Jpn. J. Infect. Dis., 69, 66–74, 2016

Original Article

Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16 Xiao Zhang1†, Chunyun Sun2†, Xiangqian Xiao1, Lin Pang3, Sisi Shen1, Jie Zhang2, Shan Cen4, Burton B. Yang5, Yuming Huang3, Wang Sheng1*, and Yi Zeng1 1College

of Life Science and Bioengineering, Beijing University of Technology, Beijing; Cell Engineering Center, Chinese Academy of Medical Science, Beijing; 3Beijing Ditan Hospital, Capital Medical University, Beijing; 4Department of Virology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing, China; and 5Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 2Sinocelltech,

SUMMARY: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed crossneutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage displayderived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization. potential for new viral recombinants of EV71 and CVA16 to emerge have been documented (13–15). These findings suggest that both EV71 and CVA16 should be targeted for vaccine and therapeutic development for effective control and treatment of HFMD. Immunization of maternal mice with inactivated EV71 and CVA16 protects newborn mice from lethal challenge with EV71 and CVA16, respectively, which indicates that neutralizing antibodies are critical in the protection of neonatal mice against viral infection (16,17). Two neutralizing epitopes SP55 (amino acids 163–177 and SP70 (amino acids 208–222), have been identified in the VP1 of EV71 in mice. These peptide-induced antibodies cross-neutralize EV71 and protect mice from lethal challenges with homologous and heterologous EV71 strains (18). An immunoglobulin M monoclonal antibody (mAb) targeting a highly conserved epitope (amino acids 215–219 in EV71 VP1) confers effective protection against lethal EV71 challenge in vivo when administrated at 10 mg/g body weight (19). In addition, a cross-neutralizing epitope in VP2 (amino acids 141–155) has been identified through overlapped synthetic peptide mapping. Chimeric particles consisting of both defined VP2 peptide and hepatitis B core antigen (HBcAg) induce cross-protective antibody responses against EV71. Immune sera elicited by chimeric particles provide full in vivo passive protection against lethal EV71 challenge (20). In a recent study, a neutralizing antibody targeting a conformational epitope was isolated from mice immunized with an inactivated EV71-B4 strain, and it passively protected neonatal mice against lethal EV71 infection. The potential epitope was mapped by using escape mutants (21). In a previous study, we identified an immunodominant epitope at the N-terminal of the EV71 VP4 protein (22). The ``core sequence'' of VP4, which is responsible

INTRODUCTION Enterovirus 71 (EV71) and coxsakievirus A16 (CVA16) are non-enveloped RNA viruses of the Picornaviridae family. Their genomes are positive-sense single-stranded RNAs of approximately 7,500 nucleotides encoding single, large polyprotein that can be digested by proteases into P1, P2, and P3 regions (1–3). Subsequent cleavage of the P1 regions by viral protease 3CD yields 4 capsid subunit proteins: VP1, VP2, VP3, and VP4. The virions of EV71 and CVA16 are pentameric icosahedral particles with diameters of 24–30 nm. These particles are composed of 60 copies of structural proteins organized into 12 pentamers. VP1, VP2, and VP3 are located on the virion surface, and myristoylated VP4 is located inside (4–6). EV71 and CVA16 have been identified as the major etiological agents of hand, foot and mouth disease (HFMD). Viral infections by EV71 and CVA16 result in as well as severe illnesses, such as aseptic meningitis, poliomyelitis-like paralysis, and fatal brainstem encephalitis (7–9). Numerous large outbreaks of HFMD caused by EV71 and CVA16 have been reported in the Asia-Pacific region since 2000 (10–12), and HFMD remains a common infectious illness in children in this area. Moreover, EV71 and CVA16 co-infection and the Received February 4, 2015. Accepted April 13, 2015. J-STAGE Advance Publication June 12, 2015. DOI: 10.7883/yoken.JJID.2015.060 *Corresponding author: Mailing address: College of Life Science and Bioengineering, Beijing University of Technology, 100 Pingleyuan, Chaoyang District, Beijing, China. Tel: +86-10-67392780, Fax: +86-10-67392780, E-mail: shengwang@bjut.edu.cn; danielle_zx@163.com †These authors contributed equally to this work. 66

