Maturation of Recombinant Hepatitis B Virus Surface Antigen Particles

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The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with ...
[Human Vaccines 2:4, 174-180, July/August 2006]; ©2006 Landes Bioscience

Maturation of Recombinant Hepatitis B Virus Surface Antigen Particles Research Paper

ABSTRACT

UT E

ON

Received 04/26/06; Accepted 06/09/06

Previously published online as a Human Vaccines E-publication: http://www.landesbioscience.com/journals/vaccines/abstract.php?id=3015

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KEY WORDS

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Hepatitis B virus (HBV), the prototype member of the taxonomic family hepadnaviridiae, still poses a major health threat; it causes hepatitis, liver cancer, and liver cirrhosis, with 350 million carriers (mostly asymptomatic) worldwide.1,2 The 42-nm virions of HBV, or Dane particles, are composed of three structural protein components: the lipid-associated major surface antigen (HBsAg); the PreS protein that is continuously expressed with HBsAg; and the core antigen surrounding the DNA in the core. In addition to the intact 42-nm virions, abundant noninfectious subviral 22-nm spherical particles and tubules containing only HBsAg and lipids also exist in the plasma of chronically-infected individuals. These plasma-derived 22-nm particles were used in prophylactic vaccines against HBV infection before the introduction of a yeast-produced recombinant HBsAg (rHBsAg) vaccine.1 rHBsAg is a 25-kDa protein with 226 amino acids that self-assembles into immunogenic spherical particles containing host cell-derived lipids and ~100 copies of the rHBsAg molecules in a given particle. Yeast-derived rHBsAg has replaced plasma-derived HBsAg as the preferred vaccine, making it the first recombinant subunit vaccine (RECOMBIVAX HB® from Merck) licensed for human use in 1986.1-4 The detailed mechanism of assembly of monomeric rHBsAg molecules into 22-nm lipid-containing particles and the exact disulfide bond pairings in the particles remain largely unknown. The assembly of yeast-produced 22-nm lipoprotein particles is initiated by insertion of the hydrophobic segments of rHBsAg into the endoplasmic reticulum membrane of the yeast cells after expression. Release of the particles is likely through a budding process as observed for other lipid-containing particles. Intra- and intermolecular disulfide bond formation occurs in the endoplasmic reticulum and pre-Golgi compartment,5 and additional disulfide bonds are formed after cell breakage during purification and storage.6 The importance of the correct intra- and intermolecular disulfide bonds is well documented for the immunochemical and biological function of the HBsAg subviral particles: reduction of these disulfide bonds greatly decreases or abolishes both the antigenic and immunogenic properties of this protein complex.7-9

SC

ABBREVIATIONS

4,4'-dianilino-1,1'-binapthyl5,5'-disulfuric acid guanidinium thiocyanate major surface antigen of hepatitis B virus hepatitis B virus phosphate buffered saline recombinant major surface antigen of hepatitis B virus

BIO

Bis-ANS

INTRODUCTION

IEN

conformation flexibility, unfolding, atomic force microscopy, cross-linking, stopped-flow, lipoprotein particles

LA

ND

ES

GdmSCN HBsAg

06

ACKNOWLEDGEMENTS

©

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See page 180.

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*Correspondence to: Qinjian Zhao; WP42A-20; Bioprocess and Bioanalytical Research, Merck & Co. Inc.; West Point, Pennsylvania 19486 USA; Tel.: 215.652.6855; Fax: 215.993.3348; Email: [email protected]

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1Department of Bioprocess & Bioanalytical Research; 2Vaccine and Biologics Research; Merck Research Laboratories; West Point, Pennsylvania USA

HBV PBS rHBsAg

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCNinduced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturantinduced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immunogenicity of this widely-used vaccine component.

