundation Key Project of Heilongjiang ChinaNo. ZJY0701), Science and Technology Project of Heilongjiang Provincial Educationepartment (No. 11521172).∗ Corresponding author. Tel.: +86 451 87502965; fax: +86 451 86667248.

E-mail addresses: [email protected], [email protected] (Z. Qu).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.06.041

is of great significance to the molecular design of a multivalent HAdVs

© 2009 Elsevier Ltd. All rights reserved.

The major coat protein of HAdV is hexon (i.e., a homotrimer pro-tein comprising three monomers A, B and C) [14]. It has been shownthat antibodies stimulated by a hexon can neutralize an HAdV-mediated viral infection, and that this neutralization reaction istype-specific [15]. The tower region of this hexon homotrimer con-tains a large number of type-specific neutralizing epitopes (B-cellepitopes). Identifying these type-specific neutralizing epitopes isof great significance in several areas of HAdV research, includingthe molecular design of a HAdV vaccine [16–18], the developmentof a rapid HAdV diagnostic agent and preparing an antiadenovirusmedicine [19,20]. But very little is currently known about the map-ping of type-specific B-cell neutralizing epitopes of hexons in manyserotypes of HAdV. Although it is possible to locate hypervari-able regions (HVRs) [14,21] using the multiple sequence alignment(MSA) method [22], it is difficult to identify the type-specific neu-tralizing epitopes and to obtain the specific three-dimensional (3D)conformation of the epitope peptides for a specific serotype. The 3Dconformation of hexon protein can provide relevant information

about epitopes. The hexon structures of HAdV type 2 (HAdV2) andHAdV type 5 (HAdV5) [23,24] are available in the Protein Data Bank(PDB) [25], but these structures have inherent amino acid deletionsand disruption of the peptide chains. In addition, related docu-ments show that these structures are only the conserved core region
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f the HAdV hexon protein [15], and do not provide informationf the complete tower structure, which contains the type-specificeutralizing epitopes.

This study investigated two characteristics of epitopes on HAdVsexons – namely B-cell neutralizing epitopes and type-specificpitopes – using a combination of molecular simulation tech-ology [26] and bioinformatics evolutionary trace (ET) [27,28]nalysis. The 3D structure of the HAdV type 3 (HAdV3) hexonas determined using molecular simulation/homology modeling

26,29,30], and the solvent-accessible surface area (SAS) [31] ofhe model was calculated. In addition, a modified ET methodhat we named reverse ET (RET) was employed. This involved these of MSA, sites homology calculation, and a purpose-designedpitope-screening algorithm that combines the results from theAS analysis and sites homology calculation. The presence of fiveandidate epitope segments was predicted and mapped onto theD model of the hexon. Finally, the predicted epitope peptidesere synthesized. Two serological experiments were performed

o prove the correctness of epitopes prediction: (1) enzyme-linkedmmunosorbent assay (ELISA) was used to detect the affinity of epi-ope peptides and anti-HAdV3 serum; (2) Neutralization Test (NT)as used to test the neutralizing effect to HAdV3 of antipeptides

era.

. Materials and methods

.1. HAdV3 and anti-HAdV3 serum

The HAdV used in this study was an isolated strain obtainedrom clinical throat-swab specimens. In 2003 and 2004, manyhildren in the Harbin area of China contracted fever [13], and aotal of 384 throat swabs were taken from them in the Depart-

ent of Pediatrics, No. 1 Subsidiary Hospital of Harbin Medicalniversity. A strain of HAdV (namely Harbin04B) was success-

ully isolated in our laboratory, and cell culture, immunology, andorphological, PCR, and sequencing analyses of the hexon gene

dentified the virus as HAdV3, the nucleotide sequence of which haseen deposited in the NCBI GenBank database (accession number:U078562).

Anti-HAdV3 serum was obtained from a 6-week-old female Newealand rabbit that had been injected intramuscularly with 1013

AdV3 particles and then boosted subcutaneously with 1013 viralarticles emulsified in complete Freund’s adjuvant (Sigma). Bloodas collected from the ear fringe vein plexus, and serum was pre-ared for an ELISA, with preimmune serum used as a negativeontrol. The antibody titer and the HAdV3 specificity were detectedy ELISA as described previously [32].

