Virus Research 208 (2015) 66–72

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Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3 Xingui Tian a,1 , Minglong Liu a,1 , Xiaobo Su b , Zaixue Jiang a , Qiang Ma a , Xiaohong Liao a , Xiao Li a , Zhichao Zhou a , Chenyang Li a,∗ , Rong Zhou a,∗ a State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, The Affiliated First Hospital of Guangzhou Medical University, Guangzhou 510230, China b Department of Medical Genetics and Cell Biology, School of Basic Science, Guangzhou Medical University, Guangzhou 511436, China

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 30 May 2015 Accepted 1 June 2015 Available online 9 June 2015 Keywords: Human adenovirus type 3 Neutralizing Monoclonal antibody Epitope Hypervariable region 4

a b s t r a c t Human adenovirus type 3 (HAdV-3) has produced a global epidemic in recent years causing serious diseases such as pneumonia in both pediatric and adult patients. Development of an effective neutralizing monoclonal antibody (MAb) and identification of its neutralizing epitope is important for the control of HAdV-3 infection. In this study, three neutralizing MAbs were generated, of which MAb 3D7 had a high neutralization titer of 4096 (approximately 0.5 ␮g/ml) against HAdV-3 infection. In indirect enzymelinked immunosorbent assays, all three MAbs specifically recognized HAdV-3 virus particles and hexon protein, but did not react with the virus particles or the hexon protein of HAdV-7. Analyses using a series of peptides and chimeric adenovirus particles of epitope mutants revealed that all three MAbs bound to the same exposed region (amino acid positions 244–254 of hexon) in hypervariable region 4 (HVR4), which is highly conserved among global HAdV-3 strains. The amino acids T246 and G250 may be the critical amino acids recognized by these MAbs. MAb 3D7 reduced the recombinant enhanced green fluorescent protein-expressing HAdV-3 (rAd3EGFP) load recovered in the lungs of mice at 3 days post-infection. The generation of MAb 3D7 and the identification of its neutralizing epitope may be useful for therapeutic treatment development, subunit vaccine construction, and virion structural analysis for HAdV-3. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Human adenoviruses (HAdV) can cause a broad spectrum of diseases in both pediatric and adult patients, such as acute respiratory infection, acute gastroenteritis, and epidemic keratoconjunctivitis (Lenaerts et al., 2008; Sandkovsky et al., 2014). To date, seven species including more than 68 genotypes have been characterized and defined by genomics and bioinformatics (Dehghan et al., 2012; Robinson et al., 2013). Specific species genotypes are often associated with particular clinical manifestations. HAdV species C

∗ Corresponding author at: State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, 151 Yan Jiang Road, Guangzhou 510120, China. Tel.: +86 20 34281614; fax: +86 20 34281614. E-mail addresses: [email protected] (X. Tian), [email protected] (M. Liu), [email protected] (X. Su), [email protected] (Z. Jiang), [email protected] (Q. Ma), [email protected] (X. Liao), [email protected] (X. Li), [email protected] (Z. Zhou), [email protected] (C. Li), [email protected], [email protected] (R. Zhou). 1 Xingui Tian and Minglong Liu contributed equally to this work. http://dx.doi.org/10.1016/j.virusres.2015.06.002 0168-1702/© 2015 Elsevier B.V. All rights reserved.

(HAdV-1, -2, -5, and -6), species B (HAdV-3, -7, -14, and -55) and species E (HAdV-4) are most commonly found in patients with respiratory infection. Among these, HAdV-3 strains of subspecies B1 are the major epidemic strains responsible for severe respiratory disease epidemics and outbreaks worldwide (Yun et al., 2014; Lu et al., 2014; Barrero et al., 2012; Alkhalaf et al., 2015; Ampuero et al., 2012; Guo et al., 2012; Deng et al., 2013; Lai et al., 2013; Lee et al., 2015; Zhang et al., 2006). Currently, there is no effective treatment or vaccine against HAdV-3 infection. Neutralizing monoclonal antibodies (MAb) may be a promising prophylactic or therapeutic medicine against viral disease. The creation of neutralizing MAb could also be useful for identifying neutralizing epitopes, which is of great importance in the molecular design of vaccines. The adenovirus capsid icosahedron is composed of three major structural proteins: hexon, penton base, and fiber. The hexon protein is the major antigenic determinant recognized by neutralizing antibodies (NAbs) (Tian et al., 2011; Yu et al., 2013; Wu et al., 2002). Type-specific epitopes on hexons have been proposed to reside within seven highly variable regions (HVRs), of which HVR7 can be further subdivided into three

