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A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71.

Xu L, He D, Yang L, Li Z, Ye X, Yu H, zhao H, Li S, Yuan L, Qian H, Que Y, Shih JW, Zhu H, Li Y, Cheng T, Xia N - Sci Rep (2015)

Bottom Line: We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not.Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16.These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Diagnostics and Vaccine Development in infectious diseases, School of Public Health, Xiamen University, Xiamen 361102, PR China.

ABSTRACT
Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

No MeSH data available.


Related in: MedlinePlus

Analysis of chimeric VLPs.(A) Schematic presentation of the chimeric HBc protein construct. (B) SDS-PAGE and Western blot analyses of the E. coli-expressed HBc fusion proteins. Lane M, molecular mass marker; Lane 1, HBc-E1/2; lane 2, HBc-E1; lane 3, HBc-E2; lane 4, HBc (aa1-149). (C) Electron microscopy of HBc-E1/2, HBc-E1, HBc-E2 and HBc(aa1-149) VLPs. (D) Molecular modeling of chimeric VLPs and the VP1(aa208-222) and VP1(aa141-155) epitopes located on the surface of HBc VLPs colored by red and green, respectively. These two epitopes are also shown in the mature EV71 virion (PDB ID:3VBS).
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f1: Analysis of chimeric VLPs.(A) Schematic presentation of the chimeric HBc protein construct. (B) SDS-PAGE and Western blot analyses of the E. coli-expressed HBc fusion proteins. Lane M, molecular mass marker; Lane 1, HBc-E1/2; lane 2, HBc-E1; lane 3, HBc-E2; lane 4, HBc (aa1-149). (C) Electron microscopy of HBc-E1/2, HBc-E1, HBc-E2 and HBc(aa1-149) VLPs. (D) Molecular modeling of chimeric VLPs and the VP1(aa208-222) and VP1(aa141-155) epitopes located on the surface of HBc VLPs colored by red and green, respectively. These two epitopes are also shown in the mature EV71 virion (PDB ID:3VBS).

Mentions: Previous studies reported that immunization with the synthetic peptide SP70 of EV71-VP1 elicited cross-neutralizing antibodies in vitro and that vaccination with this peptide confers cross-protection in vivo against homologous and heterologous EV71 strains in suckling BALB/c mice1623. We showed that immunization with the HBc-VP2 (aa141-155) particles conferred 100% in vivo passive protection against EV71 infection21. These two epitopes are located in the GH loop of VP1 and EF loop of VP2, respectively, which are exposed on the surface of the EV71 mature virus structure (PDB: 3VBS) (Fig. 1D). We sought to determine whether a bivalent chimeric VLPs vaccine presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 would elicit a stronger immunogenic response than that elicited by VLPs containing a single epitope. In this study, the EV71-VP2 epitope (aa 141-155) and EV71-VP1 epitope (aa 208-222) were linked by two copies of a flexible decapeptide linker (G4SG4S), which was inserted into the HBc protein (amino acids 1–149) at the aa 78 and 83 sites, and expressed in E. coli. Accordingly, three constructs, designated HBc-E1, HBc-E2 and HBc-E1/2, were generated (Fig. 1A). To determine whether chimeric VLPs expressed the VP1 and VP2 epitopes, purified recombinant proteins were evaluated by Western blot analysis with nMAb BB1A5 and H3B10 (Fig. 1B), as well as by negative staining electron microscopy (Fig. 1C). SDS-PAGE analyses show that the molecular mass of HBc-E1/2 is slightly higher than those of HBc-E1 and HBc-E2 (Fig. 1B). Furthermore, the chimeric HBc-E1/2 VLPs were precipitated by both nMAbs BB1A5 and H3B10, suggesting the efficient presentation of VP1 and VP2 epitopes. As expected, specific reactivity with nMAb BB1A5 or H3B10 was detected for the HBc-E1 or HBc-E2 proteins, respectively (Fig. 1B). To directly confirm the efficient particle formation, the HBc-E1/2, HBc-E1, HBc-E2 and HBc (aa1-149) preparations were subjected to negative staining electron microscopy (EM). Empty particles with a diameter of 30 nm were observed for all proteins (Fig. 1C). In addition, these recombinant particles, whose structure was constructed using the crystal structure of the HBV capsid (PDB: 4G93) as a template, were located on the surface of HBc VLPs (Fig. 1D). These data demonstrate that HBc-E1/2, HBc-E1 and HBc-E2 fusion proteins self-assemble into chimeric VLPs presenting VP1 (aa208-222) and VP2 (aa141-155) epitopes.


