In a screen for ribavirin resistance, a novel high-fidelity variant of human enterovirus 71 (EV71) with the single amino acid change L123F in its RNA-dependent RNA polymerase (RdRp or 3D) was identified. Based on the crystal structure of EV71 RdRp, L123 locates at the entrance of the RNA template binding channel, which might form a fidelity checkpoint. EV71 RdRp-L123F variants generated less progeny in a guanidine resistance assay and virus populations with lower mutation frequencies in cell culture passage due to their higher replication fidelity. However, compared with wild-type viruses, they did not show growth defects. In vivo infections further revealed that high-fidelity mutations L123F and G64R (previously reported) negatively impacted EV71 fitness and greatly reduced viral pathogenicity alone or together in AG129 mice. Interestingly, a variant with double mutations, RG/B4-G64R/L123F (where RG/B4 is an EV71 genotype B4 virus constructed by reverse genetics [RG])showed higher fidelity in vitro and less virulence in vivo than any one of the above two single mutants. The 50% lethal dose (LD 50 ) of the double mutant increased more than 500 times compared with the LD 50 of wild-type RG/B4 in mice. The results indicated that these high-fidelity variants exhibited an attenuated pathogenic phenotype in vivo and offer promise as a live attenuated EV71 vaccine. IMPORTANCEThe error-prone nature of the RNA-dependent RNA polymerase (RdRp) of RNA viruses during replication results in quasispecies and aids survival of virus populations under a wide range of selective pressures. Virus variants with higher replication fidelity exhibit lower genetic diversity and attenuated pathogenicity in vivo. Here, we identified a novel high-fidelity mutation L123F in the RdRp of human enterovirus 71 (EV71). We further elucidated that EV71 variants with the RdRp-L123F mutation and/or the previously identified high-fidelity mutation RdRp-G64R were attenuated in an AG129 mouse model. As EV71 has emerged as a serious worldwide health threat, especially in developing countries in the Asia-Pacific region, we urgently need EV71 vaccines. Learning from the poliovirus vaccination, we prefer live attenuated EV71 vaccines to inactivated EV71 vaccines in order to effectively control EV71 outbreaks at low cost. Our results imply a new means of attenuating EV71 and reducing its mutation rate at the same time.

The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection. Particularly, variations within the amino acid sequences of major neutralizing epitopes of influenza virus hemagglutinin (HA) hindered the development of universal vaccines against H5N1 lineages. Based on distribution analyses of the identified major neutralizing epitopes of hemagglutinin, we selected three vaccine strains that cover the entire variants in the neutralizing epitopes among the H5N1 lineages. HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the preclinical efficacy of the vaccine formulations was evaluated in a mouse model. The combination of three selected vaccine strains could effectively neutralize viruses from clades 1, 2.1, 2.2, 4, 7, and 8 of influenza H5N1 viruses. In contrast, a vaccine formulation containing only adjuvanted monovalent BacHA (mono-BacHA) or a single strain of inactivated whole viral vaccine was able to neutralize only clade 1 (homologous), clade 2.1, and clade 8.0 viruses. Also, the trivalent BacHA vaccine was able to protect 100% of the mice against challenge with three different clades (clade 1.0, clade 2.1, and clade 7.0) of H5N1 strains compared to mono-BacHA or inactivated whole viral vaccine. The present findings provide a rationale for the development of a universal vaccine against H5N1 lineages. Furthermore, baculoviruses displaying HA will serve as an ideal choice for a vaccine in prepandemic or pandemic situations and expedite vaccine technology without the requirement of high-level-biocontainment facilities or tedious protein purification processes.

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the “knob” region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

BackgroundHuman Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization.Methodology/Principal FindingIn the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains.ConclusionOur results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns.