Identification and characterization of a monoclonal antibody recognizing the linear epitope RVADVI on VP1 protein of enterovirus 71

Abstract

Several large outbreaks of hand-foot-mouth disease (HFMD) have occurred in the Asian-Pacific region since 1997, with Enterovirus 71 (EV71) and/or Coxsackievirus A16 (CAV16) as the main causative agents. Despite the close genetic relationship between the two viruses, only EV71 is associated with severe clinical manifestations and deaths. Effective antiviral treatment and vaccines are not available. High-quality monoclonal antibodies (mAbs) are necessary to improve the accuracy of the diagnosis of EV71. In this study, a mAb (designated 1D9) was generated using EV71 C5 strain virus particles as immunogens. Examined by indirect immunofluorescence assay (IFA) and Western blotting, 1D9 detected successfully all 11 subgenotypes of EV71 and showed no cross-reactivity to the four selected subgenogroups of Coxsackieviruses CAV4, CAV6, CAV10, and CAV16. A linear motif, R(3) VADVI(8), which is located at the N-terminus of the EV71 VP1 protein, was identified as the minimal binding region of 1D9. Alignment and comparison of the 1D9-defined epitope sequence against the listed sequences in the NCBI EV71 database indicated that this epitope R(3) VADVI(8) was highly conserved among EV71 strains, while no significant similarity was observed when blasted against the Coxsackieviruses. This suggests that the mAb 1D9 may be useful for the development of a cost-effective and accurate method for surveillance and early differentiation of EV71 from CAV16 infection.

Monoclonal Antibodies for Diagnosis of Enterovirus 71

“…The results were comparable to other findings in which neutralization of EV71 infection can be mediated by either linear epitopedriven MAbs (8,(14)(15)(16)(17)22,23) or conformational epitope-dependent ones. (24) According to the cross-reactivities of MAbs 22 and 24 to many EV71 subgenotypes (MAb 24 could react with B4, B5, C2, and C4, while MAb 22 could react to B5, C2, and C4), both MAbs 22 and 24 are valuable in the development of vaccine candidates.…”

“…VP1, the major outer surface protein with 297 residues on EV71 virion, has been thought to be the major protein of EV71 for boosting the neutralizing antibodies and within it several linear epitopes were identified. (8,(11)(12)(13)(14)(15)(16)(17) However, except for MAb 27, none of these clones was applicable in Western blot with the reduced specimens, implying that they may recognize the conformational epitopes, instead of the linear ones identified previously. The finding was also confirmed by capture ELISA, where EV71 particles captured by rabbit anti-EV71 polyclonal antibodies could be recognized by individual monoclonal antibodies, respectively (Table 1).…”

“…(8) Consistent with the Western blot analysis results, only MAb 27 showed positive for the linear epitope DVIESSIGDSVSRAL, which was located at the N-terminus (a.a. 6-20) of EV71 VP1 protein and had also been identified and recognized by MAb 4, (17) MAb 1D9, (16) and N16 MAb, (21) respectively. The BLAST analysis revealed that this epitope is highly conserved among all EV71 genotypes and relatively specific for EV71 organism, which implies that MAb 27 recognizing this epitope might have great value in the laboratory diagnosis of EV71 infection.…”

VP2 Dominated CD4+ T Cell Responses against Enterovirus 71 and Cross-Reactivity against Coxsackievirus A16 and Polioviruses in a Healthy Population

“…Moreover, a collection of studies demonstrate that VP1 is the dominant target of neutralizing Abs in EV71, and thus, VP1 has been broadly explored for subunit vaccine development, priming strong humoral immunity in a mouse model (15,52,53). However, compared with an N-terminal-biased neutralizing determinants distribution in humans, distribution of neutralizing Ab epitopes in mouse model reveals no N-or C-terminal bias, and numbers of epitopes are identified in the C-terminal of VP1 in mouse model (52,54,55). Besides, the pathology of EV71 infection in mice and humans is also quite different (56).…”

Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

“…However, VP1 21–100 , truncated 20 aa from N-terminus, reacted weakly. Recently, two non-neutralizing linear epitopes were found at positions 3–8 and 12–19 aa of VP1 (Lim et al, 2013; Man-Li et al, 2012). The truncated proteins (VP1 6–100 , VP1 11–100 ) do not contain a complete copy of the 3–8 epitope.…”