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The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine—formed through the structural clustering of HPV epitopes—confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens., An effective vaccine for human papillomavirus (HPV) needs to protect from several genotypes. Here, Wang et al. provide a strategy to produce single capsomere-hybrid virus-like particle (chVLP) composed of capsomers of different genotypes and show that a nona-type chVLP induces similar levels of neutralizing antibodies as an approved HPV vaccine.

In this study, a toroidal quartz ( $20\overline{2}3$ ) crystal is designed for monochromatic X-ray imaging at 72.3°. The designed crystal produces excellent images of a laser-produced plasma emitting He-like Ti X-rays at 4.75 keV. Based on the simulations, the imaging resolutions of the spherical and toroidal crystals in the sagittal direction are found to be 15 and 5 μm, respectively. Moreover, the simulation results show that a higher resolution image of the source can be obtained by using a toroidal crystal. An X-ray backlight imaging experiment is conducted using 4.75 keV He-like Ti X-rays, a 3 × 3 metal grid, an imaging plate and a toroidal quartz crystal with a lattice constant of 2d = 0.2749 nm. The meridional and sagittal radii of the toroidal α-quartz crystal are 295.6 and 268.5 mm, respectively. A highly resolved image of the microgrid, with a spatial resolution of 10 μm, is obtained in the experiment. By using similar toroidal crystal designs, the application of a spatially resolved spectrometer with high-resolution X-ray imaging ability is capable of providing imaging data with the same magnification ratio in the sagittal and meridional planes.

Human papillomavirus (HPV) types 16 and 18 account for approximately 70% of cervical cancer worldwide. Neutralizing HPV prophylactic vaccines offer significant benefit, as they block HPV infection and prevent subsequent disease. However, the three licensed HPV vaccines that cover these two genotypes were produced in eukaryotic cells, which is expensive, particularly for low-income countries where HPV is highest. Here, we report a new HPV16 and -18 bivalent candidate vaccine produced from Escherichia coli. We used two strategies of N-terminal truncation of HPV L1 proteins and soluble non-fusion expression to generate HPV16 and HPV18 L1-only virus-like particles (VLPs) in a scalable process. Through comprehensive characterization of the bivalent candidate vaccine, we confirm lot consistency in a pilot scale-up of 30L, 100L and 500L. Using cryo-EM 3D reconstruction, we found that HPV16 and -18VLPs present in a T=7 icosahedral arrangement, similar in shape and size to that of the native virions. This HPV16/18 bivalent vaccine shares comparable immunogenicity with the licensed vaccines. Overall, we show that the production of a HPV16/18 bivalent vaccine from an E. coli expression system is robust and scalable, with potentially good accessibility worldwide as a population-based immunization strategy.

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50–60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates. • 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 9

Great efforts have been made to create a bright K-shell source using the thin metal cylindrical cavities in the past few decades. Several metal materials such as titanium (Ti), iron, and so on have been tested for high x-ray conversion efficiency mainly at the OMEGA and NIF laser facilities. Recently, x-ray sources in Ti K-shell transition energy range were investigated at the Shenguang-III prototype laser facility with ∼5 kJ laser energy. The experiments were aimed to reproduce the previous ones at OMEGA, but with an extraordinarily small volume of cylindrical cavity and detailed characterizations of the x-ray source. The cavities were 800 μm inner diameter, 800 μm length, and 30 μm thick plastic tubes supporting 1 μm thick Ti. Seven laser beams were focused to 200 μm diameter. The combination of the small cavity volume and the focused laser spots is intended to improve the electron temperature with limited laser energy since the electron temperature is a key issue for high x-ray conversion efficiency. Thomson scattering was adopted to experimentally probe the electron temperatures at special time and space zones as well as the average temperature obtained from the Ti K-shell spectrum. The evolutions of the electron temperature and density are predicted by the radiation hydrodynamic simulation. A top view and two photon energy bands of x-ray source images provide a way to directly observe the plasma movement toward the cavity axis and distinguish the different emission mechanisms between the Ti K-shell and lower energy x rays. Six Higher-energy x-ray detectors located at different angles were used to record the Ti K-shell x-ray emission and demonstrate its isotropic feature. The characteristics of the x-ray radiate intensity including the time evolution, the angular distribution, and the total yields for both the photon energy regions above and below 4 keV are compared between the cavity and planar targets. Obviously, different behaviors were found between the two photon energy regions and the two types of targets. The x-ray conversion efficiency of the Ti cylinder was determined to be ∼ 4 % and ∼ 21 % in 4 π sr in the Ti K-shell (4–7 keV) and

