Physical characterization and formulation development of a recombinant pneumolysoid protein-based pneumococcal vaccine.

Streptococcus pneumoniae is a major cause of death in children worldwide. There are more than 90 known pneumococcus serotypes that vary by geographical location. Pneumolysin is a protein toxin produced by virtually all invasive strains of S. pneumoniae and is considered an important virulence factor. Pneumolysin is immunogenic and has the potential to be a new vaccine antigen offering broad serotype-independent coverage. To develop a stable vaccine formulation, the conformational stability of a recombinant pneumolysin mutant (pneumolysoid L460D) was characterized by various techniques. Three data visualization diagrams were constructed to summarize the biophysical data of the L460D pneumolysoid; the protein is most stable in solution at pH 6-7, and loses conformational integrity above 48°C. Excipient screening assays were performed and sugars such as trehalose and sucrose stabilized the pneumolysin mutant with respect to improving thermal transition temperatures and minimizing aggregation. In addition, the protein antigen showed efficient binding to aluminum hydroxide adjuvant. The conformational stability of the L460D pneumolysoid on the surface of alhydrogel adjuvant was little affected by adsorption, either with or without excipients. These studies provide important preformulation characterization information useful for the development of a stable pneumolysin mutant-based vaccine.

Preformulation characterization of an aluminum salt-adjuvanted trivalent recombinant protein-based vaccine candidate against Streptococcus pneumoniae.

The preformulation of a trivalent recombinant protein-based vaccine candidate for protection against Streptococcus pneumoniae is described both in the presence and in the absence of aluminum salt adjuvants. The biophysical properties of the three protein-based antigens, fragments of pneumococcal surface adhesion A (PsaA), serine-threonine protein kinase (StkP), and protein required for cell wall separation of group B streptococcus (PcsB), were studied using several spectroscopic and light scattering techniques. An empirical phase diagram was constructed to assess the overall conformational stability of the three antigens as a function of pH and temperatures. A variety of excipients were screened on the basis of their ability to stabilize each antigen using intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. Sorbitol, sucrose, and trehalose stabilized the three proteins in solution. The addition of manganese also showed a drastic increase in the thermal stability of SP1650 in solution. The adsorption and desorption processes of each of the antigens to aluminum salt adjuvants were evaluated, and the stability of the adsorbed proteins was then assessed using intrinsic fluorescence spectroscopy and Fourier transform infrared spectroscopy. All the three proteins showed good adsorption to Alhydrogel. PsaA was destabilized when adsorbed onto Alhydrogel® and adding sodium phosphate showed a stabilizing effect. PcsB was found to be stabilized when adsorbed to Alhydrogel®, and no destabilizing or stabilizing effects were seen in the case of StkP.

Formulation development of a plant-derived H1N1 influenza vaccine containing purified recombinant hemagglutinin antigen.

Influenza is a prevalent, highly contagious and sometimes fatal respiratory disease. Vaccination provides an effective approach to control the disease, but because of frequent changes in the structure of the major surface proteins, there is great need for a technology that permits rapid preparation of new forms of the vaccine each year in sufficient quantities. Recently, using a safe, simple, time- and cost-effective plant viral vector-based transient expression system, the hemagglutinin antigen of H1N1 influenza A strain (HAC1), an H1N1 influenza vaccine candidate, has been produced in Nicotiana benthamiana plants. As a step toward the generation of a commercially viable subunit influenza vaccine, we developed HAC1 formulations in the presence and absence of an aluminum salt adjuvant (Alhydrogel(®)), analyzed their properties, and assessed immunogenicity in an animal model. Biophysical properties of HAC1 were evaluated using several spectroscopic and light scattering techniques as a function of pH and temperature combined with data analysis using an empirical phase diagram approach. Excipients that were potent stabilizers of the recombinant protein were identified using intrinsic fluorescence spectroscopy. The adsorptive capacity and thermal stability of the protein on the surface of Alhydrogel(®) were then examined in the presence and absence of selected stabilizers using UV absorbance after centrifugation and intrinsic fluorescence spectroscopy, respectively. Immunogenicity studies conducted in mice demonstrated that the highest level of serum immune responses (hemagglutination-inhibiting antibody titers), with a 100% seropositive rates, were induced by HAC1 in the presence of Alhydrogel(®), and this response was elicited regardless of the solution conditions of the formulation.

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca2+-ATPase: dependence on enzyme and Ca2+ concentrations.

Calmodulin (CaM) is a Ca2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the Kd for the interaction of CaM with the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca2+ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K(d) in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K(d) value of CaM with PMCA (5.8 +/- 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K(d) = 9.8 +/- 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca2+ to the binding sites in CaM.

Interchange of autoinhibitory domain conformations in plasma-membrane Ca2+-ATPase-calmodulin complexes.

The Ca2+ signaling protein calmodulin (CaM) stimulates Ca2+ pumping in the plasma-membrane Ca2+-ATPase (PMCA) by binding to an autoinhibitory domain, which then dissociates from the catalytic domain of PMCA to allow full activation of the enzyme. We measured single-molecule fluorescence trajectories with polarization modulation to track the conformation of the autoinhibitory domain of PMCA pump bound to fluorescently labeled CaM. Interchange of the autoinhibitory domain between associated and dissociated conformations was detected at a physiological Ca2+ concentration of 0.15 microM, where the enzyme is only partially active, but not at 25 microM, where the enzyme is fully activated. In previous work we showed that the conformation of the autoinhibitory domain in PMCA-CaM complexes could be monitored by the extent of modulation of single-molecule fluorescence generated with rotating excitation polarization. In the present work, we determined the timescale of association and dissociation of the autoinhibitory domain with the catalytic regions of the PMCA. Association of the autoinhibitory domain was rare at a high Ca2+ concentration (25 microM). At a lower Ca2+ concentration (0.15 microM), conformations of the autoinhibitory domain interchanged with a dissociation rate of 0.042 +/- 0.011 sec(-1) and an association rate of 0.023 +/- 0.006 sec-1. The results indicate that the response time of PMCA upon a reduction in Ca2+ is limited to tens of seconds by autoinhibitory dynamics. This property may reduce the sensitivity of PMCA to transient reductions in intracellular Ca2+. We suggest that the dynamics of the autoinhibitory domain may play a novel role in regulating PMCA activity.

Tyrosyl rotamer interconversion rates and the fluorescence decays of N-acetyltyrosinamide and short tyrosyl peptides.

It has long been recognized that the fluorescence lifetimes of amino acid residues such as tyrosine and tryptophan depend on the rotameric configuration of the aromatic side chain, but estimates of the rate of interchange of rotameric states have varied widely. We report measurements of the rotameric populations and interchange rates for tyrosine in N-acetyltyrosinamide (NATyrA), the tripeptide Tyr-Gly-Gly (YGG), and the pentapeptide Leu-enkephalin (YGGFL). The fluorescence lifetimes were analyzed to determine the rotameric interchange rates in the context of a model incorporating exchange among three rotameric states. Maximum entropy method analysis verified the presence of three fluorescence decay components for YGGFL and two for YGG and NATyrA. Rotameric exchange between the gauche(-) and trans states occurred on the nanosecond time scale, whereas exchange with the gauche(+) state occurred on a longer time scale. Good agreement was obtained with rotameric populations and exchange rates from molecular dynamics simulations. Quenching by iodide was used to vary the intrinsic fluorescence lifetimes, providing additional constraints on the determined interchange rates. The temperature dependence was measured to determine barriers to exchange of the two most populated rotamers of 3, 5, and 7 kcal/mol for NATyrA, YGG, and YGGFL, respectively.