.2. Homology modeling

Homology modeling, energy minimization (EM) and molecu-ar dynamics (MD) simulations were performed using a molecularimulation software package InsightII 2005 (Accelrys Inc., Saniego, USA). The consistent-valence force field (CVFF) [33–35] wasmployed for EM and MD simulations. The HAdV3 hexon aminocid sequence was deduced from the corresponding nucleotideequence derived from Harbin04B and was named by HEX3.

.2.1. Molecular modeling and structure refinementThe InsightII/Homology module was applied to build the 3D

tructure of the HAdV3 hexon. The web-FASTA tool [36] of the

DB (http://www.rcsb.org) was used to search for an appropri-te template for the homology modeling using HEX3 as a probe.he chimpanzee adenovirus 68 (AdC68) [15] hexon (PDB code:obe) at 2.1 Å resolution exhibited the highest degree of homol-gy at 85.6% higher than HAdV2 hexon (PDB code: 1p2z) [23], and

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HAdV5 hexon (PDB code: 1p30) [24], and was thus considered tobe the most appropriate template. MSA based on the Needlemanand 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 theinitial model of HEX3.

To refine the structure, the EM and MD simulations were exe-cuted in the InsightII/Discover 3 module. The entire process wasdivided into the following steps: first, all the hydrogen atomsand side chains were optimized in a vacuum by a 500-stepsteepest-descent (SD) minimization followed by conjugate gra-dient (CG) minimization until the final convergence was lowerthan 0.01 kcal mol−1 Å−1. Second, the loop regions were optimizedby fixing all atoms except for those in the tower region, andthen performing 500 steps of SD and CG until the final con-vergence was lower than 0.01 kcal mol−1 Å−1. Because EM cannotsolve the energy-barriers problem [38], a MD simulation wasperformed (1000 ps at 310 K) to achieve the stable conformationfor the tower region (residues: 115–310 and 400–510). Third, CGminimization of the full protein was performed until the final con-vergence was lower than 0.01 kcal mol−1 Å−1. This step improvedthe quality of the initial model of the HEX3 homotrimer. Theabove procedure produced the 3D model of the HAdV3 hexonhomotrimer.

The structure was further checked using the InsightII/Profiles 3D and InsightII/ProStat programs. The Profiles 3D pro-gram was used to examine the compatibility of an amino acidsequence with a known 3D protein structure [39]. The ProStat pro-gram investigated the secondary structural of the 3D model basedon 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 homotrimermodel was calculated with the InsightII/Access Surf program. Aprobe radius of 1.4 Å was used for all calculations. The differencein the SAS of each residue in the HEX3 model was determined. Tworesidue groups were created: (1) an exposed group, whose residueswere greater than 25% of the maximum SAS; and (2) a buried group,whose residues were less than 10% of the maximum SAS. The SASdata were used in the subsequent epitopes screening.

2.3. Reverse evolutionary trace analysis (RET)

ET analysis can extract the information obtained from the MSAof homologous proteins onto a certain 3D molecule and therebyinvestigate which amino acid residues are likely to be crucialfor certain functions [27,28]. Because the neutralizing epitopes ofHAdV3 Hexon are all type-specific, we utilized a special modifiedET method to map the candidate type-specific neutralizing epitopesderived from a epitope-screening algorithm onto the 3D model ofHEX3, which we called reverse evolutionary trace (RET) analysisincluding MSA, sites homology calculation and a designed candi-date 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 includ-ing all serotypes of species A, B, C, F and a few typical serotypesof species D. The sequence data were all derived from NCBI Gen-Bank 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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Fig. 1. Phylogenetic relationships of deduced amino acid sequences of HAdV hexonprA

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Table 1Synthesized polypeptides and the location of S1–S5 and two control polypeptidesP1 and P2.