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Qinjian Zhao1,* Yang Wang1 Daniel Freed2 Tong-Ming Fu2 Juan A. Gimenez1 Robert D. Sitrin1 Michael W. Washabaugh1

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rHBsAg expressed in yeast or mammalian cells also spontaneously forms disulfide-linked dimers and higher-order oligomers during expression, and the cross-linking continues during downstream purification and storage.6 Studies on the conformational flexibility and dynamics of this lipidassociated oligomeric protein complex and its morphological characterization throughout the maturation process have not been reported. In order to quantitatively analyze these changes, three different samples with different degrees of maturation/aging (shown in Fig. 1 and Table 1) were studied: Purified rHBsAg antigen preparation before KSCN treatment (Pre-KSCN, Sample A) and rHBsAg sample after overnight 3 M KSCN treatment followed by buffer exchange into PBS (Post-KSCN, Sample B). Sample C, a fully-“aged” antigen of Sample B, was obtained by storing Sample B in PBS at 37˚C for approximately one week, then at 4˚C for approximately 15 months prior to analysis. We present here some experimental results elucidating the changes in conformational rigidity/flexibility and in relative accessibility of hydrophobic areas of mature rHBsAg by millisecond time scale unfolding kinetics of the lipoprotein particles as well as by direct visualization of the rHBsAg structure and particle morphology by atomic force microscopy (AFM).

Figure 1. Recombinant HBsAg samples for cross-linking studies, morphological analysis by AFM, and conformational flexibility by unfolding kinetics. Those changes are qualitatively depicted at the bottom of the figure. The purification process from lysate to Sample A was previously described (Wampler et al., 1985), and the two downstream treatment and storage steps for further maturation of the rHBsAg particles in Samples B and C are listed and explained in Table 1.

MATERIALS AND METHOD

Materials. Guanidinium thiocyanate (GdmSCN), potassium thiocyanate (KSCN), and other buffer salts of highest purity were purchased from Sigma (St. Louis, MO). 4,4'-dianilino-1,1'binapthyl-5,5'-disulfuric acid (Bis-ANS) was purchased from Molecular Probes (Eugene, OR). Recombinant HBsAg. rHBsAg was overexpressed in yeast (Saccharomyces cerevisae) and purified according to previously published procedures.6 The purified rHBsAg was used at concentrations in the range of 40 to 400 µg/mL and was stored or diluted in phosphate-buffered saline (PBS). Protein concentrations were determined by a Lowry method with commercially available reagents (Pierce). SDS-NuPAGE. NuPAGE gels were run in a cold room (~4˚C) to maximize the visualization of cross-linked species as described previously.10 Approximately 4 µg of rHBsAg from three samples (Samples A–C in Fig. 1) were brought to equal volume and mixed with NuPAGE LDS buffer without any reducing reagent (Invitrogen). The samples were incubated at ambient temperature for 10 min before loading onto a 4–12% Bis-Tris gradient NuPAGE gel (Invitrogen) with MultiMark protein standards (Invitrogen). After running at 4˚C under constant voltage (200 V), the gel was washed in de-ionized water three times (for five minutes each) and stained with Imperial Blue (Pierce) for 2 h followed by overnight destaining in de-ionized water. The gel image was obtained using a digital scanner with the software of ImageQuant from Molecular Dynamics (now part of Amersham Biosciences Inc., Sunnyvale CA ). Atomic force microscopy. For comparison purpose, Samples A and B were studies with AFM in solution under the identical experimental conditions with each other. Mica discs used as supporting www.landesbioscience.com

