Dicky Abraham

The αvβ3 Integrin Regulates α5β1-mediated Cell Migration toward Fibronectin

This study examines the interactions of αvβ3 and α5β1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express α5β1 endogenously were transfected with αvβ3 and β3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An αvβ3 blocking antibody and the αvβ3 ligand cyclic G-Pen-GRGDSPC-A inhibited α5β1-mediated migration toward Fn, but not attachment to Fn. This function was αvβ3-specific since αvβ5 transfection and αvβ5 blocking antibody did not produce this effect. Mutations introduced into the β3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation β3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation β3-S752P into the cytoplasmic domain or its truncation (β3-Δ717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by αvβ3 blocking antibody. αvβ3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (β3-Δ693). These findings demonstrate αvβ3 regulation of α5β1-mediated cell migration and suggest that the β3 transmembrane domain is essential for this function.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

BackgroundHuman papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).ResultsVLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced.Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entireHPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres.ConclusionsD/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant. In addition, we determined the three-dimensional structure of the VLPs, both alone and when interacting with neutralizing antibodies. Two modes of binding of two different neutralizing antibodies were apparent; for HPV type 11 saturated with H11.B2, 72 potential Fab binding sites were observed at the center of each capsomer, whereas for HPV 16 interacting with H16.V5, it appears that 60 pentamers (each neighboring 6 other pentamers) bind five Fabs per pentamer, for the total of 300 potential Fab binding sites per VLP.

Antioxidant defense systems in the brains of type II diabetic mice.

Abstract: The specific activities of superoxide dismutase, catalase, and glutathione S‐transferase (μ subtype) were significantly lower in the brains of mice with type II diabetes than in the brains of control mice. On the other hand, the specific activity of glutathione peroxidase was unaltered. The concentration of vitamin E, but not that of total glutathione and ascorbate, was increased in the brains of the type II diabetic mice. The relative amount of polyunsaturated fatty acids (as determined with soybean lipoxygenase) was increased in whole brains and crude synaptosomal membranes of the type II diabetic mice. Endogenous levels of thiobarbituric acid‐positive material were decreased in both whole brain homogenates and crude synaptosomal membranes of the db/db mice. Susceptibility of lipids within whole brain homogenates and crude synaptosomal membranes of mice with type II diabetes to peroxidation with iron/ascorbate was also markedly decreased compared with that of controls. Vitamin E is known to quench lipid peroxidation. Therefore, decreased lipid peroxidation in the type II mouse brain may be due to increased vitamin E content.

In-depth process understanding of RECOMBIVAX HB® maturation and potential epitope improvements with redox treatment: multifaceted biochemical and immunochemical characterization.

Recombinant Hepatitis B surface antigen virus-like particles (VLPs) produced in yeast undergo spontaneous maturation during the vaccine production process, and the biophysical characteristics of the particles with respect to maturation were described in Zhao et al. (2006) [13]. Here we report additional biochemical and immunochemical characterization by various techniques, including the use of a panel of monoclonal antibodies (mAbs) that differ in their selectivity and conformation-sensitivity, for probing surface epitope structures. Crosslinking via interchain disulfide formation and binding of conformational specific antibodies in the mature particles were shown to be progressively enhanced. We show that redox-mediated VLP maturation is superior to heat-induced maturation in terms of generating VLPs which exhibit more complete crosslinking (>95%) and 2- to 3-fold higher antigenicity as defined by conformational antibodies. Therefore, the resulting VLPs from redox treatment resemble more closely their plasma-derived counterparts. The value of using multiple mAbs for probing surface epitopes was clearly demonstrated as different mAbs showed different degrees of sensitivity to the structural changes during HBsAg VLP maturation. The rapid, label-free technology of surface plasmon resonance performed at a single antigen concentration was shown to correlate well with a sandwich ELISA using parallel line analysis, currently implemented for product release and stability testing of RECOMBIVAX HB(®). Surface plasmon resonance offers both convenience and flexibility; multiple mAbs can be tested one at a time in the same set of experiments, providing a means to assess changes to individual epitopes. Taken together, these quantitative analytical tools enable more rapid, in-depth, and comprehensive process monitoring, process optimization, and assessment of product consistency and stability.

Real time monitoring of antigenicity development of HBsAg virus-like particles (VLPs) during heat- and redox-treatment.

