Real-time stability of a hepatitis E vaccine (Hecolin®) demonstrated with potency assays and multifaceted physicochemical methods

The first prophylactic vaccine against hepatitis E virus (HEV), Hecolin®, was licensed in China. Recombinant p239 virus-like particle (VLP) is its active component with dimeric protein as the basic building block harboring the immuno dominant and neutralizing epitopes. The real time and real condition stability of the prefilled syringes for the vaccine was demonstrated using both in vivo mouse potency and in vitro antigenicity assays. A total of 12 lots of Hecolin® were assessed with a set of assays after storage at 2-8 °C for 24 months. The particle characteristics of p239 VLP recovered from the aluminum-containing adjuvant was assessed with different methods including analytical ultracentrifugation, high performance size exclusion chromatography and transmission electron microscopy. The thermal and conformational stability of the adsorbed antigen was assessed using differential scanning calorimetry. The protein integrity of the recovered p239 antigen was demonstrated using SDS-PAGE with silvering staining, LC-MS and MALDI-TOF MS. Most importantly, the binding activity to the neutralizing antibody or vaccine antigenicity was measured using an epitope-specific and real-time SPR assay and a monoclonal antibody-based sandwich ELISA. Taken together, the overall good stability of the Hecolin® prefilled syringes was demonstrated with unaltered molecular and functional attributes after storage at 2-8 °C for 24 months

Comprehensive evaluation of natural landscapes before and after earthquake based on GST-FAHP-GIS analytical method: A case study of Jiuzhaigou Nature Reserve, China

The Jiuzhaigou earthquake caused severe damage to its natural landscape. The seismic-induced changes in landscapes represent a complex and diverse process. In order to creatively assess the extent of earthquake-induced landscape destruction and study the focal points of landscape restoration, this study proposes a comprehensive framework system that integrates multiple indicators and methods to evaluate the impact of earthquakes on the Jiuzhaigou scenic area. In comparison to previous single-method studies, our framework system offers a more holistic explanation and predictive capacity regarding the trends and characteristics of post-earthquake landscape changes. Within this approach, we amalgamate Grey Statistical Technique (GST), Fuzzy Analytic Hierarchy Process (FAHP), Delphi Technique, and Geographic Information System (GIS). By selecting 24 landscape evaluation indicators, we establish five levels of landscape assessment, ranging from broad to fine-grained. Areas with higher visibility sensitivity are categorized as high visual sensitivity regions, indicating the need for focused restoration efforts. Based on the degree of landscape visual sensitivity, we introduce four visual sensitivity evaluation factors: slope gradient, slope aspect, relative distance, and vegetation coverage. The result demonstrates that the overall landscape grade of Jiuzhaigou decreased by 15% after the earthquake; the aesthetic indicator of the landscape within geological hazard experienced the greatest decline, decreasing by 73.3%, while the landscape's resilience indicator showed relatively minor changes. Notably, regions with high visual sensitivity exhibit a distinctive “Y”-shaped distribution pattern, with 56.8% of environmental hazard points possessing the highest visual sensitivity. The study also underscores the concentrated presence of geological hazard points and high visual sensitivity between the Nuorilang Waterfall and the scenic area entrance, necessitating prioritized restoration efforts in this zone. This research innovatively provides a method for assessing the damage caused by earthquakes to natural landscapes, and it holds significant guiding implications for relevant national decision-making bodies in policy formulation and implementation

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of proteinprotein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH4)2SO4], sodium sulfate (Na2SO4), sodium chloride (NaCl), and ammonium chloride (NH4Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH4)2SO4 was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu372, Leu375, and Leu395of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions

Homology model and potential virus-capsid binding site of a putative HEV receptor Grp78

National Natural Science Foundation [30925030, 30600106, 30870514, 30972826]; Project 863 [2006AA020905, 2006AA02A209]; great project on infectious diseases and new drug, People's Republic of China [2009ZX09102-230, 2009ZX10004-704, 2008ZX10404]P239, a truncated construct of the hepatitis E virus (HEV) ORF2 protein, has been proven able to bind with a chaperone, Grp78, in both an in vitro co-immune precipitation test and an in vivo cell model. We previously solved the crystal structure of E2s-the C-terminal domain of p239 involved in host interactions. In the present study, we built a 3D structure of Grp78 using homology modeling methods, and docked this molecule with E2s using the Zdockpro module of the InsightII software package. The modeled Grp78 structure was deemed feasible by profile 3D evaluation and molecular dynamic simulations. The docking result consists of six clusters of distinct complexes and C035 was selected as the most reasonable. The interacting interface of the predicted complex is comprised of the Grp78 linker region and nucleotide binding domain along with the E2s groove region and surrounding loops. Using energy, hydrogen bond and solvent accessible surface analyses, we identified a series of key residues that may be involved in the Grp78:E2s interaction. By comparing with the known structure of the Hsp70:J complex, we further concluded that the interaction of Grp78 and E2s could interrupt binding of Grp78 with the J domain, and in turn diminish or even eliminate the binding ability of the Grp78 substrate binding domain. The predicted series of key residues also provides clues for further research that should improve our understanding of the fundamental molecular mechanisms of HEV infection

Expression and purification of soluble HIV-1 envelope glycoprotein gp160 mutant from Saccharomyces cerevisiae

