Catherine Manin

Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy ☆

Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core.

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.

The structure of HBsAg particles is not modified upon their adsorption on aluminium hydroxide gel.

Current Hepatitis B vaccines are based on recombinant Hepatitis B surface antigen (HBsAg) virus-like particles adsorbed on aluminium (Al) gel. These particles exhibit a lipoprotein-like structure with about 70 protein S molecules in association with various types of lipids. To determine whether the adsorption on Al gel affects HBsAg structure, we investigated the effect of adsorption and mild desorption processes on the protein and lipid parts of the particles, using various techniques. Electron microscopy showed that the size and morphology of native and desorbed HBsAg particles were comparable. Moreover, infrared and Raman spectroscopy revealed that the secondary structure of the S proteins was not affected by the adsorption/desorption process. Affinity measurements with Surface Plasmon Resonance showed no difference between native and desorbed HBsAg for HBsAg-specific RF-1 monoclonal antibody. Steady-state and time-resolved fluorescence data of the intrinsic fluorescence of the S proteins further indicated that the adsorption/desorption of HBsAg particles on Al gel did not modify the environment of the most emitting Trp residues, confirming that the conformation of the S proteins remains intact. Moreover, using environment-sensitive 3-hydroxyflavone probes, no significant changes of the lipid core and lipid membrane surface of the HBsAg particles were observed during the adsorption/desorption process. Finally, the ratio between lipids and proteins in the particles was found to be similar before and after the adsorption/desorption process. Taken together, our data show that adsorption on Al gel does not affect the structure of the HBsAg particles.

Characterization of the structural modifications accompanying the loss of HBsAg particle immunogenicity

The aim of this work was to further understand the relationship between the immunogenicity and the structure of Hepatitis B surface antigen (HBsAg) particles used in Hepatitis B vaccines. To reach this aim, we compared by using a large range of techniques, the structure and properties of untreated particles with those of particles stored for 3 weeks at +60°C, a treatment which resulted in a loss of HBsAg antigenicity (toward RF-1 mAb) and immunogenicity (in mice). While untreated particles imaged by electron microscopy and atomic force microscopy appeared as isolated nanoparticles of ∼ 20nm, heated particles appeared as long chains of particle aggregates with a partial loss of their protein protrusions. Moreover, infrared spectroscopy and circular dichroism revealed that the secondary structure of the S proteins was significantly affected, with a loss of 10% of their α-helix content. Steady-state and time-resolved fluorescence data further revealed strong modifications of the most emitting Trp residues at the particle surface, confirming significant changes in the conformation of the S proteins. Moreover, modifications in the organization of both the lipid core and lipid membrane surface of the heated particles were evidenced by environment-sensitive 3-hydroxyflavone probes. Taken together, our data evidenced a clear relationship between the bona fide S protein structure and lipid organization notably at the particle surface and the particle immunogenicity.

Characterization of different strains of poliovirus and influenza virus by differential scanning calorimetry

Vaccines against poliomyelitis and influenza contain inactivated forms of poliovirus and influenza virus. These antigens are generated on an industrial scale from the purified active viruses that have been analysed in this study by DSC (differential scanning calorimetry). Multiple unfolding transitions are seen for influenza virus A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Shangdong/7/97. These data, combined with previously reported data on other influenza viruses, indicates that each influenza virus strain has a characteristic unfolding behaviour. Only minor changes were seen in the thermogram of βPL (β‐propiolactone)‐inactivated influenza virus, which is consistent with the proposition that βPL reacts mainly with the nucleotide fraction of the virus. We demonstrate that a peak annotation of the thermogram of the native virus is possible using bromelain‐treated virus and virosomes. At pH 1.5–2.5, poliovirus of type I unfolds in a single unfolding event with respective Tm (midpoint of protein unfolding transition) values between 34 and 45 °C. At pH 2, polioviruses of type II unfold equally in a single event, but, compared with the type I virus, with a Tm value increased by 3.7 °C. At neutral pH, the DSC thermogram of type I poliovirus was very ‘noisy’. Data obtained offer the possibility of precisely characterizing and identifying different viral strains.

