Atomic-Scale Description of Interfaces between Antigen and Aluminum-Based Adjuvants Used in Vaccines by Dynamic Nuclear Polarization (DNP) Enhanced NMR Spectroscopy.

The addition of aluminum-based adjuvants in vaccines enhances the immune response to antigens. The strength of antigen adsorption on adjuvant gels is known to modulate vaccine efficacy. However, a detailed understanding of the mechanisms of interaction between aluminum gels and antigens is still missing. Herein, a new analytical approach based on dynamic nuclear polarization (DNP) enhanced NMR spectroscopy under magic angle spinning (MAS) is implemented to provide a molecular description of the antigen-adjuvant interface. This approach is demonstrated on hepatitis B surface antigen particles in combination with three aluminum gels obtained from different suppliers. Both noncovalent and covalent interactions between the phospholipids of the antigen particles and the surface of the aluminum gels are identified by using MAS DNP NMR 27 Al and 31 P correlation experiments. Although covalent interactions were detected for only one of the formulations, dipolar recoupling rotational echo adiabatic passage double resonance (REAPDOR) experiments reveal significant differences in the strength of weak interactions.

Treatment of influenza virus with beta-propiolactone alters viral membrane fusion.

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000µM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.

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.

Mimicking influenza virus fusion using supported lipid bilayers.

Influenza virus infection is a serious public health problem in the world, and understanding the molecular mechanisms involved in viral replication is crucial. In this paper, we used a minimalist approach based on a lipid bilayer supported on mica, which we imaged by atomic force microscopy (AFM) in a physiological buffer, to analyze the different steps of influenza fusion, from the interaction of intact viruses with the supported bilayer to their complete fusion. Our results show that sialic acid recognition and priming upon acidification are sufficient for a complete fusion with the host cell membrane. After fusion, a flat and continuous membrane was observed. Because of the fragility of the viral membrane that was removed by the tip, most probably due to the disorganization of the matrix layer at acidic pH, fine structural details of ribonucleoproteins (RNP) were obtained. In addition, AFM topography of intact virus in interaction with the supported lipid bilayer confirms that hemeagglutinin and neuraminidase can form isolated clusters within the viral membrane.

SEM3De: image restoration for FIB-SEM.

Motivation: FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. Results: With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. Availability and implementation: SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

Three dimensional morphology of rabies virus studied by cryo-electron tomography.

The rabies virus (RABV) continues to be a worldwide health problem. RABV contains a single-stranded RNA genome that associates with the nucleoprotein N. The resulting ribonucleoprotein complex is surrounded by matrix protein M, lipid bilayer and glycoprotein G. RABV was reported to organize in bullet-like virions, but the role of each viral component in adopting this morphology is unclear. We present here a cryo-electron tomography study of RABV showing additional morphologies consisting in bullet-like virions containing a tubular, lipidic appendage having G-protein at its apex. In addition, there was evidence for an important fraction of pleomorphic particles. These pleomorphic forms differed in the amount of membrane-associated M-, M/N-protein providing interesting insight into its role in viral morphogenesis. In the absence of membrane-associated M-, M/N-protein viral morphology was almost spherical. Other images, showing straight membrane portions, correlate with the M-protein recruitment at the membrane independently of the presence of the G-protein. The viral membrane was found to contain a negative net charge indicating that M-, M/N-protein-membrane charge attraction drives this interaction.

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 k(a) and the dissociation constant k(d) . A significant reduction in k(a) 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 k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) 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 k(d) 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 k(d) values. The use of DFS as a technique to study ultratight binding is discussed.

Effect of the ß-propiolactone treatment on the adsorption and fusion of influenza A/Brisbane/59/2007 and A/New Caledonia/20/1999 virus H1N1 on a dimyristoylphosphatidylcholine/ganglioside GM3 mixed phospholipids monolayer at the air-water interface.

The production protocol of many whole cell/virion vaccines involves an inactivation step with ß-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosis into the endosome. In the presence of the native virus the pH decrease caused a reduction in domain size, whereas lipid layer thickness and surface pressure were increased. These observations are consistent with a fusion of the viral membrane with the lipid monolayer. Importantly, this fusion was not observed with adsorbed inactivated virus, which indicates that BPL treatment inhibits the first step of virus-membrane fusion. Our data also indicate that BPL chemically modifies hemagglutinin, which mediates the interaction with GM3.