A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71.

Xu L, He D, Yang L, Li Z, Ye X, Yu H, zhao H, Li S, Yuan L, Qian H, Que Y, Shih JW, Zhu H, Li Y, Cheng T, Xia N - Sci Rep (2015)

Analysis of chimeric VLPs.(A) Schematic presentation of the chimeric HBc protein construct. (B) SDS-PAGE and Western blot analyses of the E. coli-expressed HBc fusion proteins. Lane M, molecular mass marker; Lane 1, HBc-E1/2; lane 2, HBc-E1; lane 3, HBc-E2; lane 4, HBc (aa1-149). (C) Electron microscopy of HBc-E1/2, HBc-E1, HBc-E2 and HBc(aa1-149) VLPs. (D) Molecular modeling of chimeric VLPs and the VP1(aa208-222) and VP1(aa141-155) epitopes located on the surface of HBc VLPs colored by red and green, respectively. These two epitopes are also shown in the mature EV71 virion (PDB ID:3VBS).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525384&req=5

f1: Analysis of chimeric VLPs.(A) Schematic presentation of the chimeric HBc protein construct. (B) SDS-PAGE and Western blot analyses of the E. coli-expressed HBc fusion proteins. Lane M, molecular mass marker; Lane 1, HBc-E1/2; lane 2, HBc-E1; lane 3, HBc-E2; lane 4, HBc (aa1-149). (C) Electron microscopy of HBc-E1/2, HBc-E1, HBc-E2 and HBc(aa1-149) VLPs. (D) Molecular modeling of chimeric VLPs and the VP1(aa208-222) and VP1(aa141-155) epitopes located on the surface of HBc VLPs colored by red and green, respectively. These two epitopes are also shown in the mature EV71 virion (PDB ID:3VBS).
Mentions: Previous studies reported that immunization with the synthetic peptide SP70 of EV71-VP1 elicited cross-neutralizing antibodies in vitro and that vaccination with this peptide confers cross-protection in vivo against homologous and heterologous EV71 strains in suckling BALB/c mice1623. We showed that immunization with the HBc-VP2 (aa141-155) particles conferred 100% in vivo passive protection against EV71 infection21. These two epitopes are located in the GH loop of VP1 and EF loop of VP2, respectively, which are exposed on the surface of the EV71 mature virus structure (PDB: 3VBS) (Fig. 1D). We sought to determine whether a bivalent chimeric VLPs vaccine presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 would elicit a stronger immunogenic response than that elicited by VLPs containing a single epitope. In this study, the EV71-VP2 epitope (aa 141-155) and EV71-VP1 epitope (aa 208-222) were linked by two copies of a flexible decapeptide linker (G4SG4S), which was inserted into the HBc protein (amino acids 1–149) at the aa 78 and 83 sites, and expressed in E. coli. Accordingly, three constructs, designated HBc-E1, HBc-E2 and HBc-E1/2, were generated (Fig. 1A). To determine whether chimeric VLPs expressed the VP1 and VP2 epitopes, purified recombinant proteins were evaluated by Western blot analysis with nMAb BB1A5 and H3B10 (Fig. 1B), as well as by negative staining electron microscopy (Fig. 1C). SDS-PAGE analyses show that the molecular mass of HBc-E1/2 is slightly higher than those of HBc-E1 and HBc-E2 (Fig. 1B). Furthermore, the chimeric HBc-E1/2 VLPs were precipitated by both nMAbs BB1A5 and H3B10, suggesting the efficient presentation of VP1 and VP2 epitopes. As expected, specific reactivity with nMAb BB1A5 or H3B10 was detected for the HBc-E1 or HBc-E2 proteins, respectively (Fig. 1B). To directly confirm the efficient particle formation, the HBc-E1/2, HBc-E1, HBc-E2 and HBc (aa1-149) preparations were subjected to negative staining electron microscopy (EM). Empty particles with a diameter of 30 nm were observed for all proteins (Fig. 1C). In addition, these recombinant particles, whose structure was constructed using the crystal structure of the HBV capsid (PDB: 4G93) as a template, were located on the surface of HBc VLPs (Fig. 1D). These data demonstrate that HBc-E1/2, HBc-E1 and HBc-E2 fusion proteins self-assemble into chimeric VLPs presenting VP1 (aa208-222) and VP2 (aa141-155) epitopes.

Bottom Line: We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not.Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16.These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Diagnostics and Vaccine Development in infectious diseases, School of Public Health, Xiamen University, Xiamen 361102, PR China.

ABSTRACT
Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

No MeSH data available.


Related in: MedlinePlus