A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

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.

Intranasal immunization of baculovirus displayed hemagglutinin confers complete protection against mouse adapted highly pathogenic H7N7 reassortant influenza virus.

BACKGROUND: Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA). The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n.) or subcutaneously (s.c.) with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs. CONCLUSION: Our results indicated that protection from high pathogenic H7N7 (NL/219/03) virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections.

Immune responses to baculovirus-displayed enterovirus 71 VP1 antigen.

The increased distribution and neurovirulence of enterovirus 71 is an important health threat for young children in Asia Pacific. Vaccine design has concentrated on inactivated virus with the most advanced undergoing Phase III clinical trials. By using a subunit vaccine approach, production costs could be reduced by lowering the need for biocontainment. In addition, novel mutations could be rapidly incorporated to reflect the emergence of new enterovirus 71 subgenogroups. To circumvent the problems associated with conventional subunit vaccines, the antigen can be displayed on a viral vector that conveys stability and facilitates purification. Additional advantages of viral-vectored subunit vaccines are their ability to stimulate the innate immune system by transducing cells and the possibility of oral or nasal delivery, which dispenses with the need for syringes and medical personnel. Baculovirus-displayed VP1 combines all these benefits with protection that is as efficient as inactivated virus.

Recombinant baculovirus associated with bilosomes as an oral vaccine candidate against HEV71 infection in mice.

BACKGROUND: Human enterovirus 71 (HEV71) is one of the major pathogen responsible for hand, foot and mouth disease (HFMD). Currently no effective vaccine or antiviral drugs are available. Like poliovirus, EV71 is transmitted mainly by the feco-oral route. To date the majority of the studied EV71 vaccine candidates are administered parenterally. Injectable vaccines induce good systemic immunity but mucosal responses are often unsatisfactory, whereas mucosal vaccines provide both systemic and mucosal immunity. Therefore, oral immunization appears to be an attractive alternative to parenteral immunization. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we studied the efficacy of an orally administered vaccine candidate developed using recombinant baculovirus displaying VP1 (Bac-VP1) in a murine model. Gastrointestinal delivery of Bac-VP1 significantly induced VP1-specific humoral (IgG) and mucosal (IgA) immune responses. Further, we studied the efficacy of the Bac-VP1 associated with bilosomes and observed that the Bac-VP1 associated with bilosomes elicited significantly higher immune responses compared to bilosomes non-associated with Bac-VP1. However, mice immunized subcutaneously with live Bac-VP1 had significantly enhanced VP1 specific serum IgG levels and higher neutralizing antibody titers compared with mice orally immunized with live Bac-VP1 alone or associated with bilosomes. CONCLUSION: Bilosomes have been shown to possess inherent adjuvant properties when associated with antigen. Therefore Bac-VP1 with bilosomes could be a promising oral vaccine candidate against EV71 infections. Thus, Bac-VP1 loaded bilosomes may provide a needle free, painless approach for immunization against EV71, thereby increasing patient compliance and consequently increasing vaccination coverage.

Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

BACKGROUND: Human 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 mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

RNA polymerase I-driven reverse genetics system for enterovirus 71 and its implications for vaccine production.

BACKGROUND: Enterovirus 71 (EV71) is a virus that causes from mild hand, foot and mouth disease (HFMD) to severe neurological complications and deaths in infants and young children. Effective antiviral agents and vaccines against EV71 are not available. However, Vero cell-based chemically inactivated EV71 vaccines could be developed soon based on the success of inactivated polio vaccine. Like poliovirus, EV71 has a positive single-stranded RNA genome of about 7400 nucleotides which contains a single open reading frame (ORF) flanked by conserved and untranslated regions at both the 5' and 3' ends. RESULTS: The universal amplification of the full length genome of EV71 regardless of its genetic diversity, and the subsequent construction of a human RNA polymerase I-driven reverse genetics (RG) system to produce pure virus stocks in Vero cell within 10 days were described. The rescued viruses were characterized by DNA sequencing, cytopathic effect (CPE) and indirect fluorescent assay (IFA) in comparison with the wild-type viruses. Moreover, the rescued viruses grew to high titers and retained the same immunogenicity as the wild-type viruses. CONCLUSION: We have established a simplified method to rescue RG EV71 virus from diverse clinical isolates with detailed genetic information and to prepare virus stocks in only 10 days. This method could accelerate EV71 vaccine development.

Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice.

Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein.

BACKGROUND: Enterovirus 71 (EV71) has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. RESULTS: We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP) of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain) did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. CONCLUSIONS: Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non-neutralizing epitope, spanning amino acids 142-146 which are located in the VP2 protein's E-F loop. The S/T(144) mutation in this epitope confers a loss of VP2 antigenicity to some newly emerged EV71-C4 strains from China. The corresponding monoclonal antibody 7C7 was used successfully in an AC-ELISA and did not cross-react to coxsackieviruses 4, 6, 10, and 16 in immunofluorescence assay and Western blots. 7C7 is the first monoclonal antibody described, that can differentiate Coxsackievirus 16 from Enterovirus 71.

Characterization of a monoclonal antibody against the 3D polymerase of enterovirus 71 and its use for the detection of human enterovirus A infection.

Over the last decade, frequent epidemic outbreaks of hand, foot and mouth disease have been observed in the Asia-Pacific region. Hand, foot and mouth disease is caused by different viruses from the enterovirus family, mainly coxsackievirus A16 and enterovirus 71 (EV71) from the human enterovirus A family. Severe disease and neurological complications are associated more often with EV71 infection, and can lead occasionally to fatal brain stem encephalitis in young children. The rapid progression and high mortality of severe hand, foot and mouth disease makes the direct detection of antigens early in infection essential. The best method for virus detection is the use of specific monoclonal antibodies. The generation and characterization of a monoclonal antibody specific for the 3D polymerase of human enterovirus A and the development of a virus detection dot blot assay are described. A recombinant 3CD protein from EV71 C4 strain was used as an immunogen to generate monoclonal antibodies (MAbs). Screening of hybridoma cells led to the isolation of monoclonal antibody 4B12 of the immunoglobulin IgG1 isotype. MAb 4B12 recognizes the linear epitope DFEQALFS close to the active site of the 3D polymerase, corresponding to amino acid positions 53-60 of 3D and 1784-1791 of enterovirus 71 polyprotein. The presence of 3D polymerase and its precursor 3CD proteinase in purified virus particles was confirmed. MAb 4B12 was used successfully to detect all enterovirus 71 subgenotypes in a denaturing dot blot assay with a sensitivity of 10 pg of 3D protein and 10(4) tissue culture infective dose of virus particles.

Display of VP1 on the surface of baculovirus and its immunogenicity against heterologous human enterovirus 71 strains in mice.

BACKGROUND: Human 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 FINDING: In 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. CONCLUSION: Our 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.

Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos.

TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development.

Identification, tissue distribution and developmental expression of tjp1/zo-1, tjp2/zo-2 and tjp3/zo-3 in the zebrafish, Danio rerio.

The tight junction (TJ) or zona occludens (ZO) proteins TJP1/ZO-1, TJP2/ZO-2 and TJP3/ZO-3 belong to the membrane associated guanylate kinase-like (MAGUK) protein family and link TJ integral membrane proteins to the actin cytoskeleton. TJPs also serve as scaffolds for signaling proteins and transcription factors that regulate vesicular traffic and cell proliferation and differentiation. Here, we report the identification of two tjp1/zo-1 (tjp1.1 and tjp1.2) and tjp2/zo-2 (tjp2.1 and tjp2.2) genes each and one tjp3/zo-3 gene, and characterize their tissue specific distribution and developmental expression in zebrafish embryos. Transcripts for all five TJPs are maternally supplied and localized expression due to embryonic transcription is observed following the midblastula transition stage of development. In addition to a widespread distribution, individual genes show tissue specific expression patterns and a dynamic regulation during the developmental stages from 2 cells to 4 dpf analyzed. The most noticeable differences in expression patterns are observed in the posterior part of the embryo during somitogenesis. While all TJPs are expressed in the pronephric ducts and epidermis by 18 hpf, tjp1.1 is highly expressed in the hypochord and blood island, tjp1.2 in the somites and the posterior part of the notochord, tjp2.1 in the somites and the ventral part of the spinal cord, and tjp2.2 in the somites only. Individual TJPs are also strongly expressed in different layers of the eye and, at later stages, in central nervous system (CNS) tissues. Interestingly, the differential tissue and developmental expression of the two tjp1 and tjp2 genes indicates that the duplicated genes have been adapted for distinct transcriptional regulations during evolution.