Organization of influenza A virus envelope at neutral and low pH.

Fusion of the influenza A H1N1 virus envelope with the endosomal membrane at low pH allows the intracellular delivery of the viral genome and plays an essential role in the infection process. Low pH induces an irreversible modification of the virus envelope, which has so far resisted 3D structural analysis, partly due to the virus pleiomorphy. This study showed that atomic force microscopy (AFM) in physiological buffer could be used to image the structural details of the virus envelope, both at neutral pH and after a low-pH treatment. At low and intermediate magnification, AFM of control virions confirmed both the pleiomorphy and the existence of zones devoid of glycoprotein spikes at the virus surface, as established by electron microscopy (EM). At higher magnification, the unique vertical resolution of the AFM in 3D topography demonstrated the lateral heterogeneity in spike distribution and strongly suggested that, at least locally, the spikes can be organized in an irregular honeycomb pattern. The surface honeycomb pattern was more easily detected due to an increase in spike height following low-pH treatment at low temperature, which probably prevented disruption of the organization. This enhanced contrast associated with low-pH treatment emphasized differences in the glycoprotein distribution between virions. It was concluded that, together with EM approaches, AFM may help to establish a correlation between surface structure and influenza virus infectivity/pathogenicity.

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses.

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.

Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy.

The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.

PrP(d) accumulation in organs of ARQ/ARQ sheep experimentally infected with BSE by peripheral routes.

To study the pathogenesis of bovine spongiform encephalopathy infection in small ruminants, two Lacaune sheep with the AA136RR154QQ171 and one with the AA136RR154RR171 genotype for the prion protein, were inoculated with a brain homogenate from a French cattle BSE case by peripheral routes. Sheep with the ARQ/ARQ genotype are considered as susceptible to prion diseases contrary to those with the ARR/ARR genotype. The accumulation of disease-associated prion protein (PrP(d)) was analysed by biochemical and immunohistochemical methods. No PrP(d) accumulation was detected in samples from the ARR/ARR sheep 2 years post inoculation. In the two ARQ/ARQ sheep that had scrapie-like clinical symptoms, PrP(d) was found in the central, sympathetic and enteric nervous systems and in lymphoid organs. Remarkably, PrP(d) was also detected in some muscle types as well as in all peripheral nerves that had not been reported previously thus revealing a widespread distribution of BSE-associated PrP(d) in sheep tissues.

A thermodynamic study of GPI-anchored and soluble form of alkaline phosphatase films at the air-water interface.

Glycosylphosphatidyl inositol (GPI) anchored proteins are localized and clustered on the outer layer of the plasma membranes forming microdomains. Among them, mammalian alkaline phosphatases (AP-GPI) are widely distributed enzymes. They can also exist as soluble proteins without anchor (APs). Using the Langmuir film technique, we study the thermodynamic properties of monolayers for both protein forms at the air-buffer interface. The enzymatic activity is maintained at the interface but the adsorption of the two forms of AP is very different. AP-GPI presents a higher surface activity and a larger molecular area than the soluble form. The molecular area deduced for high surface pressures suggests a different organization of the monolayers for these two forms. APs molecules seem to adsorb as a multilayer at the interface while AP-GPI appear to be orientated with the major axis parallel to the interface. This orientation allows the accessibility of AP-GPI enzymatic sites that are turned in direction of the subphase as in vivo where the active sites must be turned outside of the membrane.

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles.

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.

Behavior of a GPI-anchored protein in phospholipid monolayers at the air-water interface.

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.

Study of rabies virus by Differential Scanning Calorimetry.

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.

Structural studies of virus-antibody immune complexes (poliovirus type I): Characterization of the epitopes in 3D.

The inactivated polio vaccine (IPV) contains poliovirus (PVs) 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. In a new approach, combining cryo-electron microscopy and image analysis with X-ray crystallography data available along with identification of exposed amino acids we have mapped in 3D the epitope sites recognized by five specific Fabs and one Mab and characterized precisely the antigenic sites for these Mabs. We propose this method to be used to map the entire "epitopic" surface of virus.

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.

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.

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.