RESUMEN
Biotherapeutics, molecules produced from biological systems, require rigorous purification steps to remove impurities including host cell proteins (HCPs). Regulatory guidelines require manufacturers to monitor process-related impurities along the purification workflow. Mass spectrometry (MS) has recently been considered as a complementary method to the well-established ELISA for HCPs quantification, since it has the advantage of unambiguously identifying individual HCP. In this study, we developed an innovative standard dedicated to MS-based HCP profiling analysis in order to monitor the consistency of viral vaccine intermediate purification samples. This standard, termed the HCP-PROFILER standard, is composed of a water-soluble bead (READYBEADS technology) which, after being added into the sample, releases unlabeled peptides in controlled amounts. The standard meets three desired criteria: (1) it is composed of multiple peptides, at different concentration levels, allowing construction of a calibration curve covering the dynamic range of HCPs present in the target sample, ensuring quantification accuracy; (2) it demonstrates high batch-to-batch reproducibility, ensuring quantification robustness and consistency over time; and (3) it is easy to use and avoids user-induced analytical biases. In this study, we present the use of the HCP-PROFILER standard for vaccine batches comparison and downstream process performance studies.
Asunto(s)
Espectrometría de Masas en Tándem , Vacunas Virales , Animales , Anticuerpos Monoclonales , Células CHO , Cromatografía Liquida , Cricetinae , Cricetulus , Reproducibilidad de los ResultadosRESUMEN
The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with ß-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.
Asunto(s)
Bioensayo , Vacunas Antirrábicas , Potencia de la Vacuna , Animales , Ensayo de Inmunoadsorción Enzimática , Ratones , Rabia/inmunología , Rabia/patología , Rabia/prevención & control , Vacunas Antirrábicas/química , Vacunas Antirrábicas/inmunología , Vacunación , Vacunas de Productos InactivadosRESUMEN
The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Vacunas Antirrábicas/inmunología , Potencia de la Vacuna , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Interacciones Huésped-Patógeno/inmunología , Humanos , Ratones , Rabia/inmunología , Rabia/virología , Vacunas Antirrábicas/normas , Virus de la Rabia/inmunología , Virus de la Rabia/fisiología , Reproducibilidad de los Resultados , Vacunación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
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.
Asunto(s)
Virus del Dengue/inmunología , Espectrometría de Masas , Proteínas Virales/análisis , Vacunas Virales/análisis , Animales , Chlorocebus aethiops , Vacunas Atenuadas/análisis , Células Vero , Proteínas Virales/inmunología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
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.
Asunto(s)
Membrana Celular , Microscopía por Crioelectrón , Liofilización , Antígenos de Superficie de la Hepatitis B/metabolismo , Espectroscopía de Resonancia Magnética , Antígenos de Superficie de la Hepatitis B/genética , Pichia/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/química , Complejo Antígeno-Anticuerpo/química , Bordetella pertussis/inmunología , Toxina del Pertussis/química , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Humanos , Cinética , Microscopía de Fuerza Atómica/métodos , Toxina del Pertussis/inmunología , Vacuna contra la Tos Ferina/química , Vacuna contra la Tos Ferina/inmunología , Unión Proteica/inmunología , Estadística como Asunto , Resonancia por Plasmón de Superficie/métodos , Vacunas Acelulares/inmunología , Tos Ferina/inmunología , Tos Ferina/prevención & controlRESUMEN
The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these laboratory markers relate to vaccine efficacy and safety.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Vacunas contra el Dengue/inmunología , Epítopos/inmunología , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Pruebas de Neutralización , Resonancia por Plasmón de Superficie , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/inmunología , Ensayo de Placa ViralRESUMEN
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 9â¯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.