Segment Locationa Sequence

S1 135–146 Cb IVTAGEERAVTTS2 169–178 C GKDITADNKS3 237–251 C NRKVKPTTEGGVETES4 262–272 C GREAADAFSPES5 420–434 C SKDNGWEKDDNVSKSP1 149–160 C NTFGIASMKGDNP2 469–482 C VYKYTPTNITLPAN

roteins. The evolutionary history was inferred using the MP method. Unrooted treeeflected the distance relationship of all HAdV hexon amino acid sequences in it.ccession number of reference sequences is shown in the tree.

B052911 (HAdV34), AB052912 (HAdV35), DQ149632 (HAdV37),51782 (HAdV40), DQ315364 (HAdV41), EF153473 (HAdV48),Q149643 (HAdV50). Besides the HEX3, MSA was performedsing the ClustalW1.83 program [41] with the Clustal algorithmnd adjusted manually to conform the optimized alignment ofeduced amino acid sequences. In order to show the evolutionaryelationship of these sequences, a phylogenetic tree was con-tructed by the maximum-parsimony (MP) method [42] usinghe MEGA4.0 package [43]. The phylogenetic tree is shown inig. 1.

.3.2. Sites homology calculationSites homology refers to the similarity of the same site in dif-

erent sequences aligned by MSA. It reflects how a certain sites conserved in aligned homologous protein sequences. Takinghe sites of HEX3 as standard sites, the sites homology in dif-erent hexon amino acid sequences were calculated according tohe aligned result. The calculation method was as follows: siteomology = number of conserved amino acids on same site/numberf total sequences × 100%. Then a color mapping scheme wasmployed in InsightII environment to map the information fromhe sites homology calculation onto the 3D model of HEX3, we sethree ranges of site homology values (≤90%, ≤60%, and ≤30%) toeflect the trend of sites homology.

.3.3. Candidate epitopes screeningBecause the epitopes on the hexon protein are type-specific B-

ell neutralizing epitopes [4,5], we designed an epitope-screeninglgorithm to screen the candidate epitopes: site with a homology ofess than 45% were defined as hypervariable site. Segments fulfillinghe following standards were selected as candidates: (1) length ofetween 6 and 15, (2) more than half of them being hypervariableites, (3) interval between candidate sequences not shorter thanhree, and (4) 90% of residues belonging to the exposed group withesidues greater than 25% of the maximum SAS. Finally, five candi-ate epitope segments were screened out and mapped onto the 3D

EX3 model using a color mapping scheme in the InsightII envi-

onment. All above of the sites homology calculation and candidatepitopes screening were implemented in programs written by theioperl script language [44].

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

tides.

2.4. Epitope peptides synthesis and peptides ELISAs

2.4.1. Synthetic epitope peptidesThe 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 PeptideSynthesizer (Tianjin Saier Biotechnology Co., Ltd. TianJin, China).In the meanwhile two control polypeptides (P1: 149–160 andP2: 469–482) were synthesized by the same method: P1 had atype-specific property but was located in the buried group in SASanalysis, while P2 was selected from the exposed group in the SASanalysis but it was conserved among the HAdV serotypes. High-performance liquid chromatography was used to analyze the purityand correctness of the synthetic polypeptides [46]. The Cys on theN-terminal of synthetic polypeptides was added for conjugation.Each polypeptide (purity ≥85%) was then chemically linked to thecarrier protein bovine serum albumin (BSA, Sigma) and keyholelimpet hemocyanin (KLH, Sigma) by the glutaraldehyde (GA) treat-ment method [47]. The sequences of synthesized polypeptides andtheir locations are listed in Table 1.

2.4.2. Peptide ELISAsThe ELISAs were performed using streptavidin to coat the plates

and bind biotin with casein blocker [48], with the previously pre-pared HAdV3 antiserum serially diluted in PBS with dilution: 1/500,1/1000, 1/2000 and 1/4000. The ELISA plates were also coated with100 �l of PBS (pH 7.4) containing the seven synthesized polypep-tides (S1–S5, P1 and P2) coupled with BSA, BSA protein only as anegative control, and purified hexon protein (purity≥85%) preparedin our lab [32] coupled with BSA by the GA methods of HAdV3 asa positive control. After incubation for 16 h at 4 ◦C, the wells wereblocked 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 samplewas added to the wells and incubated for 1 h at 37 ◦C. The secondaryantibody was horseradish-peroxidase-conjugated antirabbit IgGgoat serum (Shanghai Sangon Biological Engineering Technology& Services Co., Ltd. Shanghai, China), followed by washes and treat-ment with H2O2 and o-phenylenediamine (Sigma). The opticaldensity (OD) was measured at 490 nm after incubation for 30 minat 22 ◦C.