media were cleaved immediately before use for sample preparation. Aqueous sample was deposited on a freshly cleaved mica surface (1/4'' diameter) at room temperature to allow passive adsorption. Then the mica surfaces were gently rinsed with de-ionized water. Additional deionized water was added to the sample on the mica surface prior to AFM analysis. AFM images were collected with Digital Nanoscope III MultiMode AFM TappingModeTM (Digital Instrument, Santa Barbara, CA) in fluid at ambient temperature. In solution AFM measurements help to preserve the native structure as opposed to those done on dried samples.11,12 The quantitative statistical assessment of the average height and diameter of individual rHBsAg particles was performed with the image software supplied by Digital Instrument after discounting obvious aggregation and agglomeration areas. Fluorescence studies of rHBsAg. The intrinsic fluorescence intensity of rHBsAg and a Bis-ANS • rHBsAg complex was studied in the presence and absence of GdmSCN. The fluorescence spectra were obtained by excitation at 280 nm and scanning the emission signals in the range 300–450 nm for rHBsAg or by excitation at 400 nm and scanning the emission signals in the range 430–550 nm for the Bis-ANS • rHBsAg complex. The fluorescence of the rHBsAg and the Bis-ANS • rHBsAg complex was also studied in the presence of GdmSCN. A stock solution of approximately 1.0 mM Bis-ANS was made in dimethyl sulfoxide and its exact concentration was determined using the extinction coefficient ε360nm = 23,000 cm-1 ⋅M-1. A final concentration of 20 µM Bis-ANS was introduced to a 1.7 µM HBsAg solution in PBS containing different amounts of GdmSCN. The final concentrations of GdmSCN were in the range of 0.2 to 3.8 M. The fluorescence intensity was measured after mixing and incubating the Bis-ANS • rHBsAg samples for at least 10 min. All fluorescence intensities were measured with a Fluorolog-22 Fluorescence Spectrophotometer (Spex Instruments, Edison, NJ).

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Table 1

The downstream treatment of rHBsAg preparations for particle maturation/aging

Treatment

Purpose

Molecular Events

3 M KSCN treatmenta

Cross-linking for better stability and immunogenicity Maturation of rHBsAg and epitope development for better antigenicity and immunogenicity

Maturation process: Sample A → B Maturation process: Sample B → C Storage at 37˚C for ~1 week and extended storage at 4˚C

Conditions

Ref./Note

Inter- and intra-molecular disulfide formation

Denaturing/4˚C

Referenceb and this work

Disulfide rearrangement to native disulfides; Inter- and intra-molecular disulfide formation; Increased fluidity of lipids

Native/37˚C and Native/4˚C

this work

aAs shown in Figure 1, the chromatographically purified Sample A is a process intermediate and the most upstream sample in the study. The subsequent treatment steps (KSCN treatment followed by dialysis back into PBS, followed by heat treatment and prolonged storage) are intended to demonstrate the different degrees of rHBsAg maturation or aging. bWampler et al., 1985.

(with both absorbance and fluorescence detections) attached, was used to mix these two solutions (Solutions A and B) in a 1:1 ratio using two syringes within the instrument to initiate the unfolding of rHBsAg in 2 M GdmSCN. The unfolding kinetics were observed by monitoring the decrease in fluorescence of bound Bis-ANS after exposure to denaturing conditions. The decrease in Bis-ANS fluorescence upon unfolding of the protein was monitored with excitation at 400 nm and total emission intensity was measured with a cut-off filter at 450 nm and above. The high-voltage setting for the detector was 700 V and slit widths for both excitation and emission were set at 2 mm. Experiments were performed at 25˚C. A total of 4,000 data points was collected in 200 ms for each run. Eight to twelve runs were performed for each sample and data points (fluorescence intensity vs. time) were averaged and exported for curve-fitting. Equation 1 was used to calculate the rate constants (k) and amplitudes (A) for the biphasic fluorescence decay: y = A0(1)⋅e - k1t + A0(2)⋅e - k2t + offset

(1)

Data analysis. Nonlinear least square fits of the fluorescence intensities vs. time with different models (including the biphasic unfolding kinetics model) were performed using Grafit.13

RESULTS Figure 2. SDS-NuPAGE gel showing the enhanced cross-linking and stability upon maturation/aging of the rHBsAg particles. Progressive increases in the oligomerization and in the resistance to SDS-induced denaturation were observed along the course of KSCN- and storage-induced maturation from Sample A to B and then to C (refer to Fig. 1 and Table 1).