A high-resolution x-ray spectrometer has been designed and constructed, using a highly oriented pyrolytic graphite Bragg crystal in von Hamos geometry. The spectrometer was designed to study x-ray fluorescence emissions of Ti, and it covers a detecting energy range from 4000 eV to 5500 eV. The factors affecting the spectral resolution and efficiency of the spectrometer were discussed. In particular, the bending of the spectral lines on the image plate was also corrected, due to the detecting plane not being parallel to the axis of the cylindrically curved crystal. Its performance has been evaluated in a laser–foil interaction experiment. The experimental results indicate that the spectrometer is of good spectral resolution and relatively high efficiency, which enables it as a suitable device for the measurements of high-resolution x-ray fluorescence spectroscopy.

Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples.

Enhancement of x-ray sources from laser-produced plasmas is significant in wide-ranging applications. Solid and foam TiO2 targets were both used to generate bright x-ray sources at the Shenguang-III prototype laser facility, with a total laser power of 3.2 TW. The new foam targets were with an ultra-low initial density of 8.1 mg/cm3 and a high Ti fraction of 33 sat. %. By absolute measurements, the multi-keV x rays of the 4 keV–7 keV band and 1.6 keV–4.4 keV band and the total x rays above 0.1 keV of the foam target have simultaneously shown conversion enhancements of 1.4, 3.1, and 2.3 times, respectively, compared with the solid target. A much larger emission volume and an average electron temperature of 3.2 keV by moving the foci of laser beams inner were obtained for the foam target, providing a good condition for multi-keV x-ray production.

An experiment has been conducted at the Shenguang-Ⅲ prototype laser facility, demonstrating the application of x-ray fluorescence imaging (XRFI) in diagnosing laser-driven hydrodynamic instability systems in planar geometry. An aluminum (Al)-foam target was used, including a modulated Al disk followed by a low density poly-4-methyl-1-pentene foam cylinder which was doped with titanium (Ti) dioxide nanoparticles to serve as the tracer layer. A blast wave was driven through the rippled interface, triggering the development of hydrodynamic instabilities and the growth of the perturbation. Vanadium plasma radiation dominated by He-α x-rays, was generated with a delay in time, inducing the emission of Ti K-shell fluorescent photons from the tracer layer. A Ross Pair Imager with matched scandium and Ti filters was employed to provide two-dimensional quasi-monochromatic fluorescent images, and the temporal evolution of the perturbation structures in the tracer layer was successfully observed. Furthermore, one-dimensional fluorescent intensity profiles across the perturbation structures were extracted and compared with the simulation results via a combination of the MULTI code, a buoyancy-drag model, a self-similar analysis and an imaging code. The boundaries of the measured profiles were shown to be connected together due to the small initial length of the tracer layer and the large boundary width caused by the curved interfaces and blurring effects, which made the absolute positions of the perturbation structures inaccessible. This research suggests that XRFI works well in qualitatively diagnosing the instability evolution and the experimental design has to be modified to further improve its performance in achieving quantitative information.

In spectral diagnostic physics experiments of inertial confinement fusion, the spectral signal is weak due to the low diffraction efficiency when using bent crystals. A spectral diagnostic instrument with high efficiency and wide spectral range is urgently needed. A multi-curvature bent crystal with multi-energy focusing ability is proposed based on the traditional conical crystal geometry. It has advantages of wide spectral range, strong focusing ability, and high spectral resolution. It also can eliminate the imaging aberration in principle due to rotational symmetry for the incoming X rays. A spectral diagnostic experiment based on a fabricated multi-curvature α-quartz crystal was accomplished using a titanium X-ray tube of the bent crystal, and the corresponding experimental data using a plane α-quartz crystal was also acquired to demonstrate the strong focusing ability. The result shows that the Kα intensity of the multi-curvature α-quartz crystal is 157 times greater than that of the plane crystal, and the corresponding energy range is about 4.51–5.14 keV. This diagnostic instrument could be combined with a streak camera at a vertical direction so as to intensify the diffracted X-ray signal with a wide spectral range.

