Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules.

We formulate a general analysis to determine the two-dimensional dissociation constant (2D Kd), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.

Mechanisms of Cellular Avidity Regulation in CD2-CD58-Mediated T Cell Adhesion.

The CD2 receptor on T lymphocytes is essential for T cell adhesion and stimulation by antigen presenting cells (APCs). Blockade of CD2 function is immunosuppressive in both model systems and humans, indicating the importance of CD2 for the cellular immune response. Although the affinity of the molecular interaction between CD2 and its counter-receptor, CD58, is relatively low when measured in solution, this interaction mediates tight adhesion within the 2D cell-cell interface. To understand the mechanisms responsible for regulating the avidity of the CD2-CD58 interaction, we measured the number, affinity, and lateral mobility of CD2 molecules on resting and activated T cells. Cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and the 2D affinity of CD2 for CD58 increased by 2.5-fold. The combination of T cell activation and CD2 ligation to CD58 decreased the laterally mobile fraction of the ligated CD2. Together, these changes would substantially enhance CD2 avidity and strengthen T cell-APC adhesion. The change in CD2 mobile fraction suggests that the cell uses cytoskeletal regulators to immobilize the receptor selectively at the site of contact with surfaces expressing CD58. Our observations are consistent with a model in which T cell activation initially induces increased CD2 2D affinity, cell surface receptor expression, and lateral mobility, allowing the CD2 molecules to diffuse to sites of contact with CD58-bearing APCs. Subsequently, T cell activation causes the CD58-bound CD2 to be recognized and immobilized at sites of cell-cell contact, thereby strengthening T cell-APC adhesion.

Molecular mechanism and thermodynamics study of plasmid DNA and cationic surfactants interactions.

The molecular mechanism and thermodynamics of the interactions between plasmid DNA and cationic surfactants were investigated by isothermal titration calorimetry (ITC), dynamic light scattering, surface tension measurements, and UV spectroscopy. The cationic surfactants studied include benzyldimethyldodecylammonium chloride, benzyldimethyltetradecylammonium chloride, cetylpyridinium chloride, and cetyltrimethylammonium chloride. The results indicate a critical aggregation concentration (cac) of a surfactant: above the cac the surfactant forms aggregates with plasmid DNA; below the cac, however, there is no detectable interaction between DNA and surfactant. Surfactants with longer hydrocarbon chains have smaller cac, indicating that hydrophobic interaction plays a key role in DNA-surfactant complexation. Moreover, an increase in ionic strength (I) increases the cac but decreases the critical micellization concentration (cmc). These opposite effects lead to a critical ionic strength (I(c)) at which cac = cmc; when I < I(c), cac < cmc; when I > I(c), DNA does not form complexes with surfactant micelles. In the interaction DNA exhibits a pseudophase property as the cac is a constant over a wide range of DNA concentrations. ITC data showed that the reaction is solely driven by entropy because both deltaH(o) (approximately 2-6 kJ mol(-1)) and deltaS(o) (approximately 70-110 J K(-1) mol(-1)) have positive values. In the complex, the molar ratio of DNA phosphate to surfactant is in the range of 0.63-1.05. The reaction forms sub-micrometer-sized primary particles; those aggregate at high surfactant concentrations. Taken together, the results led to an inference that there is no interaction between surfactant monomers and DNA molecules and demonstrated that DNA-cationic surfactant interactions are mediated by the hydrophobic interactions of surfactant molecules and counterion binding of DNA phosphates to the cationic surfactant aggregates.

Development of stable liquid formulations for adenovirus-based vaccines.

We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4 degrees C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze-thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 +/- 0.9 and 28.7 +/- 0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4 degrees C. The immunogenicity of a recombinant Ad5-based human immunodeficiency virus (HIV) vaccine candidate expressing HIV-1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4 degrees C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy.

Characterization and biological evaluation of a microparticle adjuvant formulation for plasmid DNA vaccines.

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

SYK and LYN mediate B-cell receptor-independent calcium-induced apoptosis in DT-40 lymphoma B-cells.

Here, we report that the calcium ionophore ionomycin induces a massive Ca2+-dependent apoptosis in wildtype DT-40 chicken B lymphoma cells, as well as in BTK-deficient, PLCgamma2-deficient and IP3 receptor-deficient DT-40 cells, but not in LYN- or SYK-deficient DT-40 cells. Notably, the deficiency of CSK, a negative regulator of Src-family PTK, promoted ionomycin-induced apoptosis of DT-40 cells. Reconstitution of SYK-deficient cells with wild-type SYK restored the apoptotic response of the cells to ionomycin, but the expression of FYN or LCK in LYN-deficient cells did not restore the apoptotic response of LYN-deficient cells. Taken together, our data suggests that both LYN and SYK, but not BTK, FYN or LCK, are crucial mediators of BCR-independent Ca2+-induced apoptosis in DT-40 lymphoma B cells.