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additional highly variable regions (Rux et al., 2003; Bradley et al., 2012; Yuan et al., 2009). Our previous study demonstrated that HVR1, 2, 5, and 7 of HAdV-3 contained neutralizing epitopes (Qiu et al., 2012). In this study, we report three neutralizing MAbs against HAdV3, including MAb 3D7, which has a strong neutralizing capacity. Epitope mapping revealed that all three MAbs recognize the HVR4 of HAdV-3 hexon. The findings of this study will be useful for the development of prophylactic or therapeutic treatments against HAdV-3 infection and for adenovirus structural analysis.

transfected cells were cultured at 37 ◦ C with 5% CO2 for 6–10 days and were examined daily for evidence of cytopathic effect. The cells were frozen and thawed for three cycles and fresh cultures of HEp-2 cells were infected with viral suspension. At 96 h postinfection, the viruses were harvested and designated rAd3A7R4 and rAd3H7A3R4. Finally, the mutant viruses were cultured with AD-293 cells in a total of twenty 100-mm dishes, then harvested and purified by standard CsCl gradient centrifugation as described above. The full-length modified hexon genes of the viruses were identified by sequencing.

2. Materials and methods

2.3. Virus neutralization test

2.1. Virus strains and cells

Purified rAdMHE3 virions were used to immunize BALB/c mice and to screen the resulting MAbs. Production and selection of mouse monoclonal antibodies against rAdMHE3 were performed as described previously (Liu et al., 2014). For in vitro adenovirus neutralization experiments, MAbs were serially diluted 2-fold in Dulbecco’s modified eagle’s medium (DMEM) (Gibco, China) and 50-␮l aliquots of each dilution were mixed with 50-␮l recombinant adenoviruses with 2 × 105 VPs. The antibody-virus mixtures were incubated for 1 h at 37 ◦ C and then transferred to 96-well plates containing 85–95% confluent monolayers of HEp-2 cells. Monolayers were cultured in RPMI Medium 1640 (Gibco) without phenol red and serum for 72 h. Infected cells were analyzed using a Varioskan Flash Multimode Reader (Thermo Scientific) to measure the eGFP expression. Neutralizing titers were defined as the minimum concentration of MAb that inhibited 90% of the eGFP expression.