Unfolding Kinetics of rHBsAg. An environment-sensitive probe, Bis-ANS, was used to monitor the rHBsAg unfolding kinetics in the presence of GdmSCN. A stock solution of approximately 1.0 mM Bis-ANS was prepared in dimethyl sulfoxide; the exact concentration of Bis-ANS was determined on the basis of the absorbance at 360 nm. The rHBsAg preparations were diluted to 160 nM in PBS. Subsequently, a small volume of Bis-ANS solution was introduced to the rHBsAg solution to make Solution A, containing a final concentration of 10 µM Bis-ANS. Solution A was prepared at least 10-15 min prior to the initiation of unfolding experiments. Solution B, 4.0 M GdmSCN chaotropic solution, was prepared by dissolving GdmSCN in PBS. A stopped-flow device from Applied Photophysics (Surrey, United Kingdom), with a spectrophotometer 176

Cross-linking of rHBsAg. Extensive cross-linking of monomeric HBsAg to form oligomers through disulfide bonds is known to occur in both plasma-derived HBsAg and rHBsAg. Limited cross-linking of rHBsAg occurs spontaneously during production in yeast and during the downstream purification process. Treatment of rHBsAg with 3 M KSCN was included in the purification process to promote disulfide bond formation,6 as shown by SDS-PAGE in (Fig. 2) (differences between Samples A and B). Two observations were made as a result of increased cross-linking: (1) a reduction in relative monomer content and (2) an increase in trimer or aggregates with higher molecular weights not entering the gel. This cross-linking is further enhanced by heat treatment (Sample C), rendering the particles completely resistant to SDS-induced denaturation. The lipo-protein particles in Sample C did not enter the gel, as shown in Fig. 2). Chemical modification and mutational analyses showed that most, if not all, of the eight Cys residues in the major hydrophilic region (amino acid 100-160) and four Cys residues in the first hydrophilic domain are likely involved in inter- or intramolecular disulfide bonds.7,14 Although there are no definitive assignments on

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Table 2

Statistical assessment of the rHBsAg morphological difference before and after KSCN treatment based on atomic force microscopy measurements

Average particle height (nm) (standard deviation) Average particle diameter (nm) (standard deviation) Number of rHBsAg particles averaged

Figure 3. AFM images of rHBsAg particles in Sample A and B. These images were obtained in aqueous environments to minimize the deformation of the lipo-protein particles. The lower figures show the expanded region from the specified portion of the top figures. Note that the protruded surface features of the rHBsAg particles are much better defined after KSCN-induced oxidative maturation.

the exact disulfide bond pairings in rHBsAg, C121 and C147 are proposed to be involved in intermolecular disulfides.14 Multiple intermolecular disulfides are formed in the lipid-containing particles to cross-link the adjacent monomers together to form dimers and high-order oligomers,13 coupled with structural changes within the subunits or at the interface. Higher degrees of cross-linking correlate with higher conformational stability and lower conformational flexibility in the mature or aged antigen preparations. Particle morphological changes upon maturation. Direct visualization of rHBsAg particles in the native aqueous environment was carried out with AFM for Samples A and B (pre and post- KSCN treatment). Tapping mode AFM in solution yields more faithful high-resolution images of protein surface.11,12 Distinct changes in morphology were observed upon KSCN-facilitated oxidative maturation (Fig. 3). Surface structural definition is poor prior to KSCN treatment; possibly due to the lack of cross-linking of subunits and poor lipid-protein organization precluding the optimal subunit interactions. After KSCN treatment, the rHBsAg particles are slightly larger, perhaps due to better surface epitope presentation or better overall structural definition or enhanced physical rigidity. Statistical assessment of the morphological changes supported this conclusion. As shown in Table 2, both averaged height and diameter of the individual rHBsAg particles were increased after KSCN treatment. The height increase reflects the fact that the particle is more rigid and more resistant to deformation on the mica surface. The degree of the deformation was indicated by the height on the mica surface. Fine structures of the protein subunits as shown by the enlarged images in Figure 3 are more clearly observable in Sample B after KSCN treatment; this observation supports a more rigid protein structure and better defined subunits, since poor AFM resolution is associated with molecular flexibility. However, due to the limited resolution of the AFM method, the images cannot establish whether the observed individual surface unit is a protein monomer, dimer, or higher www.landesbioscience.com

