Functional assessment and structural basis of antibody binding to human papillomavirus capsid

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.The authors acknowledge the funding support from the Chinese government: National 863 Program of China (2014AA021302), National Natural Science Fund of China (81373061 and 81471934) and Fujian Provincial Program for Construction Plan of Science and Technology Innovation Platform (2014Y2101). This work was also supported by a Senior Research Fellowship from the Welcome Trust, grant number 101908/Z/13/Z, to Y.M.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/rmv.186

Modeling dust sources, transport, and radiative effects at different altitudes over the Tibetan Plateau

Mineral dust plays an important role in the climate of the Tibetan Plateau (TP) by modifying the radiation budget, cloud macro- and microphysics, precipitation, and snow albedo. Meanwhile, the TP, with the highest topography in the world, can affect intercontinental transport of dust plumes and induce typical distribution characteristics of dust at different altitudes. In this study, we conduct a quasi-global simulation to investigate the characteristics of dust source contribution and transport over the TP at different altitudes by using a fully coupled meteorology–chemistry model, the Weather Research and Forecasting model with chemistry (WRF-Chem), with a tracer-tagging technique. Generally, the simulation reasonably captures the spatial distribution of satellite-retrieved dust aerosol optical depth (AOD) at different altitudes. Model results show that dust particles are emitted into atmosphere through updrafts over major desert regions and then transported to the TP. The East Asian dust (mainly from the Gobi and Taklamakan deserts) is transported southward and is lifted up to the TP, contributing a mass loading of 50 mg m−2 at a height of 3 km and 5 mg m−2 at a height of 12 km over the northern slope of the TP. Dust from North Africa and the Middle East are concentrated over both of the northern and southern slopes below 6 km, where mass loadings range from 10 to 100 and 1 to 10 mg m−2 below 3 km and above 9 km, respectively. As the dust is transported to the north and over the TP, mass loadings are 5–10 mg m−2 above a height of 6 km. The dust mass flux carried from East Asia to the TP is 7.9 Tg yr−1, mostly occurring at heights of 3–6 km. The dust particles from North Africa and the Middle East are transported eastward following the westerly jet and then are carried into the TP at the west side with dust mass fluxes of 7.8 and 26.6 Tg yr−1, respectively. The maximum mass flux of the North African dust mainly occurs at 0–3 km (3.9 Tg yr−1), while the Middle Eastern dust occurs at 6–9 km (12.3 Tg yr−1). The dust outflow occurs on the east side (−17.89 Tg yr−1) and south side (−11.22 Tg yr−1) of the TP, with a peak value (8.7 Tg yr−1) at 6–9 km. Moreover, the dust (by mass) is concentrated within the size range of 1.25–5.0 µm and the dust (by particle number) is concentrated in the size range of 0.156–1.25 µm. Compared with other aerosols, the dust contributes to more than 50 % of the total AOD over the TP. The direct radiative forcing induced by the dust is −1.28 W m−2 at the top of the atmosphere (cooling), 0.41 W m−2 in the atmosphere (warming), and −1.68 W m−2 at the surface (cooling). Our quantitative analyses of the dust contributions from different source regions and the associated radiative forcing can help us to better understand the role of dust on the climate over the TP and surrounding regions

Functional assessment and structural basis of antibody binding to human papillomavirus capsid.

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.The authors acknowledge the funding support from the Chinese government: National 863 Program of China (2014AA021302), National Natural Science Fund of China (81373061 and 81471934) and Fujian Provincial Program for Construction Plan of Science and Technology Innovation Platform (2014Y2101). This work was also supported by a Senior Research Fellowship from the Welcome Trust, grant number 101908/Z/13/Z, to Y.M.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/rmv.186

A novel vaccine candidate based on chimeric virus-like particle displaying multiple conserved epitope peptides induced neutralizing antibodies against EBV infection.