The following strains of adenoviruses used in this study were obtained as previously described (Tian et al., 2011; Qiu et al., 2012; Zhang et al., 2009): recombinant adenovirus rAd3EGFP encoding a HAdV-3 GZ-01 genome and an enhanced green fluorescent protein (eGFP) with an E3 region deletion, hexon chimeric adenovirus rAd3egf/H7 generated by replacing the HAdV-3 hexon gene of the rAd3EGFP with the hexon gene from HAdV-7 GZ08 strain, epitope mutants (rAd3H7R1, rAd3H7R2, rAd3H7R5, and rAd3H7R7) from rAd3egf/H7 replaced with corresponding HAdV-3 epitopes, and epitope chimeric mutant rAdMHE3 from rAd3EGFP replaced with HVR5 of HAdV-7. All the adenoviruses were cultured in HEp2 cells or AD293 cells as previously described (Tian et al., 2011). Adenovirus particles were purified by standard CsCl gradient centrifugation as previously described (Wu et al., 2002). The virus particle (VP) titers were determined by spectrophotometry using a conversion factor of 1.1 × 1012 VPs per absorbance unit at 260 nm (Tian et al., 2011). 2.2. Generation of the HVR4 mutants rAd3A7R4 and rAd3H7A3R4 The plasmid pBRAdE3GFP encoding a HAdV-3 GZ-01 genome (Genbank accession no. DQ099432) and eGFP with an E3 region deletion was constructed, as previously described (Zhang et al., 2009). The hexon-chimeric adenovirus vector pAd3egf/H7, in which the HAdV-3 hexon gene in the pBRAdE3GFP vector was replaced with the hexon gene from HAdV-7, was constructed as previously described (Tian et al., 2011). The shuttle vector pBRLR was also constructed as previously described (Qiu et al., 2012). In this study, the HVR4 mutants rAd3A7R4 and rAd3H7A3R4 were obtained using the same strategy as previously described (Qiu et al., 2012). Briefly, the mutated fragment H7A3R4 was produced by overlapping PCR extension mutagenesis with primer pairs A3HVR4u/HexD, A3HVR4r/HexU, and HexU/HexD, using pAd3egf/H7 as the DNA template. The mutated fragment H3A7R4 was produced by overlapping PCR extension mutagenesis with primer pairs A7HVR4u/HexD, A7HVR4r/HexU, and HexU/HexD, using pBRAdE3GFP as the DNA template. Then, the fragments H7A3R4 and H3A7R4 were cloned into pBRLR to generate shuttle vectors pBRLR-H7A3R4 and pBRLR-H3A7R4. Finally, the LR-H7A3R4 fragment and the LR-H3A7R4 fragment were cloned into the pAd3egf/H7 vector to generate the HAdV-7 hexon HVR4 mutagenesis vector pBRAd3-H7A3R4 and the HAdV-3 hexon HVR4 mutagenesis vector pBRAd3-A7R4, respectively, using homologous recombinant technology in Escherichia coli strain BJ5183. The successful creation of these constructs was confirmed by restriction digestion and sequencing analyses. To rescue viruses, these modified plasmids were digested with AsisI to linearize genomic DNA, then transfected into AD-293 cells grown in 30-mm dishes using Lipofectamine LTX with Plus reagents (Invitrogen, USA) according to the manufacturer’s instructions. The

2.4. Indirect enzyme-linked immunosorbent assay (ELISA) analysis HAdV-3 and HAdV-7 hexon peptides with a hexahistidine tag (designated A3H and A7nH, respectively), and the recombinant short peptides (HAdV-3 HVRs) with an N-terminal glutathione Stransferase (GST) tag were expressed and purified as described previously (Tian et al., 2013). For ELISAs, 96-well plates (Nunc Maxisorp) were coated overnight at 4 ◦ C with fusion peptides (about 2 ␮g/ml) or virus particles (about 1010 VPs/ml) in PBS (pH 7.4) and were washed once with 0.05% Tween-20 in phosphate-buffered saline (PBST) and blocked for 2 h with 2% bovine serum albumin (BSA) in PBST. Then, 100-␮l/well MAb ascites at a dilution of 1:5000 were added to each well and incubated for 1 h at 37 ◦ C. The plates were washed three times with PBST and incubated for 1 h with a 1:10,000 dilution of goat anti-mouse IgG (H + L)-HRP conjugated affinity-purified secondary antibody (Bio-Rad). After washing four times with PBST, the plates were developed with tetramethylbenzidine (TMB) substrate, the reaction was stopped with 2 M H2 SO4 , and the results were analyzed at 450 nm using an ELISA plate reader (Thermo Scientific Multiskan MK3). 2.5. Peptide competition ELISA The epitope detected by each MAb was confirmed by competitive inhibition ELISA. Optimized concentrations of the MAbs were determined by serial dilution. Briefly, the HAdV-3 GZ01 virions in PBS (pH 7.4) were used to coat 96-well plates overnight at 4 ◦ C. In separate tubes, constant concentrations of MAb at a final dilution of 1:5000 were added to increasing concentrations of competitor peptide (0, 3.125, 12.5, 50, or 200 ␮g/ml) in PBST with 2% BSA and incubated for 30 min at 37 ◦ C. The virion-coated plates were washed once with PBST and incubated with 2% BSA in PBST for 2 h at 37 ◦ C. Then, each of the MAb-peptide mixtures was added to duplicate wells, and the plates were incubated for 1 h at 37 ◦ C. The