Neutralizing Antibody against EV71 and CVA16

immunodominant epitope, which is highly conserved among EV71 and CVA16 viral strains, is highlighted in gray (Table 1). Seven days after a boost spleen cells were harvested to extract RNA with the TRIzol method (Invitrogen, Carlsbad, CA, USA). A SuperScript III First-Strand Synthesis System (Invitrogen) was used to synthesize cDNA from total RNA according to the manufacturer's instructions. Vk, Vl, and VH fragments were amplified with PCR using Vk, Vl, and VH primary primers (Table 2) (23,24). For PCR amplification, each 50-mL aliquot of reaction mixture contained 5 mL 10 × buffer, 100 ng cDNA, 2.0 U FastStart enzyme, 40 nmol of dNTP mix, and 20 pmol each of the appropriate forward and reverse primers. PCR cycling conditions consisted of 959 C for 5 min followed by 30 cycles of 959 C for 1 min, 559 C for 45 s, 729 C for 2 min, and a final 10-min extension at 729 C. Purified VL (Vk + Vl) and VH PCR products were combined randomly to synthe-

for immune stimulation, is highly conserved among CVA16 and EV71 viral strains. Immunization with the VP4 peptide elicits a broad neutralizing antibody response against various EV71 genotypes (22). In the present study, a phage display system was used to produce a recombinant mAb targeting this conserved peptide (designated VP4N20) of EV71 VP4. The neutralizing activity of the recombinant antibody against EV71 and CVA16 was evaluated with in vitro neutralization assay. MATERIALS AND METHODS Phage library construction: Six-week-old female BALB/c mice were immunized with an intraperitoneal injection of 5 mg recombinant chimeric particles composed of HBcAg and VP4N20 as described previously (22). The sequence of VP4N20 is shown in Table 1. The

Table 1. Amino acid sequence of VP4N20 peptide conserved in EV71 and CVA16 VP4 proteins EV71

Gly

Ser

Gln

Val

Ser

Thr

Gln

Arg

Ser

Gly

Ser

His

Glu

Asn

Ser

Asn

Ser

Ala

Thr

Glu

G

S

Q

V

S

T

Q

R

S

G

S

H

E

N

S

N

S

A

T

E

Gly

Ser

Gln

Val

Ser

Thr

Gln

Arg

Ser

Gly

Ser

His

Glu

Asn

Ser

Asn

Ser

Ala

Ser

Glu

G

S

Q

V

S

T

Q

R

S

G

S

H

E

N

S

N

S

A

S

E

CVA16

Table 2. The sequences of primers for the scFv-encoding gene amplification Hv Primers: MHV.BACK1 (SkiI) MHV.BACK2 (SfiI) MHV.BACK3 (SfiI) MHV.BACK4 (SfiI) MHV.BACK5 (SfiI) MHV.BACK6 (SfiI) MHV.BACK7 (SfiI) MHV.BACK8 (SfiI) MHV.BACK9 (SfiI) MHV.BACK10 (SfiI) MHV.FOR1 MHV.FOR2 MHV.FOR3 MHV.FOR4 Lv-k Primers: MKV.BACK1 MKV.BACK2 MKV.BACK3 MKV.BACK4 MKV.BACK5 MKV.BACK6 MKV.BACK7 MKV.BACK8 MKV.BACK9