Epstein-Barr virus (EBV) is the causative pathogen for infectious mononucleosis and many kinds of malignancies including several lymphomas such as Hodgkin\u27s lymphoma, Burkitt\u27s lymphoma and NK/T cell lymphoma as well as carcinomas such as nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBV-GC). However, to date no available prophylactic vaccine was launched to the market for clinical use

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

HIV-1 Membrane-Proximal External Region Fused to Diphtheria Toxin Domain-A Elicits 4E10-Like Antibodies in Mice.

The production of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an HIV-1 vaccine. The membrane-proximal external region (MPER) of gp41, which plays a critical role in the virus membrane fusion process, is highly conserved and targeted by bNAbs 2F5, 4E10, and 10E8. As such, MPER could be a promising epitope for vaccine design. In this study, diphtheria toxin domain A (CRM197, amino acids 1-191) was used as a scaffold to display the 2F5 and 4E10 epitopes of MPER, named CRM197-A-2F5 and CRM197-A-4E10. Modest neutralizing activities were detected against HIV-1 clade B and D viruses in the sera from mice immunized with CRM197-A-4E10. Monoclonal antibodies raised from CRM197-A-4E10 could neutralize several HIV-1 strains, and epitope-mapping analysis indicated that some antibodies recognized the same amino acids as 4E10. Collectively, we show that 4E10-like antibodies can be induced by displaying MPER epitopes using an appropriate scaffold. These results provide insights for HIV-1 MPER-based immunogens design

Toolbox for Non-Intrusive Structural and Functional Analysis of Recombinant VLP Based Vaccines: A Case Study with Hepatitis B Vaccine

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. Methodology: The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). Principal Findings: SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turnaround, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipidto-protei

Viral neutralization by antibody-imposed physical disruption

中和抗体是机体抵御病毒入侵的一类免疫球蛋白，也是疫苗发挥作用的主要效应分子。目前已知的中和抗体作用机制，主要包括阻断病毒-细胞相互作用和介导免疫调理作用。最近我校夏宁邵教授团队研究结果揭示了一种由抗体诱导病毒原位崩解的中和新机制。该研究首次揭示了抗体的直接物理碰撞中和机制，并提出诱导这类中和抗体的方法，有助于病毒保护性抗体和疫苗设计，适用于多种病原体，而不仅限于戊型肝炎病毒。分子疫苗学和分子诊断学国家重点实验室夏宁邵教授、李少伟教授和顾颖副教授为该论文的共同通讯作者，郑清炳博士、硕士生蒋婕、博士生何茂洲和郑子峥副教授为共同第一作者。In adaptive immunity, organisms produce neutralizing antibodies (nAbs) to eliminate invading pathogens. Here, we explored whether viral neutralization could be attained through the physical disruption of a virus upon nAb binding. We report the neutralization mechanism of a potent nAb 8C11 against the hepatitis E virus (HEV), a nonenveloped positive-sense single-stranded RNA virus associated with abundant acute hepatitis. The 8C11 binding flanks the protrusion spike of the HEV viruslike particles (VLPs) and leads to tremendous physical collision between the antibody and the capsid, dissociating the VLPs into homodimer species within 2 h. Cryo-electron microscopy reconstruction of the dissociation intermediates at an earlier (15-min) stage revealed smeared protrusion spikes and a loss of icosahedral symmetry with the capsid core remaining unchanged. This structural disruption leads to the presence of only a few native HEV virions in the ultracentrifugation pellet and exposes the viral genome. Conceptually, we propose a strategy to raise collision-inducing nAbs against single spike moieties that feature in the context of the entire pathogen at positions where the neighboring space cannot afford to accommodate an antibody. This rationale may facilitate unique vaccine development and antimicrobial antibody design.This research was supported by grants from the Natural Science Foundation of Fujian Province (Grant 2017J07005), the National Science and Technology Major Project of Infectious Diseases (Grant 2018ZX10101001-002), and the National Natural Science Foundation of China (Grants 81871247, 81991490, and 81571996).国家自然科学基金重大项目、海峡联合项目和面上项目、福建省自然科学杰出青年基金、国家传染病科技重大专项等资助了该项研究