An eight-channel x-ray flat crystal spectrometer was developed for high energy density physics research at the Shenguang-III (SG-III) laser facility. The spectrometer uses trihydroxymethylaminomethane crystals (2d = 8.78 A) to record Ti K-shell emission in the photon energy range of 4.65-5.05 keV. The spectrometer couples to an x-ray framing camera to achieve time-resolution. This has four microstrips, and each strip records two snapshots of the emission image. Based on the intersection positioning system with a dual-charge coupled device, the alignment system is easily operated and efficient. The instrument was tested and used for Au hohlraum plasma diagnosis experiments on SG-III. The He-α line and its Li-like satellites and the Ly-α line of a Ti tracer were detected, from which the spectral resolution of the instrument was analyzed. The spectral resolution E/ΔE at the Ti He-α line ranges from about 500 to 880 and mainly limited by the x-ray source size.

Human infection with highly pathogenic avian influenza A viruses causes severe disease and fatalities. We previously identified a potent and broadly neutralizing antibody (bnAb), 13D4, against the H5N1 virus. Here, we report the co-crystal structure of 13D4 in complex with the hemagglutinin (HA) of A/Vietnam/1194/2004 (H5N1). We show that heavy-chain complementarity-determining region 3 (HCDR3) of 13D4 confers broad yet specific neutralization against H5N1, undergoing conformational rearrangement to bind to the receptor binding site (RBS). Further, we show that mutating four critical residues within the RBS-Trp153, Lys156, Lys193, and Leu194-disrupts the binding between 13D4 and HA. Viruses bearing Asn193 instead of Lys/Arg can evade 13D4 neutralization, indicating that Lys193 polymorphism might be, at least in part, involved in the antigenicity of recent H5 genotypes (such as H5N6 and H5N8) as distinguished from H5N1. BnAb 13D4 may offers a template for therapeutic RBS inhibitor design and serve as an indicator of antigenic change for current H5 viruses.IMPORTANCE Infection by highly pathogenic avian influenza A virus remains a threat to public health. Our broadly neutralizing antibody, 13D4, is capable of neutralizing all representative H5N1 viruses and protecting mice against lethal challenge. Structural analysis revealed that 13D4 uses heavy-chain complementarity-determining region 3 (HCDR3) to fit the receptor binding site (RBS) via conformational rearrangement. Four conserved residues within the RBS are critical for the broad potency of 13D4. Importantly, polymorphism of Lys193 on the RBS may be associated with the antigenicity shift from H5N1 to other newly emerging viruses, such as H5N6 and H5N8. Our findings may pave the way for highly pathogenic avian influenza virus vaccine development and therapeutic RBS inhibitor design.

X-ray imaging plates are one of the most important X-ray imaging detectors and are widely used in inertial-confinement fusion experiments. However, their linear response range, which is the foundation of their quantitative data analysis, has not been sufficiently deeply investigated. In this work, we develop an X-ray fluorescer calibration system and carefully explore the linear response range of X-ray imaging plates. For the first time, nearly the entire grayscale range of the X-ray imaging plate linear response—7819–64 879 in the range of 0–65 535—has been observed. Further, we discuss the uncertainties involved in the calibration process. This work demonstrates the excellent linear response qualities of X-ray imaging plates and provides a significant foundation for expanding their quantitative applied range.

Image Plates as x-ray recorder have advantages including reusable, high dynamic range, large active area, and so on. In this work, Fuji BAS-SR type image plate combined with BAS-5000 scanner is calibrated. The fade rates of Image Plates has been measured using x-ray diffractometric in different room temperature; the spectral response of Image Plates has been measured using 241Am radioactive sealed source and fitting with linear model; the spatial resolution of Image Plates has been measured using micro-focus x-ray tube. The results show that Image Plates has an exponent decade curve and double absorption edge response curve. The spatial resolution of Image Plates with 25μ/50μ scanner resolution is 6.5lp/mm, 11.9lp/mm respectively and gold grid radiography is collected with 80lp/mm spatial resolution using SR-type Image Plates. BAS-SR type Image Plates can do high spatial resolution and quantitative radiographic works. It can be widely used in High energy density physics (HEDP), inertial confinement fusion (ICF) and laboratory astronomy physics.

Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.

Human papillomavirus (HPV)-6 and HPV11 are the major etiological causes of condylomata acuminate. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent subsequent disease. Currently, two commercially available HPV vaccines cover these two genotypes, expressed by Saccharomyces cerevisiae. Here we describe another HPV6/11 bivalent vaccine candidate derived from Escherichia coli. The soluble expression of N-terminally truncated L1 proteins was optimized to generate HPV6- and HPV11 L1-only virus-like particles (VLPs) as a scalable process. In a pilot scale, we used various biochemical, biophysical and immunochemical approaches to comprehensively characterize the scale and lot consistency of the vaccine candidate at 30L and 100L. Cryo-EM structure analysis showed that these VLPs form a T=7 icosahedral lattice, imitating the L1 capsid of the authentic HPV virion. This HPV6/11 bivalent vaccine confers a neutralization titer and antibody production profile in monkey that is comparable with the quadrivalent vaccine, Gardasil. This study demonstrates the robustness and scalability of a potential HPV6/11 bivalent vaccine using a prokaryotic system for vaccine production.

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(®) in China) against HEV-induced hepatitis in a large scale clinical trial.

The technique of x-ray fluorescence imaging (XRFI) has been applied to the high-energy-density (HED) jet experiments on the SG-III prototype laser facility. The jet was produced by the interaction of the laser driven shock with the perturbed Ti disk. The fluorescence emission of the Ti ejecta was pumped by the V He-α emission and recorded using a Ross Pair Imager with an optimized configuration. We achieved a spatial resolution of 35 μm and a temporal resolution of 1 ns. We also developed a method to infer the density distribution of the jet from the fluorescence images and discussed the conditions under which this method was accurate. These experiments provided insight into the characteristics of a HED jet and implied that XRFI is a promising way for studying HED hydrodynamics.

We present non-collective spectrally resolved X-ray scattering measurements from isochorically heated graphite samples. The elastic and inelastic scattering spectra for the titanium He-α x-ray line at 4.75 keV yield characterization of the temperature and ionization state of the samples. It is found that the plasma theories employing a screened Coulomb potential fail to describe the ion structure of warm dense carbon plasmas and only the models accounting for the effects of the electronic configuration show good agreement with the experimental result. This agreement further suggests that the short-lived chemical bonds play an important role in determining the ion structure of warm dense graphite systems with a temperature of several electron volts.

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 protein–protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368–606), 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 (aa368–395) 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.

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 24months. 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 24months.

X-ray Thomson scattering technique for diagnosing dense plasma was demonstrated on Shenguang-II laser facility. Laser plasma x-ray source of titanium He-α lines (~4.75 keV), generated by laser beam (1.5 kJ/527 nm/2 ns) heated titanium thin foil, was used as x-ray probe beam. The x-ray probe was then scattered by cold CH foam column of 1 g/cm3 density. The scattered radiation at 90° was diffracted by polyethylene terephthalate (PET) crystal and recorded on x-ray charge-coupled device. Well-defined scattering spectra were obtained with good signal to noise ratio.

An experimental method has been developed to measure the X-ray polarization of the Beamline 4B7 on Beijing Synchrotron Radiation Facility in the X-ray energy range 2100-5600 eV by using a PET crystal diagnostic system and rotating the system around the incident X-ray beam by 90 degrees. The experimental results show that the measured X-ray polarization is more than 0.88 in the photon energy range 2100-3600 eV with maximum of 0.99 at 2707.3 eV, and quickly decreases to about 0.38 at 4719.2 eV with photon energy increasing from 3600 to 4719.2 eV, then it keeps almost constant as the photon energy increases further to 5600 eV. (C) 2010 Elsevier B.V. All rights reserved.