2.5. Antipeptides sera and Neutralization Tests

2.5.1. Preparation of antipeptides seraFourteen 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 sevensynthesized polypeptides (epitope peptides: S1–S5; and two con-trol peptides: P1 and P2) coupled with KLH were used for animalimmunization (two rabbits for each coupled peptide), with preim-mune serum used as a negative control.
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The immunization schedule was as follows: 14 rabbits were sub-utaneously immunized with a 600-�g dose of coupled peptides1 ml) emulsified in 1 ml of complete Freund’s adjuvant (Sigma). Allabbits were boosted with the same method at 14 and 28 days afterhe primary immunization with a 400-�g dose of coupled peptides1 ml) emulsified in 1 ml of incomplete Freund’s adjuvant (Sigma).lood samples were taken from the ear fringe vein plexus at 0 dpipreimmune), 21 dpi, and 35 dpi, stored overnight at 4 ◦C, and cen-rifuged at 1200 g relative centrifugal force to obtain clarified sera.ntibody titers were detected by ELISA using peptides coupled withSA. The titers of the 21- and 35-dpi sera were 1:8000 and 1:10,000,espectively. The sera were then prepared for Neutralization TestsNTs).

.5.2. Neutralization Tests (NTs)NTs were performed to test the neutralizing effect of seven

ntipeptides sera to HAdV3. All of the antipeptides sera (anti-S1,nti-S2, anti-S3, anti-S4, anti-S5, anti-P1, and anti-P2), preimmuneerum as a negative control, and anti-HAdV3 serum as a positiveontrol were serially diluted in PBS, and 25-�l aliquots of eachilution were mixed with 25 �l of HAdV3 with 100 TCID50. Thentibody–virus mixtures were incubated for 1 h at 37 ◦C in the pres-nce of 5% CO2 and then transferred to 96-well plates containingearly confluent (85–95%) monolayers of HELA cells. Monolayersere incubated for 48 h in the presence of 5% CO2, after which infec-

ion was monitored using fluorescence microscopy by identifyingositive sera that inhibited 50% of the cytopathic effect (CPE).

. Results

.1. Homology modeling of HEX3

The HAdV3 hexon monomer encoded by hexon gene contains

37 amino acids, and results obtained with the web-FASTA tool havehown that the AdC68 hexon in the PDB (code: 2obe) has the high-st sequence homology, at 85.6%. However, this high homology isot balanceable, since in the SCR it is more than 95%, whereas in theower region (residues: 115–310, 400–510) it is only about 66%, and

ig. 2. The final 3D structure of the HEX3 homotrimer (A), and its monomer A ribbonimulation. The random coil, sheet, helix and turn are represented by green, yellow, red anegend, the reader is referred to the web version of the article.)

Fig. 3. Profile 3D verification result of the HEX3 model, with residues exhibitingreasonable folding. A score >0 indicates residues are compatible, and a score <0indicates 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 automatedhomology modeling program Modeler, then MM optimization andMD simulation were performed to refined the model. The final sta-ble structure of HEX3 and one of its monomer A are displayed inFig. 2.

The overall quality of the final structure was further evaluatedusing the Profiles 3D program, which is normally used to quan-tify the compatibility of an amino acid sequence with a 3D proteinstructure and especially to check the validity of a hypothetical pro-tein structure. The result is shown in Fig. 3. Note that compatibilityscores above zero are considered acceptable, and regions of the pro-tein for which the score approaches zero or becomes negative arelikely to be misfolded if a surface patch of a protein shows a low

score or becomes negative, this might indicate that the surface isinteracting with other proteins and should be buried internally.From Fig. 3, we can see that the negative scores were obtainedin the regions of residues 186–191, 228–235 and 345–355 of eachmonomer. The closest approximation was to template 2obe, which

structure (B). The structure obtained by a series of energy minimization and MDd blue color respectively. (For interpretation of the references to color in this figure

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hows that they are regions where the three hexon monomers inter-ct with each other. The Profiles 3D verification scores were alligher in the tower region than in the base region, indicating thathe conformation of the tower was acceptable.