Hepatitis B subvirus particles display both a fluid bilayer membrane and a strong resistance to freeze drying: a study by solid-state NMR, light scattering, and cryo-electron microscopy/tomography

Hepatitis B surface antigen (HBsAg) subvirus particles produced from yeast share immunological determinants with mature viruses, which enable the use of HBsAg as a potent antigen for human vaccination. Because the intimate structure of such pseudoviral particles is still a matter of debate, we investigated the robustness of the external barrier and its structure and dynamics using the noninvasive solid‐state NMR technique. This barrier is made of 60% proteins and 40% lipids. Phospholipids represent 83% of all lipids, and chain unsaturation is of 72%. Dynamics was reported by embedding small amounts of deuterium chain‐labeled unsaturated phospholipid into the external barrier of entire subviral particles, while controlling particle integrity by cryoelectron microscopy, tomography, and light scattering. Variable preparation modes were used, from mild incubation of small unilamellar vesicles to very stringent incorporation with freeze‐drying. A lipid bilayer structure of 4‐ to 5‐nm thickness was evidenced with a higher rigidity than that of synthetic phospholipid vesicles, but nonetheless reflecting a fluid membrane (50–52% of maximum rigidity) in agreement with the elevated unsaturation content. The HBsAg particles of 20‐ to 24‐nm diameter were surprisingly found resistant to lyophilization, in such a way that trapped water inside particles could not be removed. These dual properties bring more insight into the mode of action of native subviral particles and their recombinant counterparts used in vaccines.—Grélard, A., Guichard, P., Bonnafous, P., Marco, S., Lambert, O., Manin, C., Ronzon, F., and Dufourc, E. J., Hepatitis B subvirus particles display both a fluid bilayer membrane and a strong resistance to freeze drying: a study by solid‐state NMR, light scattering, and cryo‐electron microscopy/tomography. FASEB J. 27, 4316–4326 (2013). www.fasebj.org

Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance.

An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant ka and the dissociation constant kd. A significant reduction in ka was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in kd. The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the kd values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the kd value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate kd values. The use of DFS as a technique to study ultratight binding is discussed. Copyright

Method for the simultaneous assay of the different poliovirus types using surface plasmon resonance technology.

The inactivated polio vaccine (IPV) contains viral samples that belong to serotypes 1, 2 and 3. We report here a surface plasmon resonance (SPR)-based technique that permits the simultaneous assay of the individual viral types in the IPV as well as in different bulk intermediates from the industrial vaccine production process. Monoclonal antibodies specific to each of the 3 viral types along with a negative control antibody are captured via an anti-IgG antibody on the surface of the 4 flow cells of the SPR instrument. The viral samples are then injected over these flow cells and the increase in resonance units as a result of virus binding is measured. The method was calibrated by an analysis of the European Working Standard (EWS) for poliovirus vaccines. We show that the antibodies used recognize viruses with functional affinities in the picomolar range permitting an effective capture of the antigen. In addition we demonstrate that the antibodies are highly specific to a given virus type and that the heat induced destruction of the D-antigen abolishes antibody recognition entirely. The technique was found to be reproducible and robust and its response was linear to the antigen concentration. Due to the rapidity of analysis this technique permits an almost real-time follow-up of the industrial production process and may present an alternative to the established ELISA assay for the analysis of the intermediates and the final product.

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Mapping of the epitopes of poliovirus type 2 in complex with antibodies.

The inactivated polio vaccine (IPV) contains poliovirus (PV) samples that belong to serotypes 1, 2 and 3. All three serotypes contain the D-antigen, which induces protective antibodies. The antigenic structure of PVs consists of at least four different antigenic sites and the D-antigen content represents the combined activity of multiple epitopes (Ferguson et al., 1993; Minor, 1990; Minor et al., 1986). The potency of IPV vaccines is determined by measuring the D-antigen content. Several ELISA methods have been developed using polyclonal or monoclonal antibodies (Mabs) in order to quantify the D-antigen content. Characterization of the epitopes recognized by the different Mabs is crucial to map the entire virus surface and ensure the presence of epitopes able to induce neutralizing antibodies. Using a new approach that we developed to study the interaction between monoclonal antibodies and poliovirus type 2, which combines cryo-electron microscopy, image analysis and X-ray crystallography along with identification of exposed amino acids, we have mapped in 3D the epitope sites recognized by three specific Fabs at the surface of poliovirus type 2 (PV2) and characterized precisely the antigenic sites for these Fabs.