Impact of aluminum adjuvants on the stability of pneumococcal polysaccharide-protein conjugate vaccines.

Development of a vaccine drug product requires formulation optimization to ensure that the vaccine's effectiveness is preserved upon storage throughout the shelf-life of the product. Although aluminum adjuvants have been widely used in vaccine formulations to safely and effectively potentiate an immune response, careful attention must be directed towards ensuring that the type of aluminum adjuvant does not impact the stability of the antigenic composition. PCV15 is a polysaccharide-protein conjugate vaccine comprising the pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F), each individually conjugated to the protein carrier CRM197. PCV15 was formulated with either amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS) or aluminum phosphate adjuvant (AP) and examined for both stability and immunogenicity. Using a collection of methods to evaluate vaccine stability, it was discovered that certain PCV15 serotypes (e.g., 6A, 19A, 19F) formulated with AAHS resulted in a reduction of immunogenicity in vivo and a reduction in recoverable dose as tested by an in vitro potency assay. The same polysaccharide-protein conjugates formulated with AP were stable regarding all measures tested. Moreover, the reduction in potency of certain serotypes correlated with chemical degradation of the polysaccharide antigen caused by the aluminum adjuvant as measured by reducing polyacrylamide gel electrophoresis (SDS-PAGE), High-Pressure Size Exclusion Chromatography coupled with UV detection (HPSEC-UV) and ELISA immunoassay. This study suggests a formulation, which includes AAHS, may negatively impact the stability of a pneumococcal polysaccharide-protein conjugate vaccine that contains phosphodiester groups. This decrease in stability would likely result in a decrease in the "active" concentration of antigen dose, and herein, it is shown that such instability directly compromised vaccine immunogenicity in an animal model. The results presented in this study help to explain critical degradation mechanisms of pneumococcal polysaccharide-protein conjugate vaccines.

Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine.

Phospholipid-containing antigens, such as Hepatitis B Surface Antigen (HBsAg), adsorb to aluminum-containing adjuvants by ligand exchange of a phosphate group for a hydroxyl group on the adjuvant surface. In this study, a tightness of binding (TOB) assay was developed to characterize the strength of binding between HBsAg and aluminum hydroxyphosphate sulfate adjuvant containing two levels of phosphate. Antigen desorption was induced using either fluoride or phosphate as a competing ion. HBsAg, formulated as a monovalent or combination vaccine, showed decreased tightness of binding when the amount of phosphate in the adjuvant composition increased, indicating that there was less ligand exchange between HBsAg and the adjuvant. Furthermore, the physicochemical property of TOB was related to enhanced immunogenicity in a murine model. These data show that tightness of binding can be a useful characterization tool, and potential predictor of immunogenicity, during development of vaccines that adsorb to aluminum adjuvants via ligand exchange.

Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response.

Hepatitis B surface antigen (HBsAg) is known to adsorb to aluminum hydroxide adjuvant (AH) by ligand exchange between its accessible phosphate groups and surface hydroxyl groups of the adjuvant. To study the effect of the binding strength, five vaccines were prepared with AH or four samples of AH that were modified by pretreatment with different concentrations of potassium dihydrogen phosphate. The adsorptive coefficients ranged from 3660 to 250mL/mg based on the Langmuir adsorption isotherm and degrees of elution ranged from 1 to 31% when the vaccines were exposed to interstitial fluid in vitro. When tested in mice the four vaccines containing phosphate-treated AH (PTAH) induced significantly greater antibody responses than the vaccine containing AH, which had the highest adsorptive coefficient and the smallest degree of elution of HBsAg. The results indicated that antibody production is reduced when the antigen is adsorbed too strongly. Thus, the strength of adsorption of the antigen to an aluminum-containing adjuvant can affect the immunogenicity of the vaccine and should be optimized during vaccine formulation.

Loss of PECAM-1 function impairs alveolarization.

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.