A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice
Introduction
Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortality in young children under the age of six [1], [2], [3]. EV71 was first isolated in mainland China in 1987 [4]. From 2008 to 2011, HFMD claimed 1893 lives in mainland China, and these deaths were caused mainly by EV71 infection [5], [6], [7]. Three candidate vaccines against EV71 have been developed and evaluated in phase III trials in mainland China, demonstrating good safety profiles and abilities to mediate protective effects [8], [9], and antiviral drug studies have also been reported [10], [11], [12], [13], [14], [15]. However, no vaccine against EV71 is currently available [16].
EV71 is a small, non-enveloped, positive-stranded RNA virus with a genome size of about 7.4 kb, which is encapsidated within an icosahedral capsid, and the four viral structural proteins (VP1, VP2, VP3 and VP4) function as a single structural subunit, the protomer. Five protomers in turn form a pentamer, twelve of which can self-assemble into a naturally native virion [16], [17], [18], [19]. VP1, VP2 and VP3 are arranged at the surface of the virion and therefore are exposed to immune pressure, while VP4 is located internally [20], [21].
Analysis of the crystal structure of EV71 to 3.8 Å resolution demonstrated that the EV71 virion has a quasi-T = 3 symmetry with β-sandwich “jelly-roll” folds like other picornaviruses. The most variable regions of picornavirus virions are the loops exposed on the virion surface that are also important neutralizing immunogenic sites [22], [23], [24]. For example, Foo et al. reported that residues 208–222 (GH loop) of VP1, which is the most surface exposed viral protein, as an important neutralizing epitope of EV71 [25]. Additionally, Xu et al. identified that residues 141–150 of VP2, alongside the VP2 EF loop, as an EV71 neutralization epitope [26]. Therefore, a peptide-based vaccine containing a neutralizing epitope is considered an attractive and promising approach to stimulate an effective and specific protective immune response [25]. However, small synthetic peptides containing linear B-cell epitopes are usually poorly immunogenic [27], [28]. Virus-like particles (VLPs), such as Hepatitis B core antigen (HBc) VLPs, human papillomavirus (HPV) VLPs and modular VLPs, as well as norovirus P particles [16], [29], [30], [31], [32], [33], [34], display morphologies similar to natural virions and have proven to be optimal delivery systems for the presentation of epitopes to the immune system. With the ability to efficiently interact with antigen-presenting cells, VLPs can display heterologous epitopes at high density and maximize the immunogenic and protective potential of neutralizing epitopes.
The capsid protein of norovirus, belonging to the Caliciviridae family, is composed of a shell (S) domain and protrusion (P) domain [35], [36]. When expressed alone, the P domain can assemble spontaneously into P particles with a T = 1 octahedral (24-mers) symmetry. As a new platform for displaying foreign antigen epitopes, norovirus P particles offer several advantages. They can be produced in Escherichia coli and Pichia pastoris (yeast) easily and stably [20], [37]. P particles also can enhance immune responses to the inserted foreign antigen [29], [36], [38], [39]. Conceivably, P particles can be applied as a platform for novel vaccine development against various viral and bacterial pathogens.
Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) both belong to the genus Enterovirus within the family Picornaviridae [40], but EV71 vaccines have failed to prevent CVA16 infections [41]. By protein alignment of these two viruses, we found differences in amino acids concentrated on particular regions in different loops based on the crystal structure [24], [42]. Therefore, we hypothesized that the regions of differential amino acids concentrated on the surface-exposed loops may be immunodominant and antigenic sites. This hypothesis was confirmed by different genotypes of HPV [43], [44]. In this study, conserved regions of EV71 were selected based on alignment of EV71 and CVA16 P1 amino acid sequences [7], [45], [46], as well as the reported major surface-exposed loop domains of EV71 [42]. P particle-EV71 epitope chimeric vaccines were constructed by insertion of selected EV71 epitopes, including the previously identified neutralizing linear epitope SP70 (208–222 of VP1), into loop 2 of the norovirus P particle [25]. We evaluated the potential of the norovirus P particle as a carrier for delivery of EV71 epitopes to achieve enhanced immunogenicity and protection against viral infection in the mouse model. Analysis of sera from immunized mice led to the identification of the neutralizing epitope 71-6 (residues 176–190) within the VP3 protein of EV71. Study of the crystal structure of EV71 have demonstrated that the 71-6 epitope of the GH loop in VP3 could generate potent neutralizing antibodies [24], [47]. Immunized mice developed significantly increased immune responses, and 100% of those animals survived after a lethal challenge with the EV71 virus. These results led to the identification of anew cross-neutralizing epitope (residues 176–190) of VP3 utilizing a P particle carrier.
Section snippets
Cell lines, media and viruses
Human rhabdomyosarcoma (RD) cells, RD-SCARB2 (RDS) and 293-SCARB2 (293S) cell lines were cultured and EV71 C4 strain was propagated as described previously [46], [48], [49].
Peptides, inactivated EV71 and VLP vaccines
Keyhole Limpet Hemocyanin (KLH) conjugated synthetic 71-6 peptide (designated here as pep. 71-6) from GL Biochem. Ltd. (Shanghai, China) was dissolved in PBS to 1 mg/ml [49] and stored at −80 °C. Production and purification of inactivated EV71 and VLP vaccines were described previously [46].
Selection of epitopes and construction of expression vectors
The expression vector pET28a (+)-P
Expression and purification of chimeric P particles displaying EV71 epitopes
All purified P particle–EV71 epitope proteins (i.e., norovirus chimeric proteins) were subjected to SDS-PAGE analysis, which showed that their inferred molecular masses (∼37 kDa) were slightly higher than that of the unmodified P particles (∼35 kDa) (Figure S2B). Concentrations of the chimeric proteins (yields ranging 5–10 mg/L) were determined by using the Odyssey Infrared Imaging System (Fig. S2C), and all proteins reacted with the anti-His mAb in Western blot assays (Fig. S2D).
Antibody response following PP-EV71 epitope protein immunization
Groups of five
Discussion
In previous studies, a cross-protective vaccine against EV71 infections was developed based on the peptide selection method, and two conserved linear neutralizing epitopes (SP70 and SP55) belonging to the VP1 subunit were identified [25], [61]. Another VP2 peptide also was demonstrated as a potential neutralization site. However, based on the EV71 crystal structural analysis, the VP3 subunit was thought to have major antigen clusters, but it was not described as having neutralizing antibody
Conclusions
We have successfully identified residues 176–190 within the VP3 capsid protein of EV71 as a conformational neutralizing epitope. Our results suggested that the chimeric protein PP-71-6 might provide an opportunity for the design of a dual vaccine candidate against both EV71 and norovirus. Simultaneously, the discovery of the amino acids 176–190 of VP3 as a conformational neutralizing epitope provides valuable information for the production of mAbs and development of a vaccine for HFMD.
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