Sample A

Sample B

3.3 ± 1.3

5.2 ± 2.5

21.1 ± 4.3

23.4 ± 5.6

741

1136

oligomer. It is likely that, inside the yeast cell, the insertion of rHBsAg into the lipid membrane and the subsequent budding out may not be optimized. As a result, fewer protein subunits would be packed in a given lipid particle. Limited conformational search process is allowed in the yeast cells with high yield production of the same over expressed protein, leading to sub-optimal protein conformation and/or the subunit-subunit or lipid-protein interactions. After cell breakage and during purification, the rHBsAg insertion or exchange process into the lipid membrane was allowed to re-equilibrate during the 3 M KSCN treatment. This treatment is essentially a partial disassembly and reassembly process with the rHBsAg particles being exposed to the strong chaotrope for 16–18 h, promoting subunit refolding and exchange, followed by the removal of the chaotrope. High degree conformational stability of lipid-embedded rHBsAg. The purified rHBsAg particles are highly resistant to both thermal and chemical denaturation, and to proteolysis. Even in the presence of the commonly-used denaturants guanidinium chloride or urea, no clear two-state unfolding titration curves could be obtained for rHBsAg. In this regard, rHBsAg is unlike most properlyfolded globular proteins.15,16 Therefore, more drastic conditions using GdmSCN (with both cation and anion being strongly chaotropic) were employed to disrupt the native structure of rHBsAg. The intrinsic fluorescence of tryptophan residues in rHBsAg was initially explored for its sensitivity to structural changes. When excited at 280 nm, the maximal emission wavelength is 332 nm for rHBsAg under native conditions in PBS, and this maximal emission wavelength was red-shifted to 348 nm in the unfolded state in 3.8 M GdmSCN, indicating more solvent exposure of the buried tryptophan residues, consistent with most folded proteins. Eight out of a total of twelve tryptophan residues are located either in or near the transmembrane helices.17 The fluorescence intensity at 332 nm decreases as a function of GdmSCN concentration, appearing to be in a biphasic mode. Therefore, the kinetics of unfolding of the protein complex could be followed by monitoring the decay of fluorescence intensity upon exposure to chaotropic environments, although the sensitivity of the intrinsic fluorescence change is limited at the concentration where the highly hydrophobic rHBsAg lipoprotein particles are soluble. Structural integrity and accessible hydrophobic areas of rHBsAg. To enhance the sensitivity of the fluorescence intensity change upon HBsAg unfolding, a few environment-sensitive fluorescence probes were tested. Bis-ANS was found to be most sensitive for probing the structural changes of rHBsAg particles. Bis-ANS is a dimer of ANS and exhibits a very weak fluorescence in polar solvents due to solvent quenching. Bis-ANS binds reversibly to the hydrophobic areas of proteins, and this greatly enhances its fluorescence, making it useful as a conformational indicator for accessible hydrophobic cavities in proteins or for folding/unfolding studies.18,19

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Figure 4. The fluorescence intensity of the sample mixture containing 20 µM Bis-ANS and 1.7 µM rHBsAg as a function of GdmSCN concentration. The fluorescence intensity (million counts per minute or mcpm) was measured at ambient temperature (22–25˚C) with the excitation at 400 nm and the emission at 486 nm. The Bis-ANS binding peaked at ~0.5 M GdmSCN.

Figure 5. An example of an unfolding kinetic profile of rHBsAg in 2.0 M GdmSCN in the presence of Bis-ANS at 25˚C for rHBsAg in Sample C. The unfolding of rHBsAg (as indicated by the loss of accessible hydrophobic areas) displayed biphasic kinetics and obeyed Equation 1. Amplitudes of fluorescence changes and rate constants for different product stages are shown in Table 3.

