Novel TFG mutation causes autosomal-dominant spastic paraplegia and defects in autophagy.

BACKGROUND: Mutations in the tropomyosin receptor kinase fused (TFG) gene are associated with various neurological disorders, including autosomal recessive hereditary spastic paraplegia (HSP), autosomal dominant hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) and autosomal dominant type of Charcot-Marie-Tooth disease type 2. METHODS: Whole genome sequencing and whole-exome sequencing were used, followed by Sanger sequencing for validation. Haplotype analysis was performed to confirm the inheritance mode of the novel TFG mutation in a large Chinese family with HSP. Additionally, another family diagnosed with HMSN-P and carrying the reported TFG mutation was studied. Clinical data and muscle pathology comparisons were drawn between patients with HSP and patients with HMSN-P. Furthermore, functional studies using skin fibroblasts derived from patients with HSP and patients with HMSN-P were conducted to investigate the pathomechanisms of TFG mutations. RESULTS: A novel heterozygous TFG variant (NM_006070.6: c.125G>A (p.R42Q)) was identified and caused pure HSP. We further confirmed that the well-documented recessively inherited spastic paraplegia, caused by homozygous TFG mutations, exists in a dominantly inherited form. Although the clinical features and muscle pathology between patients with HSP and patients with HMSN-P were distinct, skin fibroblasts derived from both patient groups exhibited reduced levels of autophagy-related proteins and the presence of TFG-positive puncta. CONCLUSIONS: Our findings suggest that autophagy impairment may serve as a common pathomechanism among different clinical phenotypes caused by TFG mutations. Consequently, targeting autophagy may facilitate the development of a uniform treatment for TFG-related neurological disorders.

Preparation of pectin-coated and chitosan-coated phenylethanoside liposomes: Studies on characterization, stability, digestion and release behavior.

In this paper, the effects of extrusion, ultrasound on physicochemical properties of liposomes were studied, and the liposomes were prepared by ethanol injection combined with extrusion-ultrasound. In addition, the quality of PhGs lips, pectin-coated PhGs lips (P-lips) and chitosan-coated PhGs lips (C-lips) was evaluated by the average particle size, encapsulation efficiency (EE) and other indicators, which indicated that the nanoparticles had been successfully prepared. Compared with extrusion or ultrasonic operation alone, the EEs of ethanol injection combined with extrusion-ultrasonic increased by 8 % and 18 % respectively. Subsequently, transmission electron microscopy, Fourier transform infrared spectroscopy and DSC thermal analysis showed that PhGs in PhGs lips may produce hydrogen bonding forces with phospholipids, and pectin and chitosan in P-lips and C-lips were not only coated on the surface of PhGs lips, but also might have some interaction between them. Cell experiments showed that PhGs lips, P-lips and C-lips can effectively improve the bioavailability of PhGs. In addition, the storage stability of P-lips and C-lips was not significantly improved compared to PhGs lips, but their digestive stability was significantly improved, and the final retention rate in simulated intestinal fluid was about 25 % higher than that of PhGs lips.

Polyampholytes and Their Hydrophobic Derivatives as Excipients for Suppressing Protein Aggregation.

Protein aggregation, which occurs under various physiological conditions, can affect cell function and is a major issue in the field of protein therapeutics. In this study, we developed a polyampholyte composed of ε-poly-l-lysine and succinic anhydride and evaluated its protein protection efficacy. This polymer was able to protect different proteins from thermal stress and its performance significantly exceeded that of previously reported zwitterionic polymers. In addition, we synthesized derivatives with varying degrees of hydrophobicity, which exhibited remarkably enhanced efficiency; thus, the polymer concentration required for protein protection was very low. By facilitating the retention of protein enzymatic activity and stabilizing the higher-order structure, these polymers enabled the protein to maintain its native state, even after being subjected to extreme thermal stress. Thus, such polyampholytes are extremely effective in protecting proteins from extreme stress and may find applications in protein biopharmaceuticals and drug delivery systems.