Here we report the expression of HIV-1 gp160 and its mutated proteins in Saccharomyces cerevisiae. Two strong hydrophobic regions, aa 511-537 and aa 679-703, were predicted by GCG Wisconsin Package software and removed to investigate the solubility of the mutated gp160 (gp160 Delta 12). The results showed that gp160 Delta 12 assumes high solubility as to be present in supernatant of cell lysate exclusively. The mutant exists as trimeric form in solutions via some inter-molecule disulfide bonds, which can be associated to monomer with the reduced reaction of DTT. The fermentation procedure was optimized to get high cell density yield and expression level as similar to 10 mg/L After purification with electro elution, gp160 Delta 12 was checked as glycosylation form by Endo-H deglycosylating catalysis. The ELISA performed with a panel of human sera suggests that the purified gp160 Delta 12 shares some determinants with gp120 and gp41, but exposes some distinct epitopes that react with early HIV-infected antibody. Thus, we may provide a novel antigen for immunodetection assay, vaccine candidate, and other relative research purposes. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.National Natural Science Foundation of China [30500092]; National Science and Technology "863" Program [2006AA020905]; Education Ministry of Chin [705031]; New Century Excellent Scholar Innovation Project of Education Ministry of China [NCET-05-0567

Identification of Broad-Genotype HPV L2 Neutralization Site for Pan-HPV Vaccine Development by a Cross-Neutralizing Antibody.

Human Papillomavirus (HPV), a non-enveloped, double-stranded DNA virus, is responsible for 5% of human cancers. The HPV capsid consists of major and minor structural proteins, L1 and L2. L1 proteins form an icosahedral shell with building blocks of the pentameric capsomere, and one L2 molecule extends outward from the central hole of the capsid. Thus, L2 is concealed within L1 and only becomes exposed when the capsid interacts with host cells. The low antigenic variation of L2 means that this protein could offer a target for the development of a pan-HPV vaccine. Toward this goal, here we describe an anti-L2 monoclonal antibody, 14H6, which broadly neutralizes at least 11 types of HPV, covering types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58 and 59, in pseudovirion--based cell neutralization assay. The mAb 14H6 recognizes a minimal linear epitope located on amino acids 21 to 30 of the L2 protein. Alanine scanning mutagenesis and sequence alignment identified several conserved residues (Cys22, Lys23, Thr27, Cys28 and Pro29) that are involved in the 14H6 binding with L2. The epitope was grafted to several scaffolding proteins, including HPV16 L1 virus-like particles, HBV 149 core antigen and CRM197. The resultant chimeric constructs were expressed in Escherichia coli and purified with high efficiency. Immunization with these pan-HPV vaccine candidates elicited high titers of the L2-specific antibody in mice and conferred robust (3-log) titers of cross-genotype neutralization, including against HPV11, 16, 18, 45, 52, 58 and 59. These findings will help in the development of an L2-based, pan-HPV vaccine

Robust manufacturing and comprehensive characterization of recombinant hepatitis E virus-like particles in Hecolin (R)

China Ministry of Science and Technology via the Major Project [2012AA02A408]; National Science Fund [81373061]; Fujian Provincial Science Fund for Distinguished Young Scholars [2011J06015]; Institute Reconstruction Fund [2011FU125Z04]The hepatitis E virus (HEV) vaccine, Hecolin (R), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated REV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30 nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-REV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin (R). (C) 2014 Elsevier Ltd. All rights reserved

N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro

Highlights • N-terminal truncations promote the soluble expression of HPV L1 proteins in E. coli and their self-assembly of T = 7 icosahedral particle in vitro • An HPV 9-valent vaccine candidate was formulated with E. coli-derived HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 VLPs, and conferred comparable immunogenicity with Gardasil

K-shell photoabsorption edge of strongly coupled aluminum driven by laser-converted radiation

The first observation of the K-shell photoabsorption edge of strongly coupled aluminum generated by intense x-ray radiation-driven shocks is reported. By using a “dog bone” gold hohlraum as an x-ray converter, colliding shocks compression and preheating shielding are achieved to generate an unexplored state with a density of 5.5 g/cm3 and temperature of 0.43 eV (the ion-ion coupling parameter $\Gamma_{ii}$ is around 240). The time-resolved K-shell photoabsorption edges are measured with a crystal spectrometer using a short x-ray backlighter. The broadenings and redshifts of the edges are studied by using the slope fitting of the edge and quantum molecular dynamics calculations. This work shows that the K-edge of aluminum driven by laser-converted radiation provides a novel capability to probe WDM at extended conditions

Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes

National High-tech R&D Program (863 Program) [2012AA02A408]; National Science Fund of China [81373061]; National International Cooperation Program [2011DFG33050]; Fujian Provincial Science Fund for Distinguished Young Scholars [2011J06015]The protein encoded by ORF2 in hepatitis E virus (HEV) is the only capsid protein for this single-stranded RNA virus. It was previously shown that 148 aa (aa 459-606) was needed for dimer formation, whereas 239 aa (aa 368-606) was necessary to form virus-like particles (VLPs). The self-assembled VLPs of p239 were characterized with a series of methods including high performance size-exclusion chromatography to demonstrate the particulate nature of purified and properly refolded p239. A neutralizing and protective mouse monoclonal antibody (mAb) 8C11 was previously shown to bind three discontinuous peptide segments in the dimer. In addition to the good binding activity to recombinant dimeric form, E2s or E2, and VLP form p239, we demonstrated that 8C11 was able to capture the authentic HEV virions. The capability of virus capturing was demonstrated with a titration curve from 10(5) to 10(7) HEV genome copies, making binding activity to 8C11 a surrogate marker of virion-like epitopes on recombinant VLPs as well as vaccine efficacy in eliciting protective and neutralizing antibodies. Taken together, it was demonstrated that Escherichia coli expressed pORF2 proteins, p239 in particular, maintain the virion-like epitopes on VLP surface. This is consistent with the fact that p239 was demonstrated to be an effective prophylactic vaccine (recently licensed as Hecolin (R) in China) against HEV-induced hepatitis in a large scale clinical trial. (C) 2014 Elsevier Ltd. All rights reserved