Abstract
Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and young children (<5 years). HFMD outbreaks occur frequently in the Asia-Pacific region, and these outbreaks are associated with enormous healthcare and socioeconomic burden. There is currently no specific antiviral agent to treat HFMD and/or the severe complications that are frequently associated with the enterovirus of serotype EV71. Therefore, the development of a broadly effective and safe anti-enterovirus agent is an existential necessity. In this study, human single-chain antibodies (HuscFvs) specific to the EV71-internal capsid protein (VP4) were generated using phage display technology. VP4 specific-HuscFvs were linked to cell penetrating peptides to make them cell penetrable HuscFvs (transbodies), and readily accessible to the intracellular target. The transbodies, as well as the original HuscFvs that were tested, entered the enterovirus-infected cells, bound to intracellular VP4, and inhibited replication of EV71 across subgenotypes A, B, and C, and coxsackieviruses CVA16 and CVA6. The antibodies also enhanced the antiviral response of the virus-infected cells. Computerized simulation, indirect and competitive ELISAs, and experiments on cells infected with EV71 particles to which the VP4 and VP1-N-terminus were surface-exposed (i.e., A-particles that don’t require receptor binding for infection) indicated that the VP4 specific-antibodies inhibit virus replication by interfering with the VP4-N-terminus, which is important for membrane pore formation and virus genome release leading to less production of virus proteins, less infectious virions, and restoration of host innate immunity. The antibodies may inhibit polyprotein/intermediate protein processing and cause sterically strained configurations of the capsid pentamers, which impairs virus morphogenesis. These antibodies should be further investigated for application as a safe and broadly effective HFMD therapy.
Highlights
Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and children younger than 5 years of age; it can occasionally infect adults (Lee et al, 2009; Kaminska et al, 2013)
Derived-recombinant enterovirus 71 (EV71)-VP4 was produced for use as antigen to select out the recombinant VP4 (rVP4)-bound phage clones from the HuscFv-phage display library by panning process (Kulkeaw et al, 2009)
E. coli HB2151 infected with rVP4-bound phages were screened by PCR for the presence of genes coding for HuscFvs, and 44 phage-transformed E. coli colonies were positive for the huscfvs (Figure 1C)
Summary
Foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and children younger than 5 years of age; it can occasionally infect adults (Lee et al, 2009; Kaminska et al, 2013). P2 and P3 are the precursors of seven non-structural proteins, including 2A, 2B, 2C, 3A, 3B (VPg), 3C, and 3D, which are responsible for viral protein processing, genome replication, translation and host factor interaction (Yuan et al, 2018). The externalized VP1 and VP4 of the A-particle interact with the host endosomal membrane, and the VP4 forms a membrane pore through which the viral RNA exits into the cytoplasm (genome uncoating/release) to initiate viral translation and replication, leaving behind the empty capsid shell (Shingler et al, 2013). Certain cells [e.g., Chinese hamster ovarian (CHO) K1 cells] allow EV71 to attach to the cell membrane, but no infection was observed (Tan et al, 2013) Those authors hypothesized this to be due to failure of the viral genome to enter the cytoplasm. The HuscFvs and transbodies inhibited replication of EV71 strains of different subgenotypes, as well as other enterovirus serotypes, and enhanced host innate immune response
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