Polyampholyte-Based Polymer Hydrogels for the Long-Term Storage, Protection and Delivery of Therapeutic Proteins.

Protein storage and delivery are crucial for biomedical applications such as protein therapeutics and recombinant proteins. Lack of proper protocols results in the denaturation of proteins, rendering them inactive and manifesting undesired side effects. In this study, polyampholyte-based (succinylated ε-poly-l-lysine) hydrogels containing polyvinyl alcohol and polyethylene glycol polymer matrices to stabilize proteins are developed. These hydrogels facilitated the loading and release of therapeutic amounts of proteins and withstood thermal and freezing stress (15 freeze-thaw cycles and temperatures of -80 °C and 37 °C), without resulting in protein denaturation and aggregation. To the best of our knowledge, this strategy has not been applied to the design of hydrogels constituting polymers, (in particular, polyampholyte-based polymers) which have inherent efficiency to stabilize proteins and protect them from denaturation. Our findings can open up new avenues in protein biopharmaceutics for the design of materials that can store therapeutic proteins long-term under severe stress and safely deliver them.

Lead-imprinted polyvinylidene fluoride membrane for selective removal of lead from contaminated water: material fabrication, filtration application, and mechanism study.

The drinking water has become contaminated with lead in many countries across the world. In this study, a novel lead-imprinted polyvinylidene fluoride (PVDF) membrane was successfully fabricated for selective decontamination of lead from water. First of all, the membrane fabrication process was explored and optimized. The physical and chemical properties were then studied for a better understanding of the features of the membrane. The performance of lead removal by the adsorptive membrane was evaluated by systematic batch adsorption experiments, including pH effect, kinetics, isotherm, selectivity, and regeneration studies. The results indicated that the adsorptive membrane showed a high adsorption capacity of 40.59 mg Pb/g at the optimal pH of 5.5, fast kinetics of 2 h, high selectivity towards lead, and outstanding regeneration performance. The Langmuir equation fitted the isotherm better than the Freundlich equation, while the pseudo-second-order model and pore diffusion model well described the kinetics. The adsorptive membrane showed high selectivity towards lead in the lead/zinc binary solution. In the continuous filtration study, a small piece of adsorptive membrane could treat 3.75 L of lead solution. The XPS studies revealed that the lead uptake was mainly due to the complex reaction between lead and carboxyl and hydroxyl in the membrane.

Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Effects of coarse and fine bubble aeration on performances of membrane filtration and denitrification in moving bed membrane bioreactors.

In this study, two lab-scale Moving Bed Membrane Bioreactors (MBMBR) were setup and operated in parallel to study the effect of coarse and fine bubble aeration on the performances of membrane filtration and denitrification treating domestic wastewater. The bacterial populations in the two MBMBRs were further analyzed to investigate the mechanisms involved in the different denitrification performances. The results showed that coarse bubble aeration could effectively mitigate membrane fouling by decreasing the formation of cake layer, although smaller sizes of bio-flocs were induced. In addition, coarse bubble aeration could also maintain dissolved oxygen (DO) at a relatively lower level without compromising the moving of bio-carriers, which achieved 10% higher total nitrogen removal rate due to anoxic zone created at inner layers of biofilms on bio-carriers. Accumulation of denitrifier (Thiobacillus denitrificans) on the bio-carriers was found under the coarse bubble aeration system, which can explain its superior denitrification performance.

Histo-blood group antigens as divergent factors of groups A and C rotaviruses circulating in humans and different animal species.