The parameters during unfolding of rHBsAg particles in 2.0 M GdmSCN at 25˚C

structured conformation that may resemble a molten globule. Molten globules are generally achieved under mild denaturing conditions, with the conformation being in a local energy minimum somewhere between the Sample A Sample B Sample C native and denatured states.20-22 These molten globuleInitial Fluorescenceb 3.41 3.10 1.88 like states consist of some loosely-packed hydrophobic cores,23-25 resulting in increased binding of the Bis-ANS Unfolding rate constants k1obs, s-1 (fast) 145.5 ± 0.9 125.7 ± 0.6 133.5 ± 1.1 probe. The fluorescence intensity then decreased sharply with increasing amounts of GdmSCN (Fig. 4), which -1 k2obs, s (slow) 23.5 ± 0.9 22.4 ± 0.1 17.0 ± 0.1 we attribute to loss of hydrophobic cavities in the Lost hydrophobic areas during unfolding unfolded protein upon the disruption of the secondary A1, FL Unit (fast) 0.629 0.630 0.345 and tertiary protein structures as well as, to some A2, FL Unit (slow) 0.160 0.363 0.234 degree, disruption of the lipid structure or lipid-protein A1+A2, FL Unit c (total) 0.79 0.99 0.58 interaction. Percentage of hydrophobic area lost during fast unfolding process Stopped-flow kinetics of rHBsAg unfolding. It has been reported previously for phage p22 coat protein.26 Fast unfolding (A1)%c 80 63 59 and E. coli dihydrofolate reductase.27 that comparable aObserved unfolding rate constants (k or k ) and the corresponding fluorescence changes or amplitudes (A or A , arbi1obs 2obs 1 2 kinetic parameters were observed regardless of whether trary fluorescence unit or FL Unit) of the Bis-ANS • rHBsAg complex for fast and slow unfolding processes, respectively. Samples the intrinsic tryptophan fluorescence or extrinsic enviA, B and C are described in Figure 1. All parameters and standard deviations were derived from non-linear least square fit of a data set representing an average of 10–13 independent measurements using GraFit (9). A total of 4000 data points were ronment-sensitive fluorescence probes were monitored. collected in 200 ms for each measurement. The tabulated results are from the curve fitting with the double exponential fits Therefore, we employed Bis-ANS as a conformational (Fig. 5) using the data set with averaged data points from multiple runs. The ~6 ms dead time of the instrument was taken indicator for enhanced sensitivity in monitoring the into consideration when deriving the amplitudes. This dead time does not impact the calculation of the rate constants. bInitial unfolding/denaturation of the rHBsAg particles in three fluorescence, reflecting total bound Bis-ANS concentration, was calculated from (offset + A1 + A2). cNote the decreases from Sample A to Sample C in overall fluorescence changes (A1+A2) and in the percentage of the fast unfolding population in the different antigen preparations with different degrees of maturation. The kinetic studies were performed for the biphasic unfolding kinetics as rHBsAg particles mature. structural changes from native (without GdmSCN) to almost completely unfolded (in 2.0 M GdmSCN) Figure 4 shows the Bis-ANS fluorescence intensity in the rHBsAg rHBsAg particles. solution (Sample C, KSCN- and heat-treated) as a function of To assess the conformational flexibility and accessible hydrophoGdmSCN concentration. At low GdmSCN concentrations, a slight bic areas with Bis-ANS in both the native and the denatured states increase in fluorescence intensity was observed. We attribute this of rHBsAg, GdmSCN offers much higher denaturing capacity than increase to exposure of more hydrophobic patches allowing additional other denaturants at the same molar concentrations. The loss of Bis-ANS molecules to bind. The fluorescence of the Bis-ANS ⋅ native conformation (or the accessible surface area) in 2.0 M rHBsAg complex peaked at ~0.2–0.5 M GdmSCN. This increase in GdmSCN was monitored through the fluorescence changes of the Bis-ANS binding may suggest the presence of a non-native, partially Bis-ANS ⋅ rHBsAg complex with a stopped-flow device for real-time Table 3