The Hepatitis B virus major surface antigen (HBsAg) is a cysteine-rich, membrane-bound protein which self-assembles into 22-nm spherical virus-like particles (VLPs). While this VLP based human vaccine has been demonstrated to be safe and efficacious since 1986, the structural and exact molecular basis for its antigenic determinants has not been elucidated. Maturation of the yeast-derived purified VLPs was characterized for the changes in 37 their biophysical properties. Using rapid and label-free surface plasmon resonance technique with a neutralizing monoclonal antibody - A1.2, the epitope evolution kinetics of purified VLPs was monitored in real time. Evidence supporting the mechanism that the correct disulfide bond pairing is the molecular basis for shaping up the native virion-like epitopes was obtained. At least 10-fold enhancement in antigenicity probed by A1.2 of the VLPs was achieved by heat-treatment (t(1/2) ∼ 6-10 h), and another 2- to 3-fold enhancement was obtained when they were treated with redox buffer. This antigenicity development, presumably via disulfide formation/isomerization, was shown to be inhibited by a free radical scavenger and facilitated in the presence of light. Relative antigenicity determination with surface plasmon resonance was shown to be a valuable tool for process characterization in the kinetic monitoring mode or for final VLP product assessment in the end point antigenicity testing mode.

Protein and virus-like particle adsorption on perfusion chromatography media.

The structural and protein adsorption characteristics of the perfusion chromatography matrix POROS(®) HS 50 are determined. Transmission electron microscopy shows a broad distribution of pore sizes with 100-500nm through-pores transecting a network of much smaller pores formed by aggregates of microgranules about 100nm in size. Dextran standards, proteins, and virus-like particles (VLPs) show size-exclusion behavior consistent with such a bimodal distribution of pore sizes. For non-binding conditions, the trends in height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity and molecular size are consistent with perfusion suggesting that a fraction of the mobile phase between 0.0005 and 0.0008 flows through the particles. This small fraction provides little or no enhancement of intraparticle mass transfer for relatively small proteins (lysozyme and IgG) even at 1000cm/h, but can contribute substantially to transport for large proteins (thyroglobulin) and VLPs. Intraparticle concentration profiles during transient adsorption are determined by confocal microscopy in batch and flow systems. The profiles are spherically symmetrical indicating a dominance of diffusion for smaller proteins in both batch and flow systems but become highly asymmetrical and skewed in the direction of flow for thyroglobulin at 1000cm/h. Estimates of the convective enhancement of intraparticle transport for these conditions based on the confocal measurements are consistent with estimates of the intraparticle Peclet number and previously published models. Adsorption of VLPs, however, was found to be confined to a thin layer on the outer surface of the particles indicting that bound VLPs block access to the underlying pore network and suggesting that pores larger than those present on the resin studies are needed to take advantage of the effects of perfusion for the adsorption of large VLPs.

Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media.

The resin structure, chromatographic behavior, and adsorption kinetics of proteins and virus-like-particles (VLPs) are studied for POROS HS 20 and POROS HS 50 (23 and 52 μm mean diameter, respectively) to determine the effects of particle size on perfusion chromatography and to determine the predictive ability of available models. Transmission electron microscopy (TEM) and inverse size-exclusion chromatography (iSEC) show similar structures for the two resins, both containing 200-1000 nm pores that transect a network of much smaller pores. For non-binding conditions, trends of the height equivalent to a theoretical plate (HETP) as a function of reduced velocity are consistent with perfusion. The estimated intraparticle flow fractions for these conditions are 0.0018 and 0.00063 for POROS HS 20 and HS 50, respectively. For strong binding conditions, confocal laser scanning microscopy (CLSM) shows asymmetrical intraparticle concentrations profiles and enhanced rates of IgG adsorption on POROS HS 20 at 1000 cm/h. The corresponding effective diffusivity under flow is 2-3 times larger than for non-flow conditions and much larger than observed for POROS HS 50, consistent with available models. For VLPs, however, adsorption is confined to a thin layer near the particle surface for both resins, suggesting that the bound VLPs block the pores.

Comparison of perfusion media and monoliths for protein and virus-like particle chromatography.

Structural and performance characteristics of perfusion chromatography media (POROS HS 20 and 50) and those of a polymethacrylate monolith (CIM SO3-1 tube monolith column) are compared for protein and virus-like particle chromatography using 1mL columns. Axial flow columns are used for POROS while the monolith has a radial flow configuration, which provides comparable operating pressures. The POROS beads contain a bimodal distribution of pore sizes, some as large as 0.5μm, which allow a small fraction of the mobile phase to flow within the particles, while the monolith contains 1-2μm flow channels. For proteins (lysozyme and IgG), the dynamic binding capacity of the POROS columns is more than twice that of the monolith at longer residence times. While the DBC of the POROS HS 50 column decreases at shorter residence times, the DBC of the POROS HS 20 column for IgG remains nearly twice that of the monolith at residence times at least as low as 0.2min as a result of intraparticle convection. Protein recoveries are comparable for all three columns. For VLPs, however, the eluted peaks are broader and recovery is lower for the monolith than for the POROS columns and is dependent on the direction of flow in the monolith, which is attributed to denser layer observed by SEM at the inlet surface of the monolith that appears to trap VLPs when loading in the normal flow direction.