Histo-blood group antigens (HBGAs) have been found to be important host susceptibility factors or receptors for human rotavirus (RVs) with genotype-specific host ranges, impacting the disease patterns, epidemiology, and strategy development against RV diseases in humans. However, how the glycan factors contribute to RV diversity and host ranges to different animal species remains unclear. In this study using recombinant VP8* proteins as probes to perform glycan array analyses of RVs, we observed a wide range of glycan-binding profiles, including those binding to sialic acid-containing glycans, among group A (RVA) and group C (RVC) RVs that mainly infect different animal species. A tri-saccharide glycan Galα1-3Galß1-4Glc containing a terminal α-Gal was recognised by multiple RVA/RVC genotypes, providing valuable information on RV evolution under selection of the step-wisely synthesised HBGAs in many animals before they were introduced to humans to be human pathogens. Saliva binding studies of VP8* also revealed strain-specific host ranges or species barriers between humans and these animal RV genotypes, further improved our understanding on RV host ranges, disease burdens, epidemiology, and vaccine strategy against RVs.

Dual Thermo- and pH-Responsive Behavior of Double Zwitterionic Graft Copolymers for Suppression of Protein Aggregation and Protein Release.

Graft copolymers consisting of two different zwitterionic blocks were synthesized via reversible addition fragmentation chain transfer polymerization. These polymers showed dual properties of thermo- and pH-responsiveness in an aqueous solution. Ultraviolet-visible spectroscopy and dynamic light scattering were employed to study the phase behavior under varying temperatures and pH values. Unlike the phase transition temperatures of other graft copolymers containing nonionic blocks, the phase transition temperature of these polymers was easily tuned by changing the polymer concentration. Owing to the biocompatible and stimuli-responsive nature of the polymers, this system was shown to effectively release proteins (lysozyme) while simultaneously protecting them against denaturation. The positively charged lysozyme was shown to bind with the negatively charged polymer at the physiological pH (pH 7.4). However, it was subsequently released at pH 3, at which the polymer exhibits a positive charge. Protein aggregation studies using a residual enzymatic activity assay, circular dichroism, and a Thioflavin T assay revealed that the secondary structure of the lysozyme was retained even after harsh thermal treatment. The addition of these polymers helped the lysozyme retain its enzymatic activity and suppressed its fibrillation. Both polymers showed excellent protein protection properties, with the negatively charged polymer exhibiting slightly superior protein protection properties to those of the neutral polymer. To the best of the authors' knowledge, this is the first study to develop a graft copolymer system consisting of two different zwitterionic blocks that shows dual thermo- and pH-responsive properties. The presence of the polyampholyte structure enables these polymers to act as protein release agents, while simultaneously protecting the proteins from severe stress.

Cytosolic delivery of quantum dots mediated by freezing and hydrophobic polyampholytes in RAW 264.7 cells.

Quantum dots (QDs) can be delivered efficiently inside macrophages using a freeze-concentration approach. In this study, we introduced a new, facile, high concentration-based freezing technology of low toxicity. We also developed QD-conjugated new hydrophobic polyampholytes using poly-l-lysine (PLL), a naturally derived polymer, which showed sustained biocompatibility, stability over one week, and enhanced intracellular delivery. When freeze-concentration was applied, the QD-encapsulated hydrophobic polyampholytes showed a higher tendency to adsorb onto the cell membrane than the non-frozen molecules. Interestingly, we observed that the efficacy of adsorption of QDs on RAW 264.7 macrophages was higher than that on fibroblasts. Furthermore, the intracellular delivery of QDs using hydrophobic polyampholytes was higher than those of PLL and QDs. In vitro studies revealed the efficient endosomal escape of QDs in the presence of hydrophobic polyampholytes and freeze-concentration. Collectively, these observations indicated that the promising combination of freeze-concentration and hydrophobic polyampholytes may act as an effective and versatile strategy for the intracellular delivery of QDs, which can be used for biological diagnosis and therapeutic applications.

Plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic HJT-sRNA-m7.