5?-gcggcccagccggccatggccGATGTGAAGCTTCAGGAGTC-3? 5?-gcggcccagccggccatggccCAGGTGCAGCTGAAGGAGTC-3? 5?-gcggcccagccggccatggccCAGGTTCAACTGCAGCAATC-3? 5?-gcggcccagccggccatggccCAGGTTACTCTGAAAGAGTC-3? 5?-gcggcccagccggccatggccGAGGTCCAGCTGCAACAATCT-3? 5?-gcggcccagccggccatggccGAGGTCCAGCTGCAGCAGTC-3? 5?-gcggcccagccggccatggccCAGGTCCAACTGCAGCAGCCT-3? 5?-gcggcccagccggccatggccGAGGTGAAGCTGGTGGAGTC-3? 5?-gcggcccagccggccatggccGAGGTGAAGCTGGTGGAATC-3? 5?-gcggcccagccggccatggccGATGTGAACTTGGAAGTGTC-3? 5?-tgaaccgcctccaccTGCAGAGACAGTGACCAGAGT-3? 5?-tgaaccgcctccaccTGAGGAGACTGTGAGAGTGGT-3? 5?-tgaaccgcctccaccTGAGGAGACGGTGACTGAGGT-3? 5?-tgaaccgcctccaccTGAGGAGACGGTGACCGTGGT-3? 5?-tctggcggtggcggatcgGATGTTTTGATGACCCAAACT-3? 5?-tctggcggtggcggatcgGATATTGTGATGACGCAGGCT-3? 5?-tctggcggtggcggatcgGATATTGTGATAACCCAG-3? 5?-tctggcggtggcggatcgGACATTGTGCTGACCCAATCT-3? 5?-tctggcggtggcggatcgGACATTGTGATGACCCAGTCT-3? 5?-tctggcggtggcggatcgGATATTGTGCTAACTCAGTCT-3? 5?-tctggcggtggcggatcgGATATCCAGATGACACAGACT-3 5?-tctggcggtggcggatcgGACATCCAGCTGACTCAGTCT-3? 5?-tctggcggtggcggatcgGAAATTGTGCTGACTCAATCT-3?

MKV.FORI (NotI) 5?-ctgcggccgcTTTGATTTCCAGCTTGGTGCCTCC-3? Lv-l Primers: MLV.BACK 5?-tctggcggtggcggatcgCAGGCTGTTGTGACTCAGGAA-3? MLV.FOR (NotI) 5?-ctgcggccgcTTTGATTTCCAGCTTGGTCTTGGGCTG-3? Linkers: 5?-GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG-3? 3?-CCACCTCCGCCAAGTCCGCCTCCACCGAGACCGCCACCGCCTAGC-5?

67

size genes encoding single-chain variable fragments (scFvs) by using two overlapping linkers through splicing-overlap-extension PCR (SOE-PCR) method. The linker sequences are shown in Table 2. PCR fragments were subsequently digested with SfiI and NotI restriction enzymes and subcloned into phage display vector pCANTAB5E (GE Healthcare, Piscataway, NJ, USA). The gene cloning process is summarized in Fig. 1. TG1 Escherichia coli was used for recombinant plasmid transformation. Bacterial colonies from culture plates were picked for sequencing to examine the diversity of the VH and Vk genes cloned into the vector. The final VH/Vk + Vl transformant library was scraped off and stored as glycerol stocks after suspension in Luria Broth. Antigen-specific phage selection via panning: The glycerol stocks of the scFv library were inoculated into 2 × YTAG media containing 1.6z tryptone, 1z yeast extract, 0.5z NaCl, 1z glucose, and 100 mg/mL ampicillin and incubated at 379 C with shaking until the op-

tical density at 600 nm reached 0.5. Phages were rescued with M13K07 helper phage (GE Healthcare) for 1 h, as described previously (25–28). Rescued phage particles in the supernatant were precipitated by adding a 1/5 volume of 20z polyethylene glycol in 2.5 M NaCl and incubating the mixture on ice for 30 min. The phage pellets were collected via centrifugation at 4,000 × g at 49 C for 40 min and resuspended in 1 mL of 1 × phosphate buffered saline. The obtained phage library was subjected to five rounds of selection. Panning selection of the phage was carried out in enzyme-linked immunosorbent assay (ELISA) plate wells (Nunc, Roskilde, Denmark) coated with EV71 VP4 peptide (Table 1). The ELISA plate wells were coated with antigen overnight at 49 C as described previously (22). Antigen-coated wells were blocked with 1z (w/v) bovine serum albumin (BSA) in Tris-buffered saline (TBS) at 379 C for 1 h. After the blocking solution was removed, the phage was added and incubated at 379 C for 1 h. The phage was washed with TBS, and then

