Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.

The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.

Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice.

BACKGROUND: Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection. RESULTS: In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice. CONCLUSION: Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.

Complete genome sequence analysis of human echovirus type 30 isolated in China.

We report here the complete genome sequence of a human echovirus type 30 strain ECV30/GX10/05 isolated in Guangxi, China, in 2010. Phylogenetic analysis showed that ECV30/GX10/05 was closely related to a Korean strain isolated in 2008. The sequence information will help in an understanding of the molecular epidemiology and evolution of echovirus.

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.

Human enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease in young children and infants. EV71 can cause various clinical manifestations and has been associated with severe neurological complications; it has resulted in fatalities during recent outbreaks in Asian-Pacific regions since 1997. The early and rapid detection is critical for prevention and control of EV71 infection, since no vaccine or antiviral drugs are currently available. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of EV71. The detection limit of the RT-LAMP assay was approximately 0.01 PFU per reaction mixture, and no cross-reactive amplification with other enteroviruses was observed. The assay was evaluated further with 40 clinical specimens and exhibited 92.9% sensitivity and 100% specificity. This RT-LAMP assay may become a useful alternative in clinical diagnosis of EV71, especially in resource-limited hospitals or rural clinics of China and other countries in the Asian-Pacific region.

Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo.

BACKGROUND: Human enterovirus 71 (EV71) has emerged as a significant cause of acute encephalitis and deaths in young children. The clinical manifestations caused by EV71 varied from mild hand, foot and mouth disease to severe neurological complications and deaths, but its pathogenesis remains elusive. Antibody dependent enhancement (ADE) infection has been reported in various viruses and has been shown to contribute to disease severity. RESULTS: In this study, the presence of sub-neutralizing antibody was demonstrated to enhance EV71 infection in THP-1 cells and increase the mortality of EV71 infection in a suckling mouse model. Further, a secondary infection model was established to characterize the correlation between ADE and disease severity, and primary asymptomatic EV71 infection was shown to increase the mortality of the secondary EV71 infection in suckling mice. CONCLUSIONS: Together, these in vitro and in vivo experiments strongly supported the hypothesis of ADE infection of EV71. The present findings indicate ADE might contribute to the pathogenesis of severe EV71 infection, and raise practical issues of vaccine development and antibody-based therapy.

Producing infectious enterovirus type 71 in a rapid strategy.

BACKGROUND: Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends. RESULTS: A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. CONCLUSIONS: Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method.

Pediatric Drug Nitazoxanide: A Potential Choice for Control of Zika.

Zika virus (ZIKV) infection can be the cause of congenital malformations, including microcephaly in infants and can cause other disorders such as Guillain-Barré syndrome, meningoencephalitis, and myelitis, which can also occur in some infected adults. However, at this time, there is no drug approved to treat ZIKV infection. Drug repurposing is the promptest way to obtain an effective drug during a global public health emergency such as the spread of Zika virus. In this study, we report a US Food and Drug Admistration-approved drug that is safe for pediatric use. Nitazoxanide and its bioactive metabolite, tizoxanide, have anti-ZIKV potential in vitro, and we identified that they exerts antiviral effect possibly by targeting the viral postattachment step.

Design, synthesis and biological evaluation of novel HSP70 inhibitors: N, N'-disubstituted thiourea derivatives.

As novel heat shock protein 70 (HSP70) inhibitors, N, N'-disubstituted thiourea derivatives were designed and synthesized based on the X-ray structure of the ATPase domain (nucleotide binding domain, NBD). An ATPase activity inhibition assay revealed that these compounds effectively inhibited HSP70 ATPase activity. The results revealed that the compounds 370/371/374/379/380//392/394/397/404/405 and 407 can inhibit the HSP70 ATPase turnover with high percentages of inhibition: 50.42, 38.46, 50.45, 44.12, 47.13, 50.50, 40.95, 65.36, 46.23, 35.78, and 58.37 in 200 µM, respectively. Significant synergies with lapatinib were observed for compound 379 and compound 405 in the BT474 breast cancer cell line. A structure-function analysis revealed that most of the thiourea derivatives exhibited cooperative action with lapatinib in the BT474 cancer cell line and the BT/Lap(R)1.0 lapatinib-resistant cell line. HSP70 inhibitors may be developed as synergetic drugs in drug-resistant cancer therapy.

Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening.

Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 µM without apparent toxicity (CC50 > 200 µM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 µM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 µM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 µM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.