Pulmonary fibrosis, a progressive chronic disease with a high mortality rate, has limited treatment options. Currently, lung transplantation remains the only effective treatment. Here we report that a small RNA, HJT-sRNA-m7, from a Chinese herbal medicine Hong Jing Tian (HJT, RHODIOHAE CRENULATAE RADIX ET RHIZOMA, Rhodiola crenulata) can effectively reduce the expressions of fibrotic hallmark genes and proteins both in alveolar in vitro and in mouse lung tissues in vivo. We also discovered over one hundred oil-soluble chemicals from HJT decoctions, most of which are found in lipid extracts from other Chinese herbals decoctions, including Pu Gong Ying (PGY, TARAXACI HERBA, Taraxacum mongolicum), Chuan Xin Lian (CXL, changed to "ANDROGRAPHIS HERBA, Andrographis paniculata"), and Jin Yin Hua (JYH, lonicera japonica or Honeysuckle). We identified the active component in these decoctions as two forms of phosphocholines, PC (18:0/18:2) and PC (16:0/18:2). These PCs potentially could form liposomes with small RNAs to enter human alveolar and gastric cells. Our experimental results suggest an unprecendent lipid complex route through which botanic small RNA can enter human bodies. Our results provide an innovative treatment strategy for oral delivery of siRNAs as therapeutic medication.

Hyaluronic acid modified nanostructured lipid carriers for transdermal bupivacaine delivery: In vitro and in vivo anesthesia evaluation.

For effective transdermal local anesthetic therapy, to reduce the barrier of stratum corneum and improve the antinociceptive effect, hyaluronic acid (HA) modified, bupivacaine (BPV) loaded nanostructured lipid carriers (NLCs) were designed. HA and linoleic acid (LOA) conjugated propylene glycol (PEG) was synthesized (HA-PEG-LOA). HA-PEG-LOA was added during the preparation process of NLCs, thus LOA was inserted into the NLCs, The physicochemical properties of NLCs, particle size, zeta potential, drug loading capacity, in vitro skin permeation, drug release profiles and in vivo therapeutic effect were evaluated. HA-BPV/NLCs have small particle size of 150?nm, with a zeta potential of ?40?mV. Nearly 90% high drug encapsulation efficiency and good stability were also observed. In vitro release rate of BPV from HA-BPV/NLCs was complying with a sustained behavior until 72?h of study. HA-BPV/NLCs and BPV/NLCs exhibited 2.5 and 1.6 fold of percutaneous penetration improvement than free BPV. BPV loaded NLCs produced a more prolonged antinociceptive effect when compared with free BPV. In vitro and in vivo results pointed out HA modified NLCs have the capability to act as effective drug carriers, thus prolonging and enhancing the anesthetic effect of BPV. The NLCs developed in this study might provide a useful platform for developing a sophisticated dermal delivery system for analgesic.

Antilung cancer effect of ergosterol and cisplatin-loaded liposomes modified with cyclic arginine-glycine-aspartic acid and octa-arginine peptides.

BACKGROUND: Lung cancer is one of the most important diseases threatening human health, and targeted therapy has become the main research direction. This work, therefore, aimed to develop cyclic arginine-glycine-aspartic (RGD) and octa-arginine (R8) peptide-modified ergosterol (ERG)-combined cisplatin (diamminedichloridoplatinum(II) [DDP]) liposomes (LIP) as a drug delivery system. METHODS: Soybean phospholipids (SPC) and cholesterol (Chol) were selected to prepare different LIPs: ERG-loaded LIP (ERG-LIP), DDP and ERG-LIP (DDP/ERG-LIP), R8 peptide-modified DDP and ERG-LIP (R8-DDP/ERG-LIP), and cyclic RGD and R8-DDP/ERG-LIP (RGD/R8-DDP/ERG-LIP). The quality, tumor sphere penetrating ability, in vitro cellular uptake, mechanism of cellular uptake, and in vitro cytotoxicity of RGD/R8-DDP/ERG-LIP were evaluated. RESULTS: The LIP quality evaluation revealed that RGD/R8-DDP/ERG-LIP is round with a double-layer structure. The average particle size, dispersion coefficient of the polydispersity index (PDI), and zeta potential of RGD/R8-DDP/ERG-LIP were 155.2 ±â€Š8.7 nm, 0.102, and 4.74 ±â€Š0.7 mV, respectively. Furthermore, the LIPs were stable in the serum, and obviously inhibited the growth of A549 lung cancer cells with RGD/R8-DDP/ERG-LIP exhibiting the strongest inhibitory effect. The highest cellular uptake rate, which was at 4 hours, was exhibited by RGD/R8-DDP/ERG-LIP in a concentration-dependent manner. CONCLUSION: The results showed that LIP uptake by A549 cells was mainly by the clathrin-mediated endocytosis pathway (chlorpromazine). The results also suggest that RGD/R8-DDP/ERG-LIP might be a promising drug delivery system to improve antilung cancer drug effect and tumor-targeting in vitro.

