Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

Abstract

Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre-and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCEEnterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAbbased anti-EV71 therapy. E nterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which has been prevalent in Southeast Asia (1). Patients with severe HFMD cases manifest neurological complications, such as brainstem encephalitis and pulmonary edema, resulting in death, and such cases are often associated with EV71 infection (2). No prophylactic vaccine against EV71 infection or therapeutic drug for EV71 infection is available. Even though inactivated whole-virus-based EV71 vaccines have progressed into clinical trials (3, 4), their licensure faces serious challenges (5). Thus, the development of efficacious antiviral drugs for the treatment of EV71-infected patients ...

Plant-Produced Anti-Enterovirus 71 (EV71) Monoclonal Antibody Efficiently Protects Mice Against EV71 Infection

“…There are several mAbs specific to EV71, for example, mAb specific to RNA-dependent RNA polymerase [40], mAb binding to EV71 VP1 [20,22,[24][25][26], mAb binding to EV71 VP2 [41], mAb binding to VP3 [42], and others. Among these mAbs, D5 is one of the most potent anti-EV71 neutralizing mAb.…”

“…Among these mAbs, D5 is one of the most potent anti-EV71 neutralizing mAb. It is specific to the highly conserved VP1 GH-loop of EV71 and was shown to have a broad cross-neutralization activity against all tested EV71 strains [26,27,43]. Therefore, this mAb has the potential to be an effective treatment for EV71 infection.…”

“…However, there are disadvantages to using pooled human sera, such as the risk of transmitting human pathogens, availability of donors, and batch-to-batch variability [19]. Therefore, neutralizing monoclonal antibodies (mAbs) are considered a safe alternative for passive immunization against EV71.Over the years, several EV71-neutralizing antibodies have been reported [20][21][22][23][24][25][26]. Among these mAbs, D5 is one of the most promising mAb for development as prophylactic and therapeutic for EV71 infection.…”

“…Among these mAbs, D5 is one of the most promising mAb for development as prophylactic and therapeutic for EV71 infection. D5 mAb binds specifically to SP70 peptide on VP1 GH loop of EV71 and can potently neutralize EV71 infection by blocking viral attachment and internalization and by stabilizing the virus through a bivalent binding mode [26,27]. However, the antibody production in mammalian cell culture has limitations with high production costs and scalability.Recently, plants are being used as an alternative mAb production platform with unique advantages such as low production cost, speed of production, and scalability [28].…”

“…Over the years, several EV71-neutralizing antibodies have been reported [20][21][22][23][24][25][26]. Among these mAbs, D5 is one of the most promising mAb for development as prophylactic and therapeutic for EV71 infection.…”