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unfolding kinetics (Fig. 5). The unfolding kinetics on the millisecond time-scale showed Table 4 Relative changes in the amplitudes and unfolding rate constants during GdmSCN-induced denaturation for rHBsAg samples with biphasic kinetics (with two exponential different degree of maturation/aginga components) for all rHBsAg preparations. A biphasic model fit the data well, showing Sample A Sample B Sample C random residuals/noises (indication of good fit). b The calculated rate constants in the millisecond Rel. hydrophobic areas indicated by Bis-ANS 1.8 1.6 1.0 time-scale and the amplitudes for both the Total initial FL Unit in Native State Total FL unit lost in Unfolded State (A1 + A2) 1.4 1.7 1.0 fast- and slow-unfolding events are shown in Table 3 and Figure 6 for the three different Rel. changes in unfolding rate constants 1.09 0.94 1.00 product stages. Smaller amplitudes of fluores- Rel. k1obs (fast) Rel. k2obs (slow) 1.38 1.32 1.00 cence changes with slower rates were associated aThe parameters for conformational flexibility are reported as relative values to those for Sample C based on the values from Table 2. All the with the mature antigen preparation. In addition to the millisecond time-scale measurements were carried out with normalized protein concentrations for all samples. Results were normalized to that of Sample C. bThe unfolding, there is a much slower unfolding conformational flexibility of the rHBsAg in the lipoprotein particles is reflected by the total hydrophobic accessible areas (relative initial fluorescence in Table 3 in PBS (native state) and the total hydrophobic accessible areas (relative sum of A1 and A2 in Table 3 being lost in 2 M event for rHBsAg with a 4–6 min half-life as GdmSCN (unfolded state) as probed by Bis-ANS in the stopped-flow experiments. indicated by the further decrease in fluorescence of the Bis-ANS ⋅ rHBsAg complex (data not shown). This slower unfolding process is likely to be coupled to another cascade of changes in molecular structure, such as disulfide formation/exchange coupled to lipid rearrangement, which can be triggered by partial unfolding of rHBsAg. This unfolding process was not characterized in detail because it does not reflect the intrinsic conformational flexibility of rHBsAg particles in their native state. We assume that the initial fluorescence of the Bis-ANS ⋅ rHBsAg complex (first row of data in Table 3), which reflects the amount of Bis-ANS bound to hydrophobic cavities, correlates to the total accessible hydrophobic area in the native rHBsAg preparations. The increased compactness of the particles resulting from conformational change during maturation and the increase in disulfide bonds were reflected by the decreasing amount of total accessible hydrophobic area (Row 7, Table 3). Specifically, the initial fluorescence values for rHBsAg in Sample A–C are 3.41, 3.10 and 1.88, respectively (Table 3). These data suggest that approximately 50% of the accessible hydrophobic area in Sample A is either lost or no longer accessible to Bis-ANS in Sample C (Table 3 and Fig. 6). Consistent with a loss of conformational flexibility, the hydrophobic areas lost (proportional Figure 6. rHBsAg particles become more compact and less flexible upon maturation/aging. This is reflected by the smaller overall fluorescence to the total fluorescence change or A1 + A2 in Table 3 in the unfolded change, the smaller percentage of fast unfolding phase, and the slower state also decreased from Samples A and B (1.4 ~ 1.7) as relative to unfolding rates as rHBsAg matures (from Sample A to B, and then to C). Sample C (1.0) (Table 4). Therefore, the mature form of the prod- Values are relative to those of Sample A and are plotted for initial fluorescence uct showed less hydrophobic area under native conditions, and it (total accessible hydrophobic area in native state), total fluorescence change underwent the GdmSCN unfolding process at slower rates, and with (the hydrophobic areas lost upon unfolding in 2 M GdmSCN), percent of less hydrophobic area lost during the unfolding process. Relative amplitude of fast unfolding phase in the total fluorescence intensity lost, and fast/slow amplitude ratios for unfolding phases. changes in observed amplitudes and rate constants for Samples A, B, and C are shown in (Table 4).