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subsequent processes were performed as described for indirect ELISAs (section 2.4). 2.6. In vivo neutralization A total of 60 BALB/c mice were subjected to an intranasal infection with 1 × 1010 VPs of rAd3EGFP per mouse. One hour after infection, each mouse was intraperitoneally injected with 20-␮l PBS, MAb 3D7 (30 ␮g), or MAb 1B10 (30 ␮g), with ten mice in each group. Three days later, the mice were sacrificed with CO2 inhalation and the lungs were harvested, homogenized on dry ice, lysed in lysis buffer using a Bullet Blender, and measured for Ad genome copies using a Q-PCR kit as described previously (Tian et al., 2011; Qiu et al., 2012). Procedures for these animal experiments complied with all relevant federal guidelines and institutional policies. 2.7. Statistical analyses The data are presented as the mean ± standard error. Statistical significance was determined using Prism 5.0 software. Comparisons between two groups were made with Student’s t tests. Comparisons among groups were performed by ANOVA with Bonferroni’s test to account for multiple comparisons and p values of less than 0.05 were considered statistically significant. 3. Results 3.1. Identification of neutralizing MAbs directed against HAdV-3 Purified rAdMHE3 virions were used to immunize mice and to screen the resulting MAbs. A total of twenty-two positive MAbs against rAdMHE3 virions were isolated, of which four neutralizing MAbs against HAdV-7 have been described previously (Liu et al., 2014). In this paper, three neutralizing MAbs of IgG2a isotype against HAdV-3 (1C2, 3D7, and 3E6) were identified, and MAb 1B10 without neutralizing activity against either HAdV-3 or HAdV-7 was used as a control. The results from ELISA analyses indicated that each of the three MAbs reacted with its parental antigen, whole rAdMHE3 virus particles, which suggests that the epitope recognized by these three MAbs is presented on the surface of the virion. The ascites fluid titer as determined by an ELISA against rAdMHE3 virions was 50,000 to 1,000,000. All three MAbs detected rAd3EGFP, but not rAd3egf/H7, by indirect ELISA (Fig. 1A). Thus, the three MAbs specifically recognized the hexon protein of HAdV-3. This result was also confirmed with assays using the truncated hexon peptides A3H and A7nH expressed in E. coli (Fig. 1B). The three MAbs were able to neutralize rAd3EGFP but not rAd3egf/H7. Of the three MAbs, MAb 3D7 had the highest neutralization titer of up to 4096 (about 0.5 ␮g/ml) against rAd3EGFP (Fig. 2). Western blotting analyses also demonstrated that these MAbs specifically detected the hexon protein of rAdMHE3 (Fig. 1C). These results demonstrate that the epitope recognized by these MAbs is serotype 3-specific and appears to be continuous. 3.2. Mapping of the epitope recognized by the neutralizing MAbs Hexon crystallographic and phylogenetic analyses suggest that human adenovirus serotype-specific epitopes may reside in any of the seven hypervariable regions (HVRs) (Rux et al., 2003; Bradley et al., 2012; Yuan et al., 2009). Sequence alignment between the HAdV-3 and HAdV-7 hexon proteins demonstrated variations of one or more amino acids in six of the HVRs, HVR-1, -2, -4, -5, -6, and -7 (Tian et al., 2011).