Characterization of human enterovirus71 virus-like particles used for vaccine antigens.

Human enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease (HFMD) and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs), which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9) using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by in situ immunogold detection, electron microscopy and immunofluorescence. Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.

Zirconium/polyvinyl alcohol modified flat-sheet polyvinyldene fluoride membrane for decontamination of arsenic: Material design and optimization, study of mechanisms, and application prospects.

Arsenic contamination in industrial wastewater and groundwater has become an important environmental issue. In this study, a novel zirconium/polyvinyl alcohol (PVA) modified polyvinyldene fluoride (PVDF) membrane was developed for arsenate removal from simulated contaminated water. A PVDF flat-sheet membrane was first fabricated; it was then soaked in a zirconium-PVA solution and dried, and finally reacted with a glutaraldehyde solution, by which the zirconium ions were impregnated onto the PVDF surface through the ether and hydroxyl groups according to the cross-linkage mechanism. The fabrication procedure was optimized by the Box-Behnken experimental design approach. The adsorption kinetics study showed that most of uptake occurred in 5 h and the equilibrium was established in 24 h. The acidic condition was beneficial for the arsenate removal and the optimal removal efficiency can be obtained at pH 2.0. The experimental data of the adsorption isotherm was better described by Langmuir equation than Freundlich equation. The maximum adsorption capacity of 128 mg-As/g was achieved at pH 2.0. In the filtration study, the modified membrane with an area of 12.56 cm(2) could treat 15.6 L arsenate solution (equivalent to 75,150 bed volumes) with an influent concentration of 98.6 µg/L to meet the maximum contaminate level of 10 µg/L. Several instrumental studies revealed that the removal was mainly associated with ion exchange between chloride and arsenate ions.

Treatment of lead contaminated water by a PVDF membrane that is modified by zirconium, phosphate and PVA.

Lead contamination is one of the most serious problems in drinking water facing humans. In this study, a novel zirconium phosphate modified polyvinyl alcohol (PVA)-PVDF membrane was developed for lead removal. The zirconium ions and PVA were firstly coated onto a PVDF membrane through crosslinking reactions with glutaraldehyde, which was then modified by phosphate. The adsorption kinetics study showed that most of ultimate uptake occurred in 5 h. The adsorption increased with an increase in pH; the optimal adsorption was achieved at pH 5.5. The experimental data were better described by Langmuir equation than Freundlich equation; the maximum adsorption capacity was 121.2 mg-Pb/g at pH 5.5, much higher than other reported adsorptive membranes. The membrane exhibited a higher selectivity for lead over zinc with a relative selectivity coefficient (Pb(2+)/Zn(2+)) of 9.92. The filtration study showed that the membrane with an area of 12.56 cm(2) could treat 13.9 L (equivalent to 73,000 bed volumes) of lead containing wastewater with an influent concentration of 224.5 µ g/L to meet the maximum contaminant level of 15 µ g/L. It was demonstrated that the membrane did well in the removal of lead in both simulated wastewater and lead-spiked reservoir water and had a good reusability in its applications. The XPS studies revealed that the lead uptake was mainly due to cation exchange between hydrogen ions and lead ions.