It was believed that the understanding of secondary structurallements is a foundation to investigate the large and complex struc-ures. Four types of secondary structures were observed in the HEX3

odel by ProStat program, which are denoted as random coil, sheet,elix and turn, respectively. As shown from Fig. 2, we can see thathe HAdV3 hexon homotrimer protein is a stable structure com-rising three interlinked hexon monomers which is consistent withome results in the literature. This trimer could be further dividednto three major parts: (1) the tower region (residues 115–310 and00–510) that forms the outer layer of homotrimer, and comprisinghree actual towers; (2) the base region that forms the internal layerf the capsomer, which the MSA later indicates is highly conservedmong all serotypes; and (3) the neck region comprising residuesetween the tower and base. There are many sheets and helices

n the base region, which are very important to maintaining thetability of the hexon trimer, but in the tower region the numberf turns and random coils increases rapidly, especially in the regionxposed on the surface of the protein, and these turns or coils mighte where the neutralizing epitopes are located.

The solvent accessibility surface analysis is commonly used tovaluate how deep a given residue is buried [31]. SAS of HEX3 modelas calculated by performing Access Surf program. SAS analysis

evealed two residue groups: the exposed group and buried group.he SAS data were be used in epitope screening in subsequentioinformatics analysis.

.2. Reverse evolutional trace analysis

In a routine ET method [27,28], conserved regions among homol-gous sequences were located by MSA [22] and next mapped tospecific 3D model to investigate which amino acid residues are

rucial to particular functions. However, this study utilized an espe-ially designed RET method, focusing on variable (not conserved)egions that we called reverse evolutional trace (RET). MSA waserformed with the Clustal algorithm and adjusted manually. Allerotypes of species A, B, C, E, and F were included in the MSA. Someerotypes were excluded for species D because HAdVs of species

contains too many serotypes and parts of the sequence data arencomplete [14]. Then the MP tree was created using MEGA4.0 pack-ge. The evolutionary relationship of hexons (shown in Fig. 1) is inccordance with the previous studies [14,21]. A Bioperl programas written to calculate the sites homology taking the HEX3 sites

s standard sites according to the MSA results. The sites homol-gy calculation results are shown in Fig. 4. We can see that therere regions with conserved sequences and two hypervariable loopsloop 1: residues 115–310; loop 2: residues 400–510), which isonsistent with some results in the literature. A color mappingcheme was employed to map three ranges of site-homology values≤90%, ≤60%, and ≤30%) onto the 3D model of HEX3, as shown inig. 5.

Fig. 5 indicates that the sites homology was lower closer to theower region, the base of the hexon was highly conserved, while theower was relatively variable. The conserved portion of the hexon isrucial for the replication and structural stability of an adenovirus.he variable portion of the hexon might contain mutations thatould not affect the life cycle of the adenovirus. Such mutation lays

he molecular foundation of the evolution history of the adenovirus,

hich results in a multitype virus family and a large type-specific

ntigen pool. So the tower can also be considered to be the regionhere the type-specific B-cell neutralizing epitopes are located. In

ontrast to methods used to identify HVRs based on MSA [14,21],ur study adopted a new strategy based mainly on two features of

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) aremarked.

the epitopes of the hexon protein. First, a B-cell epitope should belocated on the surface of the antigen molecule [49], and the SAS ofthe hexon epitope sites should belong to the exposed group. Sec-ond, as a B-cell epitope, it commonly comprises 6–15 amino acids,and most importantly the neutralizing epitopes of the HAdV hexonprotein are all type-specific, in that more than half of residues arehypervariable sites. We found that a homology of 45% was border-line for hypervariable sites, with this segment being type-specificwhen half the sites therein had a homology of lower than 45%, andthat this quality was lost when the homology was higher than 50%.

