Rational design of a multi-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles.

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.

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

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.

A time-gated multi-channel x-ray crystal spectrometer on the Shenguang-III laser facility.

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 Å) 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.

Structural Basis for the Broad, Antibody-Mediated Neutralization of H5N1 Influenza Virus.

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.

Calibration of the linear response range of x-ray imaging plates and their reader based on image grayscale values.

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.

Characterization of an Escherichia coli-derived human papillomavirus type 16 and 18 bivalent vaccine.

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.

Bacterially expressed human papillomavirus type 6 and 11 bivalent vaccine: Characterization, antigenicity and immunogenicity.

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.

Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli.

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.

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 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.

Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants.

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.

New two-dimensional space-resolving flux detection technique for measurement of hohlraum inner radiation in Shenguang-III prototype.

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.

Direct measurement of x-ray flux for a pre-specified highly-resolved region in hohlraum.

A space-resolving flux detector (SRFD) is developed to measure the X-ray flux emitted from a specified region in hohlraum with a high resolution up to 0.11mm for the first time. This novel detector has been used successfully to measure the distinct X-ray fluxes emitted from hot laser spot and cooler re-emitting region simultaneously, in the hohlraum experiments on SGIII prototype laser facility. According to our experiments, the ratio of laser spot flux to re-emitted flux shows a strong time-dependent behavior, and the area-weighted flux post-processed from the measured laser spot flux and re-emitting wall flux agrees with that measured from Laser Entrance Hole by using flat-response X-ray detector (F-XRD). The experimental observations is reestablished by our two-dimensional hydrodynamic simulations and is well understood with the power balance relationship.

Extreme ultraviolet spectrometer for the Shenguang III laser facility.

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.

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(®).

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(®).

Calibration of a gated flat field spectrometer as a function of x-ray intensity.

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.

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

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.

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

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.

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 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.

[Secretory expression and biological activity analysis of an anti-H5 single-chain antibody from Pichia pastoris].

In our previous study, a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies (MAbs) were generated and characterized. The 13D4, one of these MAbs, has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations, clades 1, 2.1, 2.2, and 2.3. Here, we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment. Then, the 13D4 single-chain antibody (scFv) was expressed in secretory maner in Pichia pastoris. The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation. The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography. We further investigated its binding property using hemagglutination inhibition (HI) test and blocking ELISA. The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab. In conclusion, an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.