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

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Phage Display-Derived Cross-Reactive Neutralizing Antibody Against Enterovirus 71 and Coxsackievirus A16 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,LinPang3, Sisi Shen1, Jie Zhang2, Shan Cen4,BurtonB.Yang5, Yuming Huang3, Wang Sheng1*, and Yi Zeng1 1College of Life Science and Bioengineering, Beijing University of Technology, Beijing; 2Sinocelltech, 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 SUMMARY: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviri- dae 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 cross- neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display- derived 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 INTRODUCTION CVA16 to emerge have been documented (13–15). These Enterovirus 71 (EV71) and coxsakievirus A16 findings suggest that both EV71 and CVA16 should be (CVA16) are non-enveloped RNA viruses of the targeted for vaccine and therapeutic development for ef- Picornaviridae family. Their genomes are positive-sense fective control and treatment of HFMD. single-stranded RNAs of approximately 7,500 nucleo- Immunization of maternal mice with inactivated tides encoding single, large polyprotein that can be EV71 and CVA16 protects newborn mice from lethal digested by proteases into P1, P2, and P3 regions (1–3). challenge with EV71 and CVA16, respectively, which Subsequent cleavage of the P1 regions by viral protease indicates that neutralizing antibodies are critical in the 3CD yields 4 capsid subunit proteins: VP1, VP2, VP3, protection of neonatal mice against viral infection and VP4. The virions of EV71 and CVA16 are penta- (16,17). Two neutralizing epitopes SP55 (amino acids meric icosahedral particles with diameters of 24–30 nm. 163–177 and SP70 (amino acids 208–222), have been Theseparticlesarecomposedof60copiesofstructural identified in the VP1 of EV71 in mice. These peptide-in- proteins organized into 12 pentamers. VP1, VP2, and duced antibodies cross-neutralize EV71 and protect VP3 are located on the virion surface, and myristoylat- mice from lethal challenges with homologous and heter- ed VP4 is located inside (4–6). ologous EV71 strains (18). An immunoglobulin M EV71 and CVA16 have been identified as the major monoclonal antibody (mAb) targeting a highly con- etiological agents of hand, foot and mouth disease served epitope (amino acids 215–219 in EV71 VP1) con- (HFMD). Viral infections by EV71 and CVA16 result in fers effective protection against lethal EV71 challenge in as well as severe illnesses, such as aseptic meningitis, vivo when administrated at 10 mg/g body weight (19). In poliomyelitis-like paralysis, and fatal brainstem ence- addition, a cross-neutralizing epitope in VP2 (amino phalitis (7–9). Numerous large outbreaks of HFMD acids 141–155) has been identified through overlapped caused by EV71 and CVA16 have been reported in the synthetic peptide mapping. Chimeric particles consist- Asia-Pacific region since 2000 (10–12), and HFMD ing of both defined VP2 peptide and hepatitis B core remains a common infectious illness in children in this antigen (HBcAg) induce cross-protective antibody area. Moreover, EV71 and CVA16 co-infection and the responses against EV71. Immune sera elicited by chi- meric particles provide full in vivo passive protection Received February 4, 2015. Accepted April 13, 2015. against lethal EV71 challenge (20). In a recent study, a J-STAGE Advance Publication June 12, 2015. neutralizing antibody targeting a conformational epi- DOI: 10.7883/yoken.JJID.2015.060 tope was isolated from mice immunized with an inacti- *Corresponding author: Mailing address: College of Life vated EV71-B4 strain, and it passively protected ne- Science and Bioengineering, Beijing University of Technol- onatal mice against lethal EV71 infection. The potential ogy, 100 Pingleyuan, Chaoyang District, Beijing, China. epitope was mapped by using escape mutants (21). Tel: ＋86-10-67392780, Fax: ＋86-10-67392780, E-mail: In a previous study, we identified an immunodomi- shengwang＠bjut.edu.cn; danielle_zx＠163.com nant epitope at the N-terminal of the EV71 VP4 protein †These authors