We therefore designed the epitope-screening algorithmdescribed in Section 2 to determine where the epitopes arelocated. Type-specific analysis and SAS analysis are more impor-tant in the epitope-screening algorithm. The calculations wereimplemented 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) aredisplayed in Fig. 6, which indicates that these five epitopes are alllocated in the tower region: S1–S4 and S5 are located in loops 1and 2 of the tower region, respectively, and they are all the surfaceloops that stretch to the external environment.

3.3. Peptides ELISAs

Free polypeptides are difficult to coat properly in ELISAs, so allthe synthesized polypeptides were coupled with BSA as a carrierprotein. Two control polypeptides (P1 and P2: residues 149–160 and469–482) 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 synthe-sized polypeptides (S1–S5, P1, and P2), a BSA as negative control,and a purified Hexon protein (coupled with BSA) of HAdV3 as posi-tive control. The first antibody was the prepared HAdV3 antiserumwith four dilution, and the secondary antibody was horseradish-peroxidase-conjugated antirabbit IgG goat serum.

Fig. 7 shows that synthesized peptides S1–S5 can bind to theanti-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 werehigher than that of the control peptides P1, P2 and negative controlBSA with an unchanged OD value (about 0.1) at any dilution. But thebinding was lower than the positive control (from 0.45 OD value on1:500 dilution to 0.3 OD value on 1:4000 dilution).
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These results show that the candidate epitope polypeptides areell recognized by the type-specific neutralization anti-HAdV3

erum and that the entire epitopes of the hexon protein maybe com-lex and multiple epitopes might comprise the entire antigen ofhe hexon. As a comparison, the P1 control polypeptide exhibited aype-specific property but was located at a buried group, and the P2ontrol was extracted from the exposed group but conserved among

AdV serotypes, it was found that control polypeptides P1 and P2ardly reacted with the serum, which are almost the same as theegative control. These results indicate that the S1–S5 polypeptidesith exposed features and type-specific characteristics are indeed

he type-specific neutralizing epitopes of HAdV3 hexon protein.

ig. 6. Predicted epitopes in the three towers region of hexon protein of HAdV-3 for S1–S) and their corresponding locations on the primary amino acid sequence of HEX3 hexoneferred to the web version of the article.)

red region representing ≤90% (A), ≤60% (B), and ≤30% (C) of sites homology.

3.4. Neutralization Tests

The serum-neutralizing antibody titer is the maximum serumdilution 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 CPEwere positive, with the cells becoming round to present typicalgrape-like lesion. The anti-S1, anti-S2, anti-S3, anti-S4, anti-S5, andanti-HAdV3 sera could protect HELA cells from the CPE at serum-

5 colored by black, violent, green, red and blue respectively (side view: A, top view:(C). (For interpretation of the references to color in this figure legend, the reader is

Page 7: Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein

X. Yuan et al. / Vaccine 27 (2009) 5103–5110 5109

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

Dilution 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

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

Fig. 7. Optical densities (ODs) of peptides ELISAs using synthesized polypeptides(a1B

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S1–S5, P1 and P2), BSA as the negative control and purified HAdV-3 hexon proteins the positive control with the prepared HAdV-3 antiserum (dilution: 1/500, 1/1000,/2000 and 1/4000). The OD value of S1–S5 are all higher than that of P1, P2 andSA in any dilution but less than that of the positive control.

eutralizing antibody titers of 1:300, 1:300, 1:400, 1:300, 1:300,nd 1:800, respectively, as indicated in Table 2.

It was found that five antiepitopes (anti-S1, anti-S2, anti-S3, anti-4 and anti-S5,) sera can neutralize the infection caused by HAdV3,nd protect HELA cells from the CPE, although the antibody titer isower than that for anti-HAdV3 serum. The preimmune, anti-P1, andnti-P2 sera were unable to protect the HELA cells from infectiont the titers tested. The NT results indicated the correctness of theapping of the five epitopes.