Asunto(s)
Microscopía por Crioelectrón/métodos , Vacunas contra el Dengue , Virus del Dengue/crecimiento & desarrollo , Espectrometría de Masas/métodos , Tecnología Farmacéutica/métodos , Virus del Dengue/química , Virus del Dengue/ultraestructura , Humanos , Vacunas Atenuadas , Vacunas SintéticasRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo/métodos , Epítopos/inmunología , Poliovirus/inmunología , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Microscopía por Crioelectrón , Epítopos/química , Procesamiento de Imagen Asistido por ComputadorRESUMEN
The reasons why some proteins induce a particular type of T helper (Th) response are of fundamental importance but only partially understood. In the present study, amphipatic sequence motifs were identified in N- and C-terminal domains of Helicobacter pylori (Hp) catalase, which are linked to the induction of Th1 or Th2 immune responses, respectively. Alignment of these motifs with other proteins known to induce either Th1 or Th2 responses has lead to the identification of Th1 and Th2 consensus motifs, termed modulotopes. Their immunomodulatory potential was demonstrated by immunisation experiments using recombinant proteins comprising the C-terminal domain of catalase fused with one or several modulotopes and by co-immunisations of C- or N-terminal catalase domains with peptides containing these motifs. In addition to these in vivo data, in vitro assays using Limulus extracts suggested that modulotopes might interfere with responses triggered by danger signals such as LPS. Th1 and Th2 modulotopes are characterised by a specific hydrophobic/hydrophilic pattern, which might be the structural determinant for their activity. Our data suggest that Th1 and/or Th2 motifs may generally exist on proteins, thus offering the possibility of a rational modulation of the immune response.
Asunto(s)
Proteínas Bacterianas/inmunología , Catalasa/química , Catalasa/inmunología , Helicobacter pylori/enzimología , Células TH1/inmunología , Células Th2/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos/química , Antígenos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Catalasa/farmacología , Citocinas/metabolismo , Femenino , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Inmunoglobulina G/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , VacunaciónRESUMEN
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.
RESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/inmunología , Mapeo Epitopo , Epítopos/inmunología , Poliovirus/inmunología , Aminoácidos/química , Anticuerpos Antivirales/química , Antígenos Virales/inmunología , Microscopía por Crioelectrón , Congelación , Procesamiento de Imagen Asistido por Computador , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Poliovirus/ultraestructura , Propiedades de Superficie , Vacunas de Productos Inactivados/inmunologíaRESUMEN
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.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Epítopos/química , Epítopos/inmunología , Imagenología Tridimensional , Poliovirus/química , Poliovirus/inmunología , Aminoácidos/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Poliovirus/ultraestructura , Huella de ProteínaRESUMEN
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 â¼ 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.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/química , Estructura Secundaria de Proteína , Animales , Dicroismo Circular , Epítopos/química , Epítopos/inmunología , Femenino , Antígenos de Superficie de la Hepatitis B/inmunología , Calor , Ratones , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Microscopía Electrónica , Espectrofotometría InfrarrojaRESUMEN
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.
Asunto(s)
Poliovirus/clasificación , Poliovirus/aislamiento & purificación , Resonancia por Plasmón de Superficie/métodos , Virología/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Hidróxido de Aluminio/química , Antígenos de Superficie de la Hepatitis B/química , Adsorción , Dicroismo Circular , Flavonoides/química , Vacunas contra Hepatitis B/química , Lípidos , Microscopía Electrónica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Espectrofotometría Infrarroja , Resonancia por Plasmón de Superficie , Vacunas de Partículas Similares a Virus/químicaRESUMEN
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.
Asunto(s)
Antígenos de la Hepatitis B/química , Microscopía de Fuerza Atómica/métodos , Nanotecnología/métodos , Ditiotreitol/metabolismo , Antígenos de la Hepatitis B/genética , Antígenos de la Hepatitis B/metabolismo , Microscopía Electrónica , Pichia/genéticaRESUMEN
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.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/química , Lípidos/química , Espectrometría de Fluorescencia/métodos , Proteínas Virales/química , Colorantes FluorescentesRESUMEN
To investigate the effect of the autoinducer AI-2 on protein expression in Neisseria meningitidis, a luxS mutant of strain MC58 was grown in the presence and absence of in vitro-produced AI-2, and differential protein expression was assessed by two-dimensional differential gel electrophoresis. N. meningitidis did not show a global response to AI-2 signaling activity.
Asunto(s)
Proteínas Bacterianas/análisis , Homoserina/análogos & derivados , Homoserina/fisiología , Neisseria meningitidis/metabolismo , Proteínas Bacterianas/fisiología , Liasas de Carbono-Azufre , Electroforesis en Gel Bidimensional , Lactonas , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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 betaPL (beta-propiolactone)-inactivated influenza virus, which is consistent with the proposition that betaPL 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 degrees 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 degrees 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.