The opacity of a molybdenum plasma in local thermodynamic equilibrium with open M-shell configurations was measured in the energy range from 200 eV to 800 eV, corresponding to the Δn = 1 M to N shell and Δn = 2 M to O shell transitions. The sample was heated by thermal x-ray radiation generated with a gold cavity. The electron temperature was diagnosed with the absorption spectrum of doped aluminum, giving a value of Te = (67 ± 4) eV. The density was 9.7 mg/cm3, which was deduced from the known initial area density and the plasma thickness by radiographic measurements of the expansion of the heated foil sample. The spectrally resolved transmission data were compared to a detailed-configuration-accounting model calculation and about 20% to 100% discrepancies were found.The opacity of a molybdenum plasma in local thermodynamic equilibrium with open M-shell configurations was measured in the energy range from 200 eV to 800 eV, corresponding to the Δn = 1 M to N shell and Δn = 2 M to O shell transitions. The sample was heated by thermal x-ray radiation generated with a gold cavity. The electron temperature was diagnosed with the absorption spectrum of doped aluminum, giving a value of Te = (67 ± 4) eV. The density was 9.7 mg/cm3, which was deduced from the known initial area density and the plasma thickness by radiographic measurements of the expansion of the heated foil sample. The spectrally resolved transmission data were compared to a detailed-configuration-accounting model calculation and about 20% to 100% discrepancies were found.

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 (gp160Delta12). The results showed that gp160Delta12 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 approximately 10 mg/L. After purification with electro elution, gp160Delta12 was checked as glycosylation form by Endo-H deglycosylating catalysis. The ELISA performed with a panel of human sera suggests that the purified gp160Delta12 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.

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.

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 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.

We present an experimental determination of the response of a gated flat-field spectrometer at the Shenguang-II laser facility. X-rays were emitted from a target that was heated by laser beams and then were divided into different intensities with a step aluminum filter and collected by a spectrometer. The transmission of the filter was calibrated using the Beijing Synchrotron Radiation Facility. The response characteristics of the spectrometer were determined by comparing the counts recorded by the spectrometer with the relative intensities of the x-rays transmitted through the step aluminum filter. The response characteristics were used to correct the transmission from two shots of an opacity experiment using the same samples. The transmissions from the two shots are consistent with corrections, but discrepant without corrections.

The hepatitis E virus (HEV) vaccine, Hecolin(®), 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 HEV 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 30nm 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-HEV 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(®).

The first observation of the K-shell photoabsorption edge of strongly coupled matter with an ion-ion coupling parameter of about 65 generated by intense x-ray radiation-driven shocks is reported. The soft x-ray radiation generated by laser interaction with a "dog bone" high-Z hohlraum is used to ablate two thick CH layers, which cover a KCl sample, to create symmetrical inward shocks. While the two shocks impact at the central KCl sample, a highly compressed KCl is obtained with a density of 3-5 times solid density and a temperature of about 2-4 eV. The photoabsorption spectra of chlorine near the K-shell edge are measured with a crystal spectrometer using a short x-ray backlighter. The redshift of the K edge up to 11.7 eV and broadening of 15.2 eV are obtained for the maximum compression. A comparison of the measured redshifts and broadenings with dense plasma calculations are made, and it indicates potential improvements in the theoretical description.

We describe an experimental technique to measure the drive symmetry of M-band radiation on the capsule in hohlraum. M-band radiation from the corona of the laser-produced gold plasma, especially the laser spot regions in the cavity, was used to pump x-ray fluorescence of a thin layer of Si-tracer coated on a solid CH-ball. The fluorescence images were time resolvedly recorded by an x-ray framing camera and the drive asymmetry due to M-band radiation was deduced from these fluorescence images. Moreover, a Si-doped gold cavity was used with the initial purpose of maximizing the fluorescence signal through resonance transitions. Since the Si-plasma expands more rapidly than the gold-plasma, the evolution of drive asymmetry was accelerated in Si-doped hohlraum.

A diagnostic is developed for determining the hotspot mix in inertial confinement fusion experiments. A multi-channel pinhole camera measures Bremsstrahlung emissions from implosion capsules ranging from 6 keV to 30 keV and records an image of the hotspot. Meanwhile, a planar crystal spectrometer measures Ar line emissions used to deduce the electron density of the hotspot. An X-ray streaked camera records the burn duration. With the Bremsstrahlung spectrum, electron density, hotspot volume, and burn duration, the mix quantity is determined by solving a pair of linear equations. This inferred mix amount has an uncertainty due to the uncertainty of the electron density, but with the help of the measured neutron product, the most likely mix quantity value can be determined. This technique is applied to experimental images to infer the quantity of CH ablator mix into the hotspot.