. Discussion

B-cell epitopes on antigens were initially studied by investi-ating special structures using X-ray crystal diffraction [24]. Thisethod was effective at predicting epitopes, but both X-ray crys-

al diffraction and nuclear magnetic resonance (NMR) methodsequire large and expensive equipment [26]. In the 1980s, Hoppnd Woods reported that a hydrophilicity parameter could be usedo predict B-cell epitopes [50]. Developments in bioinformationechnologies for determining solvent accessibility, secondary struc-ure and flexibility etc. [51–53] have also been used in recentears to predict B-cell epitopes on antigens. These methods are allased on the prediction from primary structure of proteins, mak-

ng their accuracy and reliability questionable. Homology modeling26,29] is the most powerful method for predicting the struc-ure of unknown proteins, and represents a new direction fortructure-based epitope-prediction technology. No previous study

as identified the homotrimer of HAdV3 hexon by homology mod-ling and MM or MD simulation.

In this study we developed a new approach for epitopes map-ing of the HAdV hexon protein by combining molecular modeling26] 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 surfaceof the hexon homotrimer; and (2) the neutralizing epitopes of theHAdV hexon protein are all type-specific. Firstly, molecular mod-eling technology was used to construct the complete 3D model ofthe HAdV3 hexon homotrimer molecule based on the sequence ofthe hexon protein of HAdV3 isolated from clinical specimens in ourlaboratory, homology modeling was used to initially model HEX3;then EM and MD simulations were performed to refine the hexonhomotrimer. The refined HEX3 model was used to calculate the SAS.Solvent accessibility is regarded the most important feature, and itis generally acknowledged that B-cell epitopes are located on thesurface of the antigen protein [49]. Secondly, a special RET methodwas designed for predicting the HEX3 epitopes that differed fromthe routine ET method. By performing MSA we looked for the vari-able region rather than the conserved region to locate type-specificsites. Then the two B-cell epitope features were taken together, andan epitope-screening algorithm mentioned previously was imple-mented by Bioperl script language, then five candidate epitopepeptides were screened out and mapped onto the 3D model ofHEX3. It was found that the five candidate type-specific B-cell epi-topes were not only type-specific among different serotypes butalso located on the surface where was possible exposure to theexternal solvent environment, increasing the probability of the epi-topes contacting the immune system.

Finally, in ELISAs and NTs, the five candidate type-specific neu-tralizing epitope polypeptides and two control polypeptides on theHAdV hexon protein were synthesized and coupled with KLH (forimmunization) and BSA (for ELISA, preventing the emergence ofanti-KLH antibodies and interference between the experiments).The peptide ELISAs showed that the affinity for the anti-HAdV3serum was higher for the five predicted epitope peptides than forthe BSA and P1 and P2 controls. Moreover, the NTs showed thatthe antiepitopes (anti-S1, anti-S2, anti-S3, anti-S4 and anti-S5) seracan neutralize the infection caused by HAdV3 and protect HELAcells from the CPE. The two serological experiments identified thecorrectness of predicting epitopes from molecular modeling andbioinformatics analysis. The type-specific neutralizing epitopes ofhexon protein of HAdV3 were then mapped accurately.

We have not only predicted and identified the precise locationand amino acid sequences type-specific B-cell neutralizing epi-topes of HAdV3 hexon, but also developed a new effective, reliableapproach for epitopes mapping of HAdVs hexon and at the sametime we also have obtained the conformation of five type-specificB-cell neutralizing epitopes of HAdV3 hexon, in next step which canbe used for MD simulation and molecular docking study with thecorresponding monoclonal antibody molecules.

Epitopes mapping of HAdVs is very important to the molecu-

lar design of a HAdVs vaccine, developing rapid HAdVs diagnosticagents, preparing anti-HAdV drugs, and studying the mechanism ofimmunity deduced by hexon. However, further study is needed tobetter understand the relationship of the antigenicity of hexon andits epitopes conformation.
Page 8: Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein

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110 X. Yuan et al. / Vacc

cknowledgments

We thank Zhiwei Yang, Cheng Xing of Key Laboratory of Forestlant Ecology, Ministry of Education, Northeast Forestry Univer-ity, for helping with the homology modeling and the MM and MDimulations. We also thank Weijun Lu and Changqing Ying of ouraboratory for help with ELISAs and NTs experiments.

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