Fig. 1. (Color online) Schematic illustration of the amplification of scFv-encoding genes and the cloning of amplified genes into the expression plasmid. 68

Neutralizing Antibody against EV71 and CVA16

Table 3. Signal peptides were subsequently added to the N-terminal of VH and VL domains via SOE-PCR amplification. PCR amplification was carried out in 50 mL of reaction mixture containing 5 mL of 10 × buffer, 100 ng of DNA fragments of the VH and VL domains and synthesized DNA oligonucleotides of signal peptides of the heavy and light chains, respectively, 2.0 U FastStart enzyme, 40 nmol of dNTP mix, and 20 pmol each of the appropriate forward and reverse primers shown in Table 3. PCR amplification was performed as described above. The VH and VL domains with signal peptides were eventually combined with CH and CL domains by using SOE-PCR amplification. The primers SH.BACK and CH.FOR. were used to amplify the heavy chain of the chimeric antibody. The light chain was amplified by using the primers SL.BACK and CL.FOR. as shown in Table 3. PCR fragments of the heavy and light chains were isolated using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian, China) and inserted into pcDNA3.1(+) and pcDNA3.1/zeo(+) plasmids (Invitrogen) to generate pcDNA3.1-H and pcDNA3.1/zeo-L constructs, respectively, after digestion with BamHI and XbaI as shown in Fig. 2. Preparation of the chimeric antibody: The recombinant plasmids pcDNA3.1-H and pcDNA3.1/zeo-L were co-transfected into CHO cells to express the chimeric antibody. The CHO cells were grown at 379 C in RPMI-1640 medium with 10z fetal bovine serum (FBS) in a humidified atmosphere with 5z CO2. CHO cells (1 × 105) were plated in 24-well plates for transfection with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. High antibodyproducing cells were selected in the presence of 450 mg/mL neomycin. The supernatant was collected and concentrated 10-fold with an Amicon Ultra-15 centrifugal filter unit (Millipore, Bedford, MA, USA) and further purified with a Bio-Scale Mini Affi-Prep protein A column (Bio-Rad, Hercules, CA, USA). The eluted fractions were concentrated 4-fold with the Amicon centrifugal filter.

100 mM triethylamine (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated at room temperature for 10 min to elute the bound phage. Eluted phage was first neutralized by adding 1 M Tris-HCl (pH 7.4) and then amplified in E. coli. Screening of VP4 peptide-specific clones: After five rounds of selection, HB2151 cells were infected with the amplified phages and cultured onto 2 × YTAG plates at 379 C overnight (29). The obtained colonies were inoculated into 2 × YTAG medium, and the expression of scFvs was induced by the addition of 1 mmol/L isopropyl-b-D-thiogalactopyranoside and incubation for 3–5 h. To extract soluble protein from the periplasm, we resuspended the cell pellet in 0.5 mL of ice-cold 1 × TES (0.2 mol/L Tris-HCl [pH 8.0], 0.05 mmol/L EDTA, and 0.5 mol/L sucrose). After being mixed with 0.75 mL 1/5 × TES on ice, the cells were vortexed for 30 s and incubated on ice for 1 h. The supernatants containing the soluble scFvs were collected via centrifugation at 12,000 × g for 15 min and then tested for binding to VP4 peptide (Table 1) in an ELISA with horseradish peroxidase-conjugated goat antimouse kappa antibody (Sigma-Aldrich) as a secondary antibody. Positive responses were identified with optical density at 450 nm readings three times that of the control (1z BSA in 1 × TBS). Plasmid DNA was isolated from the positive clones for sequencing after confirming the presence of the scFv inserts in each clone via enzyme digestion. Gene cloning of the chimeric antibody: Total RNA was extracted from B lymphocytes obtained from human peripheral blood by using the TRIzol method. The total RNA was reverse transcribed into cDNA by using a SuperScript III First-Strand Synthesis System. Human immunoglobulin heavy-chain constant domain (IgG CH) and light-chain constant domain (CL) of IgG1 subtype were amplified via PCR. PCR amplification was performed at 959 C for 5 min followed by 30 cycles of 959 C for 1 min, 559 C for 45 s, and 729 C for 2 min, followed by a 10-min extension at 729 C. The sequences of the primers are shown in Table 3. VH and VL domains were then amplified with PCR, by using the primers shown in