Fig. 1. Assessment of the binding specificities of anti-HAdV-3 MAbs. ELISAs were performed to measure the reaction of the indicated MAbs with whole virus particles (A) and recombinant truncated hexon fragments (B) of HAdV-3 and HAdV-7. 96Well plates were coated with the purified virus particles of rAd3EGFP, rAd3egf/H7, rAdMHE3, or the recombinant truncated hexon fragments of HAdV-3 or HAdV7, then reacted with the indicated MAbs. MAb 1B10 was used as a control. The antisera from mice immunized with rAdMHE3 were used as the positive controls, and the antisera from mice immunized with PBS were used as the negative controls. Each experiment was repeated independently at least three times, and the means ± standard deviations are shown. (C) Western-blot analyses of purified virions rAdMHE3 (lanes 1, 2, 3, and 4) and rAd3egf/H7 (lanes 5, 6, 7, and 8) were performed with MAb 3D7 (lanes 2 and 6), 3E6 (lanes 3 and 7), and 1C2 (lanes 4 and 8). MAb 1B10 (lanes 1 and 5) was used as a control.

To identify the region of the HAdV-3 hexon bound by the neutralizing MAbs, recombinant HVR peptides with GST tags were used in indirect ELISAs with the MAbs. The results revealed that the MAbs reacted with A3R4-GST but not with the other peptides (Fig. 3A), suggesting that the MAbs recognized HVR4 of the hexon. To access the epitope on a native virion, we used a series of recombinant adenoviruses with epitope-mutated hexons in indirect ELISAs with the MAbs (Fig. 3B). Further neutralization assays demonstrated that the MAbs could neutralize rAd3H7A3R4 but not rAd3A7R4 (Fig. 3C and D). These analyses also confirmed HVR4 as the region bound by the neutralizing MAbs, and further suggested that the amino acids T246 and G250 may be the critical amino acids recognized by these MAbs (Fig. 3E). Peptide competitive-inhibition ELISAs were performed to demonstrate the accuracy of the identified epitope for MAbs. Increasing concentrations of peptides were incubated with the MAbs. The A7R4-GST fragment was used as a negative control. The mixtures were then assayed by ELISAs with HAdV-3 virus particle-coated plates. The A3R4 fragment competed with virions for binding by the MAbs in a dose-dependent manner, while the

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Fig. 2. In vitro neutralization tests. Neutralization tests were performed with the indicated MAbs against rAd3EGFP (A) and rAd3egf/H7 (B). MAb 1B10 was used as a control.

control peptide A7R4 did not (Fig. 4). Together, these results suggest that A3R4 is the epitope recognized by these MAbs. MAb 3D7 showed a high neutralizing titer against HAdV-3 so we aimed to determine the distribution of its epitope among different

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Fig. 4. Peptide competition ELISAs. Increasing concentrations of the identified peptides A3R4 or A7R4 (as a control) were pre-incubated with a constant concentration of the indicated MAbs, which were then used to detect HAdV-3 particles bound to microtiter plates. Each experiment was repeated independently at least three times, and the means ± standard deviations are shown.

Fig. 3. Identification of the epitope for the screened MAbs. (A) Epitope mapping was performed by indirect ELISA of the indicated MAbs with seven HVR fusion fragments (A3R1-GST to A3R7-GST) that were expressed in E. coli (24). (B) Indirect ELISAs were performed to assess the reactions of the indicated MAbs with a series of recombinant adenovirus particles containing chimeric hexons. (C) and (D) Identification of the epitope for the indicated MAbs by neutralization tests. rAd3A7R4 is a chimeric HAdV-3 virus containing the HVR4 of HAdV-7, and rAd3H7A3R4 is a chimeric HAdV-3 virus containing the HAdV-7 hexon with the HVR4 of HAdV-3. (E) HVR4 sequences of HAdV-3 and HAdV-7. Each experiment was repeated independently at least three times, and the means ± standard deviations are shown.