[Spatial, Temporal Distribution Characteristics and Potential Risk of PPCPs in Surface Sediments from Taihu Lake].

Fourteen sediment samples from 15 river estuaries and six sediments from 6 drinking water resource were collected from Taihu Lake. Nine pharmaceutical and personal care products(PPCPs) in the sediments samples were measured by using the HPLC-MS/MS technique. The ranges of geometric mean values were 1.60-129 ng·g-1 and 1.36-22.0 ng·g-1, respectively. Caffeine was the dominant pollutant in fourteen sediments near the river estuary, the content of which covered 52% of amounts of 9 PPCPs. Lincomycin, trimethoprime, azithromycin, sulfamethoxazole and tylosin were the dominant pollutants in six sediments near the drinking water resource, the contents of which covered 79% of amounts 9 PPCPs. From the point of spatial distribution, the results of PPCPs in Zhushan bay and East of Yixing in the northwest and west of Taihu Lake showed higher concentration than those in other sample sites. From the composition, the origin of PPCPs was different. Municipal sewage, stock farming and aquaculture were the main sources of PPCPs in Taihu Lake. Pharmaceuticals of human use showed the dominant pollution in fourteen sediments near the river estuary and drugs of veterinary use showed the dominant pollution in six sediments near the water resource. The concentrations of PPCPs in fourteen sediments of river mouth showed high level. It suggested that PPCPs pollutants were discharged to Taihu Lake continuously. Further risk assessment results showed that the overall risk was not high except for some PPCPs compounds. The RQ exceeded 1 for acetaminophen, azithromycin and sulfamethoxazole in the surface sediments of 15 river estuaries and 6 water resources, which showed high risk. The RQ was between 0.01 to 0.1 for carbazepine in the surface sediments of 15 river estuaries and 6 water resources, which showed medium risk. The RQ was below 0.01 for caffeine, lincomycin, trimethoprim, diltiazem and tylosin in the sediments of 15 river estuaries and 6 water resources, which showed low risk.

Immunogenicity and protective efficacy of an EV71 virus-like particle vaccine against lethal challenge in newborn mice.

Enterovirus 71(EV71) has caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and pre-school children. It has become a serious public health threat, as currently there are no approved vaccines or antiviral drugs for EV71 infection. Many EV71 vaccines have been under development worldwide, however the main focus is inactivated EV71 vaccines. For example, the inactivated EV71 vaccine has recently finished phase III clinical trial in Mainland China. There have been very few studies on EV71 virus like particles (VLPs). In this study, the immunogenicity and protective potency of the EV71 VLPs produced in insect cells were evaluated in mice with different dosages. Our results showed that EV71 VLPs could elicit high titers of neutralizing antibodies (NTAbs) in a dose-dependent manner and NTAbs were sustained after the second injection with an average GMT (geometric mean titer) level from 19 to 2960 in immunized mice. Survival rates were 100%, 100%, 85%, and 40% after challenge with 15 LD50 (median lethal dose) of EV71 in these newborn mice, respectively. ED50 (50% effective dose) of VLPs was 0.20 µg/dose in newborn mice, while NTAb titer under this dosage was about 50. Passive protection was determined with 2 methods and demonstrated that the survival rates were positively correlated with NTAb titers, which at 24 and 54 induced 50% survival rates in experimental animals. The ED50 of VLP vaccines and the passive NTAb titers were also analyzed. The maternal NTAb titer was similar as the passive NTAb titer in the mouse model challenged with our lethal mouse EV71 strain. Hence, our work has provided preliminary data on the protection potency of VLPs as a vaccine candidate and would facilitate future VLP vaccine development.

Evaluation of monovalent and bivalent vaccines against lethal Enterovirus 71 and Coxsackievirus A16 infection in newborn mice.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.