Transmission measurements of warm dense iron plasma are reported over the photon energy range of 400–1200 eV, including the strong 2p–3d structures. One-dimensional hydrodynamic simulation is performed to estimate the plasma conditions: temperatures of several eV and densities of about 0.1 g/cm3. By using the simulated temperature and density, the calculations of the transmission spectra are performed and compared with the time-resolved experimental results.

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 protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), 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 (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of 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.

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.

The space-resolving measurement of X-ray flux from a specific area (laser spot, re-emitting wall, or capsule) inside the hohlraum is an ongoing and critical problem in indirectly driven inertial-confinement fusion experiments. In this work, we developed a new two-dimensional space-resolving flux detection technique to measure the X-ray flux from specific areas inside the hohlraum by using the time- and space-resolving flux detector (SRFD). In two typical hohlraum experiments conducted at the Shenguang-III prototype laser facility, the X-ray flux and radiation temperature from an area 0.2 mm in diameter inside the hohlraum were measured through the laser entrance hole (LEH). The different flux intensities and radiation temperatures detected using the SRFD from the inner area of the LEH were compared with the result measured using the flat-response X-ray detector from the entire LEH. This comparison was also analyzed theoretically. The inner area detected using the SRFD was found to be the re-emitting wall area alone. This important improvement in space-resolving X-ray flux measurement will enhance the current X-ray flux space characterization techniques, thereby furthering the quantitative understanding of X-ray flux space behavior in the hohlraum.

An extreme ultraviolet spectrometer has been developed for high-energy density physics experiments at the Shenguang-III (SG-III) laser facility. Alternative use of two different varied-line-spacing gratings covers a wavelength range of 10–260 Å. A newly developed x-ray framing camera with single wide strip line is designed to record time-gated spectra with ∼70 ps temporal resolution and 20 lp/mm spatial resolution. The width of the strip line is up to 20 mm, enhancing the capability of the spatial resolving measurements. All components of the x-ray framing camera are roomed in an aluminum air box. The whole spectrometer is mounted on a diagnostic instrument manipulator at the SG-III laser facility for the first time. A new alignment method for the spectrometer based on the superimposition of two laser focal spots is developed. The approaches of the alignment including offline and online two steps are described. A carbon spectrum and an aluminum spectrum have been successfully recorded by the spectrometer using 2400 l/mm and 1200 l/mm gratings, respectively. The experimental spectral lines show that the spectral resolution of the spectrometer is about 0.2 Å and 1 Å for the 2400 l/mm and 1200 l/mm gratings, respectively. A theoretical calculation was carried out to estimate the maximum resolving power of the spectrometer.

We study shell mix during implosion using a capsule with a Cl-doped gas-shell interface that is also filled with Ar-doped deuterium gas. The Ar and Cl K-shell emissions are recorded with a flat crystal spectrometer. The He-β complex is analyzed to infer the electron temperature and density in the hotspot. Two regions of different hydrodynamic states are observed: a region of Te ∼ 1.2 ± 0.2 keV and ne ∼ (4 ± 0.5) × 1023 cm−3 and another of Te ∼ 0.6 ± 0.2 keV and ne ∼ (4 ± 0.5) × 1022 cm−3 probed by Ar and Cl ions, respectively. The neutron yield was also recorded and found to have reduced by a factor of ten for the Cl-doped capsule. By attributing the degradation of the neutron yield to the enhanced radiation loss due to the presence of mixed Cl ions and using the conservation of hotspot internal energy, the amount of mixed Cl ions is estimated to be ∼1.4 × 1014, corresponding to a 0.1 μm thickness of the initial shell material and a mixing width of about 16 μm.

Accurate mathematical models called one-line calibration and parameter fitting are presented for wavelength calibration of a flat-field grating spectrometer. The models precisely establish the relationship between wavelength and pixel position of the detector, since geometry parameters and the grating equation of the spectrometer are taken into account. Compared with the commonly used polynomial fitting, the models presented here provide more reliable calibration results, especially in the extended region away from the calibration points. In addition to the high precision of calibration, the parameter fitting procedure provides a helpful way to obtain the actual parameters of the spectrometer.