Table 3. The sequences of primers for chimeric antibody-encoding gene amplification Heavy chain constant region primers: CH.BACK 5?-GTCACTGTCTCTGCAGCCTCCACCAAGG -3? CH.FOR. 5?-TGCTCTAGAGCATCATTTACCCGGGGACAGG-3? (XbaI) Light chain constant region primers: CL.BACK 5?-GTCGGTGGAGGCACCAAGGTGCAGATG-3? CL.FOR. 5?-TGCTCTAGAGCA CTAACACTCTCCCCTG-3? (XbaI) Heavy chain variable region primers: VH.BACK 5?-GCCACCATGGCCCAGGTTCAACTGCAGCAATC-3? (Kozak) VH.FOR. 5?-GGTGGAGGCTGCAGAGACAGTGACCAGAG-3? Light chain variable region primers: VL.BACK 5?-GCCACCATGGAAATTGTGCTGACTCAATCTC-3? (Kozak) VL.FOR. 5?-AAGATTTCCAGCTTGGTGCCTCC-3? Human heavy chain signal peptide: HHS: 5?-ATGAAACACCTGTGGTTCTTCTTCTTCCTGGTGGCAGCTCCCAGATGGGGTCCTGTCCGCCACCATGGCC-3? SH.BACK 5?-CGGGATCCCGATGAAACACCTG-3? (BamHI) Human light chain signal peptide: HLS: 5?-GGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCTGGGGCTCTGCTACTCTGGCTCCGCCACCATGGCC-3? SL.BACK 5?-CGGGATCCCGGGTCAGGACACAG-3? (BamHI)

69

Fig. 2. (Color online) Schematic illustration of the amplification and the cloning of heavy- and light-chain-encoding genes into the expression plasmids.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis: The purified fractions were subjected to 12z SDS-PAGE under reducing conditions (5z v/v b-mercaptoethanol) or 12–8z gradient SDS-PAGE under non-reducing conditions. After electrophoresis, the gels were stained overnight with Coomassie blue and destained with the solution composed of 10z acetic acid, 40z methanol, and 50z water. EV71 and CVA16 viral particles were collected from cell culture supernatants and lysed with RIPA lysis buffer (Beyotime, Jiangsu, China) for 10 min on ice. After the solution was centrifuged at 12,000 × g for 10 min, the concentration of proteins was measured by using Bradford's reagent (Bio-Rad). The protein samples were denatured via boiling for 10 min and loaded onto an SDS-PAGE (12z) gel for electrophoresis. The

proteins were then transferred onto polyvinylidene fluoride membranes, which were blocked with 2z (w/v) BSA in TBS solution for 1 h at room temperature and washed three times with TBS containing 0.05z (v/v) Tween 20. The membrane was then probed with either primary mouse anti-EV71 mAb (MAB979, Millipore) or phage display-derived mAb for 1 h at 379 C and washed three times with TBS buffer. After the membrane was incubated with the secondary goat anti-mouse or mouse anti-human antibody conjugated with the fluorescent dye: IRDye 800 CW (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) for 45 min, blotting images were acquired by using the Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, NE, USA) and analyzed with the software provided by the manufacturer. In vitro neutralization assay: EV71 BJ08 (genogroup 70