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Fig. 5. Amino acid sequence alignment of representative hexon proteins from the HAdV-3 strains available from GenBank. Only seven hypervariable regions are shown. The corresponding region of HAdV-7 hexon protein was used as a control.

subtypes of HAdV-3. An alignment of the hexon protein sequences available from GenBank revealed that this epitope is unique for HAdV-3 and that global HAdV-3 strains showed no amino acid substitutions in HVR4 (Fig. 5). 3.3. Passive immunization with neutralizing MAb 3D7 Because HAdV-3 infection typically occurs via the respiratory tract, the in vivo protection of MAb 3D7 was assessed by measuring the number of DNA copies of the Ad genome in the lungs of intranasally HAdV-3-infected mice that had received neutralizing MAb 3D7 or control treatments. As shown in Fig. 6, after 3 days, the

Fig. 6. Passive immunization of MAb 3D7 in mice. Six groups of ten mice were challenged with an intranasal infection of 1 × 1010 VPs of rAd3EGFP or rAd3A7R4 (as a control) per mouse, then injected intraperitoneally with MAb 3D7, MAb 1B10, or PBS. Three days later, the mice were sacrificed and their lungs were harvested for use in qPCR assays. The data are representative results of two independent qPCR experiments. Each symbol represents one mouse and the line indicates the geometric mean value of the group. MAb 1B10 and PBS were used as controls. * p < 0.05.

number of copies of the rAd3EGFP genome in mice treated with neutralizing MAb 3D7 was significantly lower than that in mice treated with control MAb 1B10 or with PBS (p < 0.05). However, there were no significant differences in the number of viral genome copies between these three groups when mice were infected with the control virus rAd3A7R4. 4. Discussion In this study, we report three neutralizing MAbs against HAdV3 that recognize the same exposed region (amino acid positions 244–254 of the hexon), corresponding to HVR4. Of these three MAb, MAb 3D7 had the highest neutralizing titer in vitro. Previous hexon crystallographic and phylogenetic analyses have suggested that serotype-specific epitopes might reside in any of the seven HVRs exposed to the viral surface (Rux et al., 2003). However, among the 52 known adenoviruses, only a few epitopes have been identified so far (Bradley et al., 2012; Yuan et al., 2009; Qiu et al., 2012). In our previous studies, we identified several epitopes of HAdV-3 and HAdV-7 with a series of MAbs (Liu et al., 2014; Tian et al., 2013; Qiu et al., 2009). We also demonstrated with experiments using epitope-incorporated recombinant adenoviruses that HVR1, 2, 5, and 7 of HAdV-3 contain the neutralizing residues (Qiu et al., 2012). In this paper, we used neutralizing MAbs to identify HVR4 of HAdV-3 as a linear neutralizing epitope. Experiments with the chimeric adenovirus rAd3A7R4, which could be neutralized by mouse anti-HAdV-7 serum, showed that HVR4 of HAdV-7 is also a neutralization region (Table 1). Alignment between the amino acid sequences of HVR4 from HAdV-7 and HAdV-3 demonstrated that the amino acids T246 and G250 may be the critical amino acids recognized by the serotype-specific neutralizing antibodies. MAbs 3D7, 1C2, and 3E6 detected and neutralized HAdV-3 but were not able to detect or neutralize HAdV-7 (Fig. 1); therefore, these MAbs may be used to distinguish HAdV-3 from other serotypes of HAdV. Our results suggest that the epitope recognized by these MAbs is unique for HAdV-3. However, it is necessary to identify the distribution of this epitope among HAdV-3 strains

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Table 1 Neutralizing antibody titers from adenovirus-immunized mice. Antiserum

Anti-HAdV-7 Anti-rAd3A7R4 Anti-rAd3EGFP Anti-rAd3H7A3R4

Neutralizing titer against viruses rAd3A7R4

HAdV-7

rAd3EGFP

rAd3egf/H7

rAd3H7A3R4

64 4096 8192 128

4096 64

Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3.

Human adenovirus type 3 (HAdV-3) has produced a global epidemic in recent years causing serious diseases such as pneumonia in both pediatric and adult...
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