DISCUSSION

Disulfide bond formation and/or shuffling and structural consolidation occur in rHBsAg lipoprotein particles, resulting in more compact surface structure and more rigid protein conformation. The unfolding kinetics of rHBsAg in the presence of a denaturant reveal a difference in the conformational flexibility before and after epitope development (via structural transformation). The matured rHBsAg particles are more compact, displaying slower unfolding rates and lower overall fluorescence changes. Furthermore, only a small fraction of the fluorescence change falls into the fast unfolding phase during the GdmSCN-induced unfolding (Tables 3 and 4). These observations are also consistent with increased rigidity of the particles after maturation as indicated with better maintained heights (i.e., greater www.landesbioscience.com

heights) when adsorbed on to mica surface (5.2 nm for Sample B vs. 3.3 nm for Sample A) in AFM studies under identical conditions (Table 2). Well-defined epitope structure and restricted conformational flexibility, often synchronized with disulfide bond formation/ exchange, could be critical in binding to neutralization antibody in vitro (antigenicity) and in eliciting neutralizing antibodies (immunogenicity) in vivo. Recently, this was demonstrated in the literature with a peptide tumor antigen MUC128 and a disaccharide antigen.29 Increased flexibility of the antigen was shown to be clearly associated with reduced immunogenicity in these studies. Maturation of the capsid on a slow time-scale (e.g., overnight or over days), involving disulfide bond formation, is common for the disulfidebonded capsid or nucleocapsid of different viruses. This has been

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observed for many viruses, including human papillomavirus,30,31 reovirus32 and respiratory syndrome virus.33 Due to the strong denaturing effect of the 3 M KSCN used in the preparation of rHBsAg, the disulfide bonds can form and rearrange, resulting in intermediate isomers that are preferred and stable under these denaturing conditions. Therefore, those disulfide bonds are likely to be different from the preferred isomers under native conditions.15 After the KSCN is removed and the solution is allowed to equilibrate in PBS and incubate at 37˚C, the protein conformation gains more native-like disulfide bonds that are energetically more stable under native conditions. Since lipids make up 25–35% of the total mass of the purified lipoprotein particles, higher lipid fluidity at 37˚C could facilitate a conformational search to yield more energetically-stable disulfide bond isomers via different disulfide bond pairing combinations. In Sample B, where a substantial amount of scrambled (or “nonnative”) disulfide bonds have been formed, disulfide exchange in rHBsAg is likely the dominant and rate-limiting process (in particular, the unscrambling of the “scrambled” isomers) during the spontaneous epitope refinement of rHBsAg. Without many preformed disulfides in Sample A, disulfide formation is likely the dominant and ratelimiting process for oxidative refolding. Therefore, the more restricted conformation of rHBsAg after maturation is likely a consequence of more complete disulfide bond formation with more native-like disulfide bond pairings. The mature form of rHBsAg particles (in Sample C) should bear a greater resemblance to the 22 nm plasmaderived or in vivo-produced34 (mouse version of ) lipoprotein HBsAg particles. In conclusion, the maturation of rHBsAg particles or the refinement of subunit and epitope structures is an intrinsic property of rHBsAg particles from yeast; this maturation occurs via disulfide bond formation and disulfide bond exchange throughout the purification process. This was demonstrated by increased cross-linking, particle uniformity and regularity, as well as decreased conformational flexibility with mature rHBsAg by unfolding kinetics and AFM studies. Substantial decrease in conformational flexibility could be important for better epitope definition, sustained antigen presentation, and increased resistance to in vivo proteolysis of the more compact particles, and could thus be potentially important for vaccine design and vaccine preparation. The observed changes in the conformational flexibility are consistent with the enhanced antigenicity (probed with conformation-sensitive antibodies) and immunogenicity for this important lipid-associated rHBsAg protein throughout the maturation process. Results for the immunochemical and immunological studies will be reported in due course. Acknowledgements

The protein in this study has the same amino acid sequence as the active ingredient of the commercial vaccine—RECOMBIVAX HB® (Merck) for the prevention of Hepatitis B virus infection. We would like to acknowledge Dr. Anna Hagen for her help in editing the manuscript and Dr. Dicky Abraham and Theresa Morley for their help with sample preparations. We would also like to thank Drs. Brent Oswald and Lorenzo Chen for their critical reading of the manuscript and helpful comments. Furthermore, we gratefully acknowledge the technical support from Dr. Michael Allen (at Biometrology, Inc.) for his effort in measuring these samples with atomic force microscopy.

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Human Vaccines

2006; Vol. 2 Issue 4