Neutralizing Antibody against EV71 and CVA16

via boiling with SDS and loaded onto SDS-PAGE gel for western blot analysis. Supernatant collected from the cultured RD cells was used as a negative control. Commercial anti-VP2 antibody was used as an antibody control. The results showed that anti-VP2 antibody recognized two protein bands of 28 kDa and 37 kDa, which corresponded to the VP2 and VP0 proteins, respectively, of EV71. These data indicated that anti-VP2 antibody recognized the VP0 protein by reacting with the VP2 protein, as VP0 is a precursor protein composed of VP2 and VP4 proteins. Interestingly, one protein band of 37 kDa, which correlated to the VP0 protein, was detected by using the isolated recombinant mAb (Fig. 4), which suggests that the recombinant mAb recognizes VP0 protein by reacting with the linear VP4N20 epitope located in the N-terminal of EV71 VP4 protein and does not cross-react with other EV71encoded viral proteins. However, the VP4 protein could not be detected by the recombinant mAb under these experimental western blot conditions owing to its low molecular weight (7 kDa). The VP4N20 peptide derived from the N-terminal sequence of EV71 VP4 protein is widely conserved among various EV71 subgenotypes and CVA16 (22). We therefore investigated whether recombinant mAb reacts with the VP4 protein of CVA16. As shown in Fig. 4, a commercial anti-VP2 antibody recognized the VP0 and VP2

C4), EV71 BrCr-TR (genogroup A), and CVA16 strain CHZH05-01, were propagated in human rhabdomyosarcoma (RD) cells. Virus titers in RD cells were determined by using the microtitration method and expressed as the 50z cell culture infective dose (CCID50) according to the Reed-Muench method. Twofold serial dilutions of sera were prepared with Dulbecco's Modified Eagle medium (Gibco-BRL, Gland Island, NY, USA) containing 2z FBS. The EV71/CVA16 stock was diluted to a working concentration of 100 CCID50/ 50 mL. The neutralization assay was conducted with 96-well plates. In each well, 50 mL of diluted mAb was mixed with 50 mL of virus at 100 CCID50 and incubated for 2 h at 379 C. A cell suspension (100 mL) containing 8,000 RD cells was further added to wells containing the virus/mAb mixture and cultured at 379 C. After 7 days, the cells were observed to evaluate the appearance of cytopathic effects by using a CCK8 kit (Beyotime). The neutralization titer was defined as the highest serum dilution that could prevent the development of cytopathic effects in 50z of the cell. RESULTS Phage display-derived mAb recognizes the VP0 proteins of both EV71 and CVA16: In our recent study, we showed that immunization with VP4N20 peptide derived from the N-terminal sequence of the VP4 protein of EV71 elicits a broad neutralizing activity against various EV71 genotypes (22). In the present study, mice were immunized subcutaneously with chimeric HBcAg particles containing VP4N20 peptide and received booster injections 3 weeks later. Splenocytes were then harvested 7 days after the boost for RNA extraction. A phage display technique was used to produce a recombinant mAb targeting VP4N20 peptide. The recombinant mAb was successfully selected with a competitive panning technique and produced in mammalian CHO cells. After purification with protein A, the purity of the isolated mAb was evaluated via densitometric analysis after staining with Coomassie blue. The light and heavy chains were visualized with SDS-PAGE gel electrophoresis under reducing and non-reducing conditions (Fig. 3). We were also interested in testing whether recombinant mAb recognizes EV71 protein. EV71 particles harvested from cell culture supernatant were denatured

Fig. 3. SDS-PAGE analysis. The phage display-derived antibody was analyzed by SDS-PAGE in non-reducing (NR; A) and reducing conditions (R; B). M: marker protein.

Fig. 4. Western blot analysis. Antibody MAB979 (A) and the phage display-derived antibody (B) were used to detect EV71 viral proteins. Antibody MAB979 (C) and the phage display-derived antibody (D) were used to detect CVA16 viral proteins. 71

Fig. 5. Neutralization assay against BrCr-TR strain of EV71. (A) RD cells without EV71 infection. (B) Cells infected by EV71 pre-mixed with phage display-derived mAb at the concentration of 0.344 mg/mL. (C) Cells infected by EV71 pre-mixed with phage display-derived mAb at the concentration of 0.043 mg/mL. (D) Cells infected by EV71. Scale bar: 50 mm.