contributed equally to this work. (22). The ``core sequence'' of VP4, which is responsible 66 Neutralizing Antibody against EV71 and CVA16 for immune stimulation, is highly conserved among immunodominant epitope, which is highly conserved CVA16 and EV71 viral strains. Immunization with the among EV71 and CVA16 viral strains, is highlighted in VP4 peptide elicits a broad neutralizing antibody gray (Table 1). Seven days after a boost spleen cells response against various EV71 genotypes (22). In the were harvested to extract RNA with the TRIzol method presentstudy,aphagedisplay system was used to (Invitrogen, Carlsbad, CA, USA). A SuperScript III produce a recombinant mAb targeting this conserved First-Strand Synthesis System (Invitrogen) was used to peptide (designated VP4N20) of EV71 VP4. The neu- synthesize cDNA from total RNA according to the tralizing activity of the recombinant antibody against manufacturer's instructions. Vk,Vl,andVH fragments EV71 and CVA16 was evaluated with in vitro neutrali- were amplified with PCR using Vk,Vl,andVH primary zation assay. primers (Table 2) (23,24). For PCR amplification, each 50-mL aliquot of reaction mixture contained 5 mL10× buffer, 100 ng cDNA, 2.0 U FastStart enzyme, 40 nmol MATERIALS AND METHODS of dNTP mix, and 20 pmol each of the appropriate for- Phage library construction: Six-week-old female ward and reverse primers. PCR cycling conditions con- BALB/c mice were immunized with an intraperitoneal sisted of 959C for 5 min followed by 30 cycles of 959C injection of 5 mg recombinant chimeric particles com- for 1 min, 559C for 45 s, 729C for 2 min, and a final posed of HBcAg and VP4N20 as described previously 10-min extension at 729C. Purified VL (Vk ＋ Vl)and (22). The sequence of VP4N20 is shown in Table 1. The VH PCR products were combined randomly to synthe- Table 1. Amino acid sequence of VP4N20 peptide conserved in EV71 and CVA16 VP4 proteins Gly Ser Gln Val Ser Thr Gln Arg Ser Gly Ser His Glu Asn Ser Asn Ser Ala Thr Glu EV71 GSQVSTQRSGSHENSNSATE Gly Ser Gln Val Ser Thr Gln Arg Ser Gly Ser His Glu Asn Ser Asn Ser Ala Ser Glu CVA16 GSQVSTQRSGSHENSNSASE Table 2. The sequences of primers for the scFv-encoding gene amplification Hv Primers: MHV.BACK1 (SkiI) 5?-gcggcccagccggccatggccGATGTGAAGCTTCAGGAGTC-3? MHV.BACK2 (SfiI) 5?-gcggcccagccggccatggccCAGGTGCAGCTGAAGGAGTC-3? MHV.BACK3 (SfiI) 5?-gcggcccagccggccatggccCAGGTTCAACTGCAGCAATC-3? MHV.BACK4 (SfiI) 5?-gcggcccagccggccatggccCAGGTTACTCTGAAAGAGTC-3? MHV.BACK5 (SfiI) 5?-gcggcccagccggccatggccGAGGTCCAGCTGCAACAATCT-3? MHV.BACK6 (SfiI) 5?-gcggcccagccggccatggccGAGGTCCAGCTGCAGCAGTC-3? MHV.BACK7 (SfiI) 5?-gcggcccagccggccatggccCAGGTCCAACTGCAGCAGCCT-3? MHV.BACK8 (SfiI) 5?-gcggcccagccggccatggccGAGGTGAAGCTGGTGGAGTC-3? MHV.BACK9 (SfiI) 5?-gcggcccagccggccatggccGAGGTGAAGCTGGTGGAATC-3? MHV.BACK10 (SfiI) 5?-gcggcccagccggccatggccGATGTGAACTTGGAAGTGTC-3? MHV.FOR1 5?-tgaaccgcctccaccTGCAGAGACAGTGACCAGAGT-3? MHV.FOR2 5?-tgaaccgcctccaccTGAGGAGACTGTGAGAGTGGT-3? MHV.FOR3 5?-tgaaccgcctccaccTGAGGAGACGGTGACTGAGGT-3? MHV.FOR4 5?-tgaaccgcctccaccTGAGGAGACGGTGACCGTGGT-3? Lv-k Primers: MKV.BACK1 5?-tctggcggtggcggatcgGATGTTTTGATGACCCAAACT-3? MKV.BACK2 5?-tctggcggtggcggatcgGATATTGTGATGACGCAGGCT-3? MKV.BACK3 5?-tctggcggtggcggatcgGATATTGTGATAACCCAG-3? MKV.BACK4 5?-tctggcggtggcggatcgGACATTGTGCTGACCCAATCT-3? MKV.BACK5 5?-tctggcggtggcggatcgGACATTGTGATGACCCAGTCT-3? MKV.BACK6 5?-tctggcggtggcggatcgGATATTGTGCTAACTCAGTCT-3? MKV.BACK7 5?-tctggcggtggcggatcgGATATCCAGATGACACAGACT-3 MKV.BACK8 5?-tctggcggtggcggatcgGACATCCAGCTGACTCAGTCT-3? MKV.BACK9 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 tical density at 600 nm reached 0.5. Phages were rescued (scFvs) by using two overlapping linkers through with M13K07 helper phage (GE Healthcare) for 1 h, as splicing-overlap-extension PCR (SOE-PCR) method. described previously (25–28). Rescued phage particles in The linker sequences are shown in Table 2. the supernatant were precipitated by adding a 1/5 PCR fragments were subsequently digested with SfiI volume of 20z polyethylene glycol in 2.5 M NaCl and and NotI restriction enzymes and subcloned into phage incubating the mixture on ice for 30 min. The phage
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