Inhibition of novel reassortant avian influenza H7N9 virus infection in vitro with three antiviral drugs, oseltamivir, peramivir and favipiravir.

BACKGROUND: A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from three patients and was identified as H7N9 in China. Antiviral agents are required to treat patients with avian influenza H7N9 virus infection. METHODS: In this study, we assessed the antiviral potential of oseltamivir, peramivir, favipiravir (T-705), amantadine and rimantadine against novel reassortant avian-origin influenza H7N9 virus in vitro. RESULTS: All three avian influenza H7N9 virus strains were sensitive to oseltamivir, peramivir and favipiravir (T-705), but resistant to amantadine and rimantadine. CONCLUSIONS: Our data show a pattern of antiviral sensitivity for this novel H7N9 strain of influenza that suggests the compounds oseltamivir, peramivir and favipiravir should be useful for therapy.

Isolation and identification of phase I metabolites of phillyrin in rats.

Phillyrin was one of the main chemical constituents of the fruit of Forsythia suspensa (Thunb.) Vahl. It showed various bioactivities including antioxidant and anti-inflammatory activities. However, the metabolism of phillyrin remained unknown. This report described the isolation and identification of phase I metabolites of phillyrin in rats. Nine metabolites including six new ones were isolated by various column chromatographies and high-performance liquid chromatography. Their structures were elucidated by extensive spectroscopic analysis. The antiviral activities of phillyrin and the metabolites were evaluated against influenza A (H3N2) virus. Among them, one metabolite M8 showed moderate activity with the IC50 value of 26.39 µM, and three metabolites (M2, M3, M9) showed weak antiviral activities at the concentration of 100 µM. Based on the structures of the metabolites, possible metabolic pathways of phillyrin in rats were also proposed.

Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice.

Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

Human IgG subclasses against enterovirus Type 71: neutralization versus antibody dependent enhancement of infection.

The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.

In vitro and in vivo characterization of a new enterovirus type 71-specific human intravenous immunoglobulin manufactured from selected plasma donors.

BACKGROUND: Enterovirus type 71 (EV71) causes large outbreaks with significant mortality among young children, and no specific antiviral treatment is currently available. Antibody-based therapy represents a promising alternative strategy for lethal EV71 infection. Our previous data has shown that anti-EV71 neutralization antibodies were present in a significant proportion of blood donors in China. OBJECTIVES: To produce a new human intravenous immunoglobulin (IVIG) product containing high titer anti-EV71 neutralizing antibodies and investigate its therapeutic efficacy against lethal EV71 infection in a murine model. STUDY DESIGN: Plasma units that contained high titer neutralizing antibodies from selected Chinese donors were pooled and processed into pharmaceutical grade IVIG preparations according to the standard procedure. The efficacy of these EV71-specific IVIG product was characterized in vitro by neutralization assay and in vivo by suckling mouse protection testing. The therapeutic effects against lethal EV71 challenge were further assayed in a suckling mouse model. RESULTS: About 12% of the normal plasma units were selected and pooled to manufacture the EV71-IVIG preparations, and in vitro and in vivo efficacy data showed that these EV71-specific IVIG preparations were enriched with neutralizing antibodies against EV71. Furthermore, treatment with EV71-specific IVIG was evidenced to confer protection against lethal EV71 challenge in a dose- and time-dependent manner in the suckling mouse model. CONCLUSIONS: This preclinical study indicates that these "tailor-made" EV71-IVIG preparations manufactured from selected plasma donors in EV71-endemic areas may represent a promising therapeutic option for the lethal EV71 infections, and further clinical trials should be warranted in the future.

CpG oligodeoxynucleotides protect against the 2009 H1N1 pandemic influenza virus infection in a murine model.

The 2009 H1N1 influenza virus pandemic poses a global public health threat, and there is a critical need for antiviral drugs for pandemic control. CpG oligodeoxynucleotides have strong immunostimulatory properties and are expected to be used as prophylactic agents to protect against microbial infections. The present study evaluated the efficacy of synthetic CpG oligodeoxynucleotide (ODN) 1826 against pandemic H1N1 virus infection in a murine model. A single injection of 15 µg ODN 1826 intraperitoneally prior to virus challenge inhibits virus replication in lungs, reduces lung lesions and prevents mortality in mice, indicating CpG ODNs as a possible strategy for future influenza pandemics control.