Fig. 6. Neutralization assay against CVA16. (A) RD cells without CVA16 infection. (B) Cells infected by CVA16 pre-mixed with phage display-derived mAb at the concentration of 0.344 mg/mL. (C) Cells infected by CVA16 pre-mixed with phage display-derived mAb at the concentration of 0.043 mg/mL. (D) Cells infected by CVA16. Scale bar: 50 mm.

of HBcAg and EV71 VP4-derived peptide neutralize homologous and heterologous EV71 strains and confer passive protection against a lethal EV71 challenge in neonatal mice (22). Thus, in the present study, we tested whether the phage display-derived recombinant mAb neutralizes the various subtypes of the EV71 virus. In vitro neutralizing experiments showed that the recombinant mAb prevented the development of cytopathic effects in RD cells. The EV71 A-type BrCr-TR and C4type Bj08 strains were used for the analysis. The median half maximal inhibitory concentration (IC50) values of the recombinant mAb against the BrCr-TR and Bj08

proteins of CVA16. By contrast, the recombinant mAb reacted only with the VP0 protein of CVA16 that contained the VP4 protein, which suggested that the phage display-derived recombinant mAb recognized the VP4 protein of CVA16 and did not cross-react with other CVA16-encoded proteins. Therefore, the phage displayderived mAb can recognize VP0 proteins of both EV71 and CVA16 by targeting the conserved VP4N20 epitope. Phage display-derived mAb neutralizes both EV71 and CVA16 in vitro: Our previous observations showed that antibodies elicited by chimeric particles consisting 72

Neutralizing Antibody against EV71 and CVA16

of both EV71 and CVA16. When used with western blot analysis, this antibody (MAB979) identified two protein bands in the isolated CVA16 particles that corresponded to the VP0 and VP2 proteins. The data indicated that CVA16 particles harvested from the cell culture were a mixture of P- and R-particles, an outcome correlated with previous observations (32). However, the phage display-derived mAb reacted only with the VP0 precursor protein of CVA16, which indicates that this antibody specifically recognizes the VP4 protein of CVA16 and does not cross-react with other capsid proteins of CVA16. The results of in vitro neutralization assays further showed that the phage display-derived anti-VP4 mAb neutralizes both EV71 and CVA16 and prevents cytopathic effects induced by either virus. Located inside virions, VP4 is a myristoylated protein composed of approximately 70 amino acids. However, the externalization of VP4 can be triggered by the receptor binding-induced conformational alteration of the capsid, which forms the channels responsible for a safe release of the picornavirus genome to the cell cytoplasm. The picornavirus capsid structure is thus more dynamic than the crystal structure suggests (33, 34). The exposure of VP4 to the outside of the capsid may potentially lead to anti-VP4 antibody-mediated neutralization against picornavirus. The results of this study are consistent with those of previous reports showing that antibodies elicited against the VP4 protein of rhinovirus neutralize infectivity in vitro (35). Although the phage display-derived anti-VP4 mAb had dual neutralizing activities against both EV71 and CVA16, the IC50 data revealed that this activity was low. Low affinity for the antigen of interest is the main problem encountered in antibody selection with phagedisplay technology. However, the affinity of a selected antibody for a specific antigen can be improved with random mutation strategies (36,37). Recently, we isolated EV71-specific mAb with high neutralizing activity from an EV71-infected infant by using a single B cell screening technique (unpublished data). Compared with antibodies obtained with phage display technology which is a complete in vitro process for antibody screening, B-cells activated by virus infection undergo affinity maturation and generate high-affinity antibody responses to external antigenic stimuli, which are fundamental processes in adaptive immunity (38). Neutralizing antibodies are both useful in the identification of neutralizing epitopes for vaccine design and important for understanding the mechanism of broadly potent cross-neutralization, a modality that has potential preventive and therapeutic value. In recent years, substantial progress has been made in the discovery of an anti-EV71 antibody, and highly potent and broad neutralizing antibodies have been isolated from a variety of animal models (19–21,39). These antibodies confer protection against EV71 lethal challenge in passively immunized animals. However, progress toward antiCVA16 neutralizing antibodies remains limited. The VP4 protein may be a potential target for neutralizing antibody development for CVA16 infection control. In this report, a recombinant mAb against the VP4 protein of EV71 was isolated via phage display. The phage display-derived mAb cross-reacted with the VP4

