Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics. IMPORTANCEIn a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

Section: Discussionmentioning

confidence: 99%

The Enterovirus 71 Procapsid Binds Neutralizing Antibodies and Rescues Virus Infection In Vitro

Shingler

Cifuente

Ashley

et al. 2015

“…RD and Vero cells were maintained as previously described (21). Jurkat T cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Passive transfer of neutralizing antiserum protects mice from lethal EV71 infection in vivo (21)(22)(23), indicating that neutralizing antibodies are major protective components in anti-EV71 immunity. The use of monoclonal antibodies (MAbs) with neutralization capabilities represents an excellent strategy in the development of antiviral drugs due to their high specificity and potency (24), as exemplified by the successful commercialization of palivizumab, a humanized MAb against respiratory syncytial virus (25).…”

mentioning

confidence: 99%

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

Shi

et al. 2015

Self Cite

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 ...

“…These features, which led to the description of VLPs as self-adjuvanting immunogen delivery systems (6,7), make VLPs attractive stand-alone vaccine candidates for many diseases (8)(9)(10)(11). In addition, VLPs can also be used as platforms for the multimeric display of foreign antigens (12)(13)(14)(15). Here we introduce a strategy for engineering chimeric VLPs for presentation of heterologous proteins to the immune system, using the capsid of the infectious bursal disease virus (IBDV).…”

mentioning

confidence: 99%

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

Pascual

Mata

Gómez-Blanco

et al. 2015

Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ϳ70-nmdiameter T‫31؍‬ IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic ␣ helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this ␣ helix assemble into genuine VLPs only when expressed with an N-terminal His 6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ϳ78,000 nm 3 ) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (ϳ240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA-and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. IMPORTANCEVirus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.