strains were 0.062 mg/mL and 0.098 mg/mL, respectively. Photographs of the neutralization experiment conducted with BrCr-TR are shown in Fig. 5. Because the phage display-derived mAb reacts with the VP0 protein of CVA16, we tested whether the recombinant mAb neutralizes the CVA16 virus. In vitro neutralizing data showed that the recombinant mAb inhibited CVA16-induced cytopathic effects in RD cells. The median IC50 of the mAb for CVA16 was 0.068 mg/mL (Fig. 6). These data suggest that the phage display-derived anti-VP4 mAb has broad cross-reactive neutralizing activity against both EV71 and CVA16, which are the major etiological factors of HFMD. DISCUSSION Phage display is a widely used technology for the high throughput screening of human therapeutic antibody candidates. It involves the establishment of an antibody library by expressing exogenous proteins on the surface of filamentous phage particles through fusion with phage surface proteins. This library can subsequently be used to select the antibodies that bind the desired target (30). Thus, in combination with antibody library screening, phage display usually allows for the rapid isolation of antigen-directed antibodies in vitro. In the current study, the VP4N20 peptide was used to immunize mice, after which mouse spleen cells were harvested to construct an antibody library and isolate a recombinant mAb targeting VP4N20 through phage display. The results of western blot analysis further showed that phage display-derived recombinant antibody recognizes the VP0 precursor protein consisting of the VP2 and VP4 proteins by binding to the VP4N20 epitope, which indicates that an antibody targeting the VP4N20 epitope was isolated using the phage display technique. Two forms of EV71 particles, E-particles and F-particles have previously identified found in the supernatant of cell cultures. EV71 E-particles are immature viral particles with low infectivity in which the P1 and VP0 polyproteins are incompletely processed, whereas Fparticles are mature forms of EV71 (31). In our experiments, EV71 viral particles were harvested from the supernatant of cell cultures and loaded onto SDS-PAGE gel, and the commercial anti-VP2 antibody MAB979 was used to monitor the expression of VP2 and VP0 via western blot. Two positive protein bands corresponding to the VP0 and VP2 proteins were detected with this system. This result is consistent with that of a previous report (31) and indicates that the collected-EV71 particles were a mixture of E- and F-particles. As expected, the VP0 precursor protein was visualized in collected EV71 particles by using the phage display-derived mAb under the applied electrophoresis conditions, and the results suggest that the isolated antibody is specific for the VP4 protein and has no cross-reactive response with other EV71 capsid proteins. Our data further show that phage display-derived mAb reacts with the VP4 protein of CVA16, which suggests that the isolated antibody can cross-reactively recognize the VP4 proteins of both EV71 and CVA16 by binding to the VP4N20 epitope that is well conserved between the two viral proteins. MAB979 is a cross-reactive antibody that recognizes the VP2 and VP0 proteins 73

proteins of both EV71 and CVA16 and cross-neutralized both EV71 and CVA16 in vitro. The results suggest that this mAb has great potential as a candidate for broad neutralizing antibody against EV71 and CVA16 infection after affinity modification and humanization.

19.

20.

Acknowledgments This work was supported by grants from International Science & Technology Cooperation (No. 2011DFG33200), National High Technology Research and Development Program of China (863 Program, No. 2012AA02A400), Project supported by the National Natural Science Foundation of China (No. 31470076).

21.

Conflict of interest